Proxy binding for screening therapeutic compounds

Abstract

Method for treating a subject for a cancer are provided. The method may include treating a subject for a cancer responsive to MYC inhibition, the method comprising administering a MYC inhibitor to the subject if the MYC inhibitor Response Signature (MiRS) score for the cancer is greater than 0.

Description

[0001]Attorney Docket No.702581.02595 (NU2023-153-02) PROXY BINDING FOR SCREENING THERAPEUTIC COMPOUNDS CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority of United States Provisional Patent Application Ser. No. 63 / 612,045, filed December 19, 2023. The contents of which is incorporated by reference in its entirety. STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT This invention was made with government support under grant numbers CA250196, CA257258 and CA180995 awarded by the National Institutes of Health. The government has certain rights in the invention. REFERENCE TO AN ELECTRONIC SEQUENCE LISTING The contents of the electronic sequence listing (70258102595.xml; Size: 23,594 bytes; and Date of Creation: December 19, 2024) is herein incorporated by reference in its entirety. BACKGROUND Intrinsically disordered proteins (IDPs) are implicated in many human diseases. However, paucity of structural insights into how small molecules bind to intrinsically disordered regions (IDRs) represents a major barrier to drug development. A prime example of a difficult to target IDP is the MYC transcription factor, which is involved in a majority of human cancers. The c-MYC oncogene is de-regulated and plays a causal role in a majority of human cancer and c-MYC inhibition profoundly affects tumor growth or survival in multiple models. MYC is the most common oncogene involved in human cancers and is overexpressed in up to half of all cancers. Therefore, developing c-MYC inhibitors is among the most attractive potential anti-cancer strategies. Unfortunately, due to the difficulty in targeting transcription factors with small molecules, c-MYC is currently regarded as “undruggable.” Thus, there remains an unmet need to develop effective small molecule compounds with biological activities toward IDPs, including MYC protein for the treatment of diseases. Attorney Docket No.702581.02595 (NU2023-153-02) SUMMARY Method for treating a subject for a cancer are provided. The method may include treating a subject for a cancer responsive to MYC inhibition, the method comprising administering a MYC inhibitor to the subject. Another aspect of the technology provides for a method for treating a subject for a cancer, the method comprising determining the binding of an MYC inhibitor to an intrinsically disordered region of MYC and one or both of a distal N-terminal region of MYC (ΔC) and an intrinsically disordered region of GCN1 and administering the MYC inhibitor if the MYC inhibitor binds to the intrinsically disordered region of MYC and one or both of the distal N-terminal region of MYC (ΔC) and the intrinsically disordered region of GCN1. Another aspect of the technology provides for a method for screening a compound for biological activity toward an intrinsically disordered protein (IDP), the method comprising determining a binding affinity of the compound to a proxy protein; and evaluating the biological activity of the compound toward the IDP based on the binding affinity of the compound to the proxy protein, wherein the proxy protein is less disordered than the IDP and comprises a binding domain with at least 80% sequence identity to an intrinsically disordered region (IDR) of the IDP; wherein the compound binds to the binding domain of the proxy protein. Another aspect of the technology provides for a method of identifying a cancer responsive to MYC inhibition, the method comprising determining a MYC inhibitor Response Signature (MiRS) score for the cancer. Another aspect of the technology provides for a method for treating a subject for a cancer responsive to ATF4 activation or induction, the method comprising administering a GCN1 ligand that activates or induces expression of ATF4. Another aspect of the technology provides for a method for sensitizing a subject having cancer to MYC inhibition treatment, the method comprising activating or inducing expression of ATF4 in the subject. BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 shows that MYCi activates the ATF4-ISR pathway. (a) ATAC-seq in MYCi975 treated cells. (b) ATF motif identified from ATAC-seq in 22Rv1, and MCF7 cells. (SEQ ID NO: 17). (c) Western blot analysis of ATF4 in 22Rv1 cells after MYCi (10μM) treatment at the indicated time points. (d) Structures of MYCi975 and its inactive analog, the regio-isomer 733. (e) Biolayer interferometry assay showing binding of MYCi975 but not Attorney Docket No.702581.02595 (NU2023-153-02) 733 to purified recombinant full length protein. (f) ATF4 and MYC protein levels in MycCaP cells after treatment with MYCi and 733 for 24hrs at indicated concentrations. (g) Heatmap representation of all ATF4 bound sites, showing ATF4 CHIP-Seq coverage in DMSO versus 16-hour MYCi975 (10 μM) treated 22RV1 cells. Figure 2 shows that MYCi activates an integrated stress response pathway, and activation of ATF4 by structurally diverse small-molecule MYC inhibitors, but not peptide inhibitor OmoMyc. (a) Heat map of unsupervised clustering representing genes whose abundances were significantly different between MYCi treated (8μM for PC3, 6μM for P493- 6, both for 24hours) and vehicle control, depicted along side genetic MYC inhibition: siRNA for PC3 and Tetracycline (Tet 0.1mg / mL for 48 hrs) for P493-6cells. Cluster 1 (C1) represents genes downregulated by both genetic and pharmacologic inhibition, while C3 are genes upregulated by both conditions relative to vehicle control. C2 and C4 represent clusters of genes for which pharmacologic inhibition resulted in opposite regulation compared to genetic inhibition. The relative abundance of each transcript is depicted as z score across rows (n=3; FDR≤0.05). (b-c) Table of HALLMARK pathways significantly enriched in Cluster 4 (genes uniquely upregulated by MYCi versus genetic controls) of PC3 (b) and P- 493 (c) cells, obtained from the unsupervised hierarchical clustering performed in “a”. (d) Gene set enrichment analysis of the ISR signature genes in PC-3, P493-6, 22Rv1 and MycCaP cells treated with MYCi975 (same conditions as in (a) for PC3 and P493-6, while 22RV1 and MycCap cells were treated with 10 μM MYCi for 24 hours). NES = normalized enrichment score. (e) ATAC-seq in 22RV1 and MCF7 cells treated with MYCi975 (10 μM).(f) ATF4 levels in cells treated with MYCi975 (10 M) for 24 hours. (g) ATF4 and MYCNprotein levels in neuroblastoma SK-N-BE (2) cells after treatment with 10μM of MYCi975 treatment for 24 hrs. (h) Plot of ATF4 induction (Fold over MYCi975) versus MYC inhibition (degradation as % of MYCi975) of 41 MYCi-related analogs. (i) Structures of stereoisomers 435 and 436. (j) MYC and ATF western in cells treated with compounds from 435, 436 and 975. (k) E-box reporter (4 hours treatment) activity of 435 vs 436 and MYCi975 positive control at the indicated concentrations in MycCaP cells stably transduced with E-box luciferase reporter. (l) Western blot for ATF4 in PC3 and MycCaP cells after treatment for 24 hours with various small-molecule MYC inhibitors - 10058-F4 (40 μM), 10074-G5 (20 μM), EN4 (10 μM), Mycro 3 (15 μM), MYCMI-6 (10 μM), KJ Pyr9 (10 μM) and KSI-3716 (8 μM). n=3 repeats, representative blot shown (m) Western blot analysis of ATF4 and MYC protein levels in 22Rv1 cells after 24 hours treatment with 10 μM OmoMYC (n = 3, a representative blot shown). OMOMYC does not lead to ATF4 activation. (n) Western blot for Attorney Docket No.702581.02595 (NU2023-153-02) MYC and ATF4 protein in PC3 cells treated for 24 hours with 435, 436 or MYCi975 at 10 μM. (o) Volcano plot displaying differentially bound ATF4 peaks after 16-hour treatment with 10 μM of MYCi975 in 22Rv1 cells. (p) Heatmap of ATF4 fold change as quantified from repeat Western blot analyses (n=3) of ATF4 protein level after treatment with structurally diverse MYC inhibitors at indicated concentrations. Figure 3 shows that (a) Western blots shows ATF4 level in MEF eIF2αS / S and eIF2αA / A cells after MYCi975 treatment for 24 hrs. (b) MycCaP cells were treated with PERK (10μM) and GCN2 (5μM) inhibitor alone or in presence of MYCi for 24 hrs. (c) GCN1 identified in both proteomics of MYCI-bound protein and by PHI-blast of MycHot region. (d) Alignment of the MYC family and GCN1 in the MycHot region (SEQ ID NOs: 18-22). (e) Biotin-975 pulldown of GCN1 and competition by soluble 975. (f) Ribosomal profiling after MYCi975 treatment (2hr). (g) ATF4 induction in GCN1 KO cells. Figure 4 shows that activated canonical EIF2 / ATF4 pathway shapes the MYC1-induced genome-wide transcriptional regulatory response. (a) Western blot for MYC and ATF4 in P493-6 cells treated with MYCi975 (10 μM) or Thapsigargin (THG, 0.1 μM) for 24 hours in the presence or absence of doxycycline (pre-treatment for 48 hours with 0.1 μg / mL). MYC suppression in P493-6 with doxycycline attenuates ATF4 induction by 975 and thapsigargin (THG). (b) MYC siRNA attenuates ATF4 induction by 975. (c) Western blot for MYC and ATF4 in rat fibroblasts (TGR-1 = WT, HO.15 = isogenic MYC KO clone) after treatment for 24 hours with MYCi975 at indicated concentrations. (d) Western blots of total and phosphorylated eIF2α and ATF4, as well as overall protein synthesis rates as measured by puromycin labeling in PC3 cells after treatment with MYCi975 (10 μM) at indicated timepoints. (e) Western blot of ATF4 and MYC in PC3 cells treated with PERK (10 μM) and GCN2 (5μM) inhibitor alone or in presence of MYCi975(10 μM) for 24 hours. (f) Western blot of ATF4 after treatment with MYCi975 (10 μM) for 24 hours in isogenic pair of MEFs (S / S = WT eIF2α, A / A = Serine51 mutated to Alanine on both alleles). (g) Western blot of ATF4 and MYC in MycCaP cells treated with PERK inhibitor (10 μM) and GCN2 (5μM) inhibitor alone or in presence of MYCi975 (10 μM) for 24 hours. (h) Heatmap of differential H3K27ac deposition in 22Rv1 cells treated with 10 μM MYCi975 for 16 hours. Differential H3K27ac regions overlapping with sites of ATF4-gain or MYC-loss are indicated by black solid bars in the adjacent single-column heatmaps. The fraction of H3K27ac sites containing overlapping ATF4-gain or MYC-loss peaks in the H3K27ac decrease or H3K27ac increase clusters are summarized in the bar charts above the ATF4- gain / MYC-loss single-column heatmaps. (i) Volcano plot of differential H3K27ac sites (FDR Attorney Docket No.702581.02595 (NU2023-153-02) < 0.01) in 10 μM-treated 22RV1 cells after 16 hours. H3K27ac regions overlapping with increased ATF4 (FDR < 0.01) are highlighted towards the right. (j) Volcano plot of differential H3K27ac sites (FDR < 0.01) in 10 μM-treated 22RV1 cells after 16 hours. H3K27ac regions overlapping with loss of MYC (FDR < 0.01) are highlighted towards the left. (k) Heatmap of all chromatin sites at which MYCi induces statistically significant ATF4 gain in 22RV1 cells after 16 hours of 10 μM treatment, compared to DMSO control. For all the gained ATF4 peaks, the differential H3K27ac signal is also plotted. (l) Violin plots of significantly differentially induced H3K27ac sites after 16 hours of 10 μM MYCi treatment in 22RV1 cells. The two plots are comparing the log2FC of MYCi-induced H3K27ac gain at sites that overlap with increased MYCi-induced ATF4 chromatin binding versus sites that do not. Figure 5 shows (a) Alpha-fold model of MycHot and surrounding region in GCN1. (b) Docking of 975 to GCN1. (c) Deletion mutant MYC pulldown by biotin-975. (d, e) Alpha-fold models of MYC and C-Linker-G MYC showing C region helices coming near the MycHot region to form a potential pocket. (f). BLI for binding of full length MYC (FL) and C region containing N terminal 1-353 amino acids of MYC. (g) BLI data for MYC proteins. Figure 6 shows that MYCi-induced ATF4 mediates anti-tumor efficacy and remodeling of the TIME. (a) Western blot for ATF4 and MYC in Doxycycline (Dox) inducible shATF4 MycCaP cells treated with MYCi975 (10 μM) for 24 hours in the presence or absence of 0.5 μg / mL Dox. (b) GSEA plot of ISR Signature in MycCaP shATF4 cells treated with MYCi975 for 24 hrs compared to MYCi975 + Dox. (c) Scheme for FVB mice bearing established MycCaP shATF4 allografts treated with MYCi975 (50 mg / kg BID i.p.) and Doxycyline (1 mg / ml in drinking water) for 3 weeks and average of tumor volumes. Tumor growth was compared between groups using linear mixed effects models with mouse as the random effect and first-order autoregressive (AR(1)) correlation structure between repeated tumor size measurements. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. (d) Representative images of H&E and IF staining for Ki67, Cleaved Caspase 3, CD3+, Nkp46, PD-L1, and FoxoP3, in MycCaP tumor tissue after MYCi and Doxycycline (Dox.) treatment from the study in “c”. Scale bar, 50μm. (e) Quantification of data from “d”. n=4-5 per graft / group. Statistical significance was analyzed by two-tailed Student’s t-test. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Figure 7 shows (a) Cell viability of shATF4 MycCaP cells after treatment with Doxycyline (Dox.) (500ng / ml) and MYCi (5μM) for 72hrs. Statistical analysis performed with two-sided Student’s t test. (b, c) Inducible knockdown of ATF4 with Doxycycline (500 Attorney Docket No.702581.02595 (NU2023-153-02) ng / mL) in 22Rv1 cells attenuates ATF4 induction and MYCi975 efficacy. (d) Heatmap of RNA-sequencing transcriptome analysis for 92 selected genes (part of ISR signature) from MYCi975 and Doxycycline (Dox.) treated MycCaP shATF4 cells for 24 hours. Difference in shades corresponds to per-gene z-score that is computed from pseudolog of DESeq2 normalized counts (after adding 0.01). Genes and samples were hierarchically clustered based on complete-linkage clustering using maximum clustering distance. Genes highlighted are referenced in the main text. From top to bottom: GOT1, DDIT4, NFIL3, SESN2, AFT4, PSPH, PAQR3, SLC7A3, TRIB3, CHAC1, GLCE, ALDH1L2, VEGFA, GTPBP2, SOAT2, CRLS1, GNPNAT1, TCEA1, EIF2S2, HAX1, SLC25A33, CYB5R1, UBR2, ATF3, STEAP1, ANGPTL6, CTH, GADD45A, MTBP, OTUB2, KRTCAP2, EIF4EBP1, MARS1, PHGDH, TGIF1, GHITM, IFRD1, LONP1, MTHFD1L, SHMT2, NOP14, FADS3, PFKP, PCK2, EIF3C, ALDH18A1, CDKN1A, MDFIC, SLC7A5, NFE2L1, SLC6A9, CLN8, DERL1, ARHGEF2, CIART, PPP1R15A, HMOX1, PVR, ATF6, NUPR1, SLC3A2, HERPUD1, TBC1D31, DDIT3, SCPEP1, MTHFD2, RHBDD1, NIBAN1, HSPA9, ASNS, SIAH2, GPT2, LEPROTL1, UHRF1BP1, PYCR1, CLIC4, SLC1A4, CEBPB, PHF10, SLC7A11, ATF5, SLC1A5, XPOT, (e) Tumor curves for Figure 6, panel c. (f) Mouse body weights for Figure 6, panel c. (g) Amount of ATP released in media after treatment of MYCCaP shATF4 cells with vehicle (Veh), Doxycyline (Dox.) (500 ng / ml), MYCi975 (10 μM) or MYCi975 and Dox together for 24 hours. Statistical significance was analyzed by two-tailed Student’s t-test. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Figure 8 shows the development of a MYC inhibitor Response Signature (MiRS). MYCi Response Signature (MiRS) score predicts in vivo MYCi efficacy. (a) Scheme for development of MiRS. (b) MiRSgenes and their known status as MYC or ATF4 target genes. (c) Correlation plot of MiRS scores of 6 different cancer cell lines and the tumor growth inhibition (TGI) percentage in vivo after MYCi treatment with 100 mg / kg / d p.o. N=10 mice per model per treatment (total 120 tumors). (d) Chi-square analysis of data from “c”. “MYCi975 Effective” is defined as tumor growth inhibition larger than 30% for each of the individual mice in the 6 in vivo studies. MiRS High is defined as a MiRS score larger than 0.2. Figure 9 shows (a) Tumor growth curves from xenografts treated with vehicle or MYCi975100mg / kd / d po as indicated (N=10 mice per group). Tumor growth was compared between groups using linear mixed effects models with mouse as the random effect and AR(1) correlation structure between repeated tumor size measurements. *P < 0.05, Attorney Docket No.702581.02595 (NU2023-153-02) **P < 0.01, ***P < 0.001, ****P < 0.0001. (b) TGI correlation to signature scores based on MYC Hallmark genes and UPR genes. Figure 10 shows ATF4 rescue with MYCi-975. Figure 10 shows the effect of GCN1 Knockout on ATF4 Induction in PC12 Cells Treated with MYCi-975. ATF4 induction was assessed in PC12 cells following GCN1 knockout (KO) using pLentiCRISPR v2. Cells were treated with MYCi-975 (20μM) for 24 and 48 hours. The results demonstrate an attenuation of ATF4 induction in GCN1 KO cells compared to Cas9 control, indicating the role of GCN1 in mediating the cellular response to MYCi-975. Cells were processed for western blot with indicated antibodies. Figure 11 shows ATF4 rescue with MYCi-975. ATF4 Attenuation in 22Rv1 cells with GCN1 Knockout (KO) and MYC Knockdown (KD) Treated with MYCi-975. ATF4 induction was evaluated in 22Rv1 cells with simultaneous GCN1 KO and MYC KD, treated with 10μM MYCi-975. The results demonstrate a significant attenuation of ATF4 compared to control cells, highlighting the combined impact of GCN1 KO and MYC KD on MYCi-975-mediated ATF4 induction. Cells were processed for western blot with indicated antibodies. Figure 12 shows that acetylation of lysine residues 148 and 157 enhances MYC C- region stability in silico and increases MY Ci binding in BLI assay. a) Highest-confidence Alpha Fold 3 model of unacetylated MYC. Blue: Basic Helix-Loop-Helix / Leucine Zipper (bHLH / LZ); Yellow: C-region (AA 128-189); Red: lysine residues 148 (K148) and 157 (K157). -c) average predicted local distance difference test (pLDDT) stability scores of each amino acid for unacetylated (blue) and acetylated (red) models of MYC across b) full protein and c) C-region. d) pLDDT score ranges for models of unacetylated and acetylated MYC at residues in the C-region where average pl DDT score becomes "high" (> 70) in acetylated models. e) Predicted aligned error (PAE) values for amino acid pairs across full-length MYC and MYC C-region. In all experiments, the top five models for unacetylated MYC and MYC acetylated at K148 and K157 according to Alpha Fold 3 Server were used. f) Binding affinity of mutant MYC proteins by BLI assay. Figure 13 shows a) Pull-down using Biotin-975 followed by western block with MYC (Y69) antibody to detected total MYC or the K148AcMYC antibody to detect MYC acetylated on lysine 148. GAP DH is included as a control. b) Quantitation of data from "a". ** P<0.01. Figure 14 shows MYC and K148Ac-MYC levels in human normal prostate and prostate cancer tissues by western blot. GAP DH is included as a control for western blot. 22RV1 prostate cancer cell lines are included as an additional control. Attorney Docket No.702581.02595 (NU2023-153-02) Figure 15 shows that MYCi preserves the function of adaptive and innate immune cells. (a) Schematic of mouse in vivo experiment for assessing effects of MYCi975 on the function of cells of the immune system. (b) Barplots depicting percentage of FACS-sorted immune cell populations from isolated mouse spleen and lymph nodes after vehicle or MYCi975 treatment. (c) Representative images of crystal-violet stained bone-marrow derived macrophages after treatment with Vehicle or MYCi975 as part of migration assay performed with either normal media (DMEM) or conditioned media. (d) Quantification of data from “c”. n = 4 separate assays set up with BMDMs isolated from each of 4 mice treated with either vehicle or MYCi975. Statistical significance was analyzed by two-tailed Student’s t-test. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. DETAILED DESCRIPTION The present disclosure relates to development of compound that have biological activities toward an intrinsically disordered protein (IDP), such as MYC transcription factor for cancer treatment. The development of approaches to target IDPs rationally with small molecules will have a major impact on the development of drugs to treat a range of currently untreatable diseases. IDPs and IDRs have a high degree of conformational flexibility. An intrinsically disordered protein or IDP is a biologically active protein that exists as ensembles of unfolded, collapsed, extended, non-globular conformations at the secondary or tertiary structural level. IDRs are protein segments of an IDP that exists as ensembles of unfolded, collapsed, extended, non-globular conformations at the secondary or tertiary structural level. As demonstrated in the Examples, analysis of small molecule binding to conserved IDRs in more ordered proteins provide helpful structural insights into small molecule binding to IDPs. The development of approaches to target IDPs rationally with small molecules may have a major impact on the development of drugs to treat a range of currently untreatable diseases. In particular, MYC is an IDP that is among the most frequently altered oncoproteins in human cancer. It regulates multiple hallmarks of cancer. There is no discernible “druggable” pocket is present in MYC. The lack of a defined structural basis for interactions between MCYi and MYC makes it unclear how these MYC-binding molecules inhibit MYC, their mechanism of action and their specificity. An IDR in the basic helix-loop-helix (bHLH) domain called MycHot is a frequent site for the binding of small molecules to MYC (1, 2). In various embodiments, the present disclosure shows that small molecule MYC inhibitors (MYCi) also target a conserved intrinsically disordered region (IDR) present in the ribosome-binding region of the amino acid stress response sensor General Control Nonderepressible 1, GCN1. Modeling MYCi Attorney Docket No.702581.02595 (NU2023-153-02) binding to GCN1 and to MYC suggests that an additional distal portion of the MYC protein contributes to MYCi binding. The present disclosure confirmed this by binding studies using full-length and MYC mutants. MYCi binding to GCN1 disrupts ribosomes and activates the eIF2α / ATF4 pathway. ATF4 activation by MYCi promotes cancer cell death and remodeling of the tumor immune microenvironment. The present studies indicate that bifunctional activity of MYC inhibitors is important for their efficacy. MCYi may also bind to a distal N-terminal region of MYC and that binding to the IDR and distal N-terminal region improves binding. In an aspect, the disclosed technology allows for the screening of therapeutic compounds to identify those capable of modulating the biological activity of an IDP by a compound by evaluating the compounds’ ability to modulate the activity of a proxy protein. A proxy protein is a protein that share sequence similarity to an IDR of the IDP. Suitably, the proxy protein may have at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with an IDR of the IDP. "Percentage of sequence identity'' or "percent similarity" is determined by comparing two optimally aligned sequences over a comparison window, wherein the portion of the polynucleotide or peptide sequence in the comparison window may comprise additions or deletions (i.e., gaps) as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions at which the identical nucleic acid base or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison and multiplying the result by 100 to yield the percentage of sequence identity. The term "substantial identity'' or "substantial similarity" of polynucleotide or peptide sequences means that a polynucleotide or peptide comprises a sequence that has at least 80% sequence identity. Alternatively, percent identity can be any integer from 80% to 100%. More preferred embodiments include at least: 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% compared to a reference sequence using the programs such as BLAST using standard parameters. These values can be appropriately adjusted to determine corresponding Attorney Docket No.702581.02595 (NU2023-153-02) identity of proteins encoded by two nucleotide sequences by taking into account codon degeneracy, amino acid similarity, reading frame positioning and the like. The method for screening the compound may comprise determining a binding affinity of the compound to a proxy protein and evaluating the biological activity of the compound toward the IDP based on the binding affinity of the compound to the proxy protein. The proxy protein is less disordered than the IDP and comprises a binding domain with at least 80% sequence identity to an intrinsically disordered region (IDR) of the IDP. The compound binds to the binding domain of the proxy protein. The biological activity of interest may comprise inhibition, activation, modulation, or regulation of the IDP by the compound. Determination of the binding activity may include methods known in the art, such as molecular modeling, pulldown assay, biolayer Interferometry (BLI) or a combination thereof. GCNI is an exemplary proxy protein. The IDR may comprise a basic helix-loop-helix (bHLH) domain. MYC is an exemplary IDP. The compound may be an inhibitor of the IDP. For example, the IDP may be MYC protein and the compound a MYC inhibitor such as any othe MYC inhibitors disclosed herein. In an aspect, the disclosed technology allows for identifying diseases or disorders responsive to the inhibition of an IDP. As demonstrated in the Examples, cancers responsive to MYC inhibition may be identified by determining a MYC inhibitor Response Signature (MiRS) score for the cancer. The MiRS can be used to identify patients that can benefit from MCYi administration. The MiRS score may be determined by gene set variance analysis of a MYC inhibition responsive gene, such as any of the genes identified herein. Exemplary genes may include, but are not limited to, ADM2, AIG1, AKNA, ANK2, ARHGEF2, ATF3, BBC3, BMF, CCNE2, CHAC1, DDIT3, DDIT4, ERN1, FYN, GADD45A, GDF15, GINS2, GTPBP2, HMOX1, KDM7A, MCM4, NUPR1, PPP1R15A, RELN, RRM2, TCP11L2, THSD7A, TK1, TR1B3, UHRF1, UNG, and YPEL2. In some embodiments, the MYC inhibition responsive gene is a MYC target or a ATF4 target. A positive MiRS score indicates a cancer responsive to MYC inhibition. Suitably, the MiRS score may be greater than 0.0, 0.5, 1.0, 1.5, or 2.0. Screening of compounds by the methods disclosed herein can be used to identify candidate compounds for use in therapies or identifying diseases or conditions that are responsive to the screened compounds. For example, MYCi screened by the present methods can be used to identify compounds having the desired properties to be effective in treatment or identify diseases or conditions that are responsive to the MYCi. Attorney Docket No.702581.02595 (NU2023-153-02) In an aspect, the disclosed technology provides for a method for treating a subject for a cancer responsive to MYC inhibition, the method comprising administering a MYC inhibitor to the subject. In some embodiment, the MYC inhibitor Response Signature (MiRS) score for the cancer is greater than 0. In some embodiment, the method further comprises determining the MiRS score for the cancer. The MiRS score may be determined by gene set variance analysis of a MYC inhibition responsive gene. The MYC inhibition responsive genes may include, but are not limited to, ADM2, AIG1, AKNA, ANK2, ARHGEF2, ATF3, BBC3, BMF, CCNE2, CHAC1, DDIT3, DDIT4, ERN1, FYN, GADD45A, GDF15, GINS2, GTPBP2, HMOX1, KDM7A, MCM4, NUPR1, PPP1R15A, RELN, RRM2, TCP11L2, THSD7A, TK1, TR1B3, UHRF1, UNG, and YPEL2. In some embodiment, the MYC inhibition responsive gene is a MYC target or a ATF4 target. In some embodiments, the cancer has acetylated MYC. The method may further comprise screening the cancer for acetylated MYC. In some embodiments, the acetylated MYC has an acetylated lysine in an extended MYC Box II domain. In some embodiments, K148 or K157 is acetylated. The MYCi may bind to an intrinsically disordered region of MYC and one or both of a distal N-terminal region of MYC (ΔC) and an intrinsically disordered region of GCN1. The IDR of MYC may comprise a MycHot motif. The MYCi may activate ATF4 or induce ATF4 expression. In some embodiments, the MYCi may comprise an MYCi disclosed herein. In an aspect, the disclosed technology provides for a method for treating a subject for a cancer, the method comprising administering a MYC inhibitor that binds to an intrinsically disordered region of MYC and one or both of a distal N-terminal region of MYC (ΔC) and an intrinsically disordered region of GCN1. The method may comprise determining the binding of an MYC inhibitor to an intrinsically disordered region of MYC and one or both of a distal N-terminal region of MYC (ΔC) and an intrinsically disordered region of GCN1. In some embodiments, the IDR of MYC may comprise a MycHot motif. The MYCi may activate ATF4 or induce ATF4 expression. In some embodiments, the MYCi may comprise an MYCi disclosed herein. In some embodiments, the MYC may be acetylated. The acetylated MYC may have an acetylated lysine in an extended MYC Box II domain. In some embodiments, K148 or K157 is acetylated. In an aspect, the disclosed technology provides for a method for treating a subject for a cancer responsive to ATF4 activation or induction, the method comprising administering a Attorney Docket No.702581.02595 (NU2023-153-02) GCN1 ligand that activates or induces expression of ATF4. In some embodiments, the GCN1 ligand binds an intrinsically disordered region of GCN1. In an aspect, the disclosed technology provides for a method for sensitizing a subject having cancer to MYC inhibition treatment, the method comprising activating or inducing expression of ATF4 in the subject. Activating or inducing expression of ATF4 in the subject may comprise administering to the subject a GCN1 ligand that activates or induces expression of ATF4. In some embodiments, the GCN1 ligand binds an intrinsically disordered region of GCN1. In some embodiments, the GCN1 ligand may be an MYC inhibitor, such as a MYC inhibitor disclosed herein.As used herein, a “subject” may be interchangeable with “patient” or “individual” and means an animal, which may be a human or non-human animal, in need of treatment. A “subject in need of treatment” may include a subject having a disease, disorder, or condition that is responsive to therapy with a compound as disclosed herein, including MYC inhibitors. For example, a “subject in need of treatment” may include a subject having a cell proliferative disease, disorder, or condition such as cancer. Examples of cancers includes, but are not limited to, breast cancer, multiple myeloma, non-small cell lung cancer, colon cancer, cancer of the central nervous system, melanoma, ovarian cancer, renal cancer, prostate cancer, and leukemia. In some embodiments, the cancer may have an MiRS score that is greater than 0. In some embodiments, the cancer tissue may possess elevated levels of lysine- acetylated MYC (e.g., K148-Ac MYC), compared to normal tissues. As used herein, the phrase “effective amount” shall mean that drug dosage that provides the specific pharmacological response for which the drug is administered in a significant number of subjects in need of such treatment. An effective amount of a drug that is administered to a particular subject in a particular instance will not always be effective in treating the conditions / diseases described herein, even though such dosage is deemed to be a therapeutically effective amount by those of skill in the art. The compounds as disclosed herein include MYC inhibitors, such as those targeting c-MYC. Examples of MYC inhibitors include, for example, the compounds disclosed in U.S. Patent Nos. 11,142,504 and 11,420,957, U.S. Publication Nos. 2020 / 0390894 and 2021 / 0395206, and international patent application PCT / US2023 / 071380, the contents of all of which are incorporated herein by reference in their entirety. Examples include, but are not limited to, Attorney Docket No.702581.02595 (NU2023-153-02) . TableNo. MYCinhibitorsStructure Function Reference1 10058-F4 MYC-MAX dimerization PMID: inhibitor 17046567 2 10074-G5 MYC-MAX dimerization PMID: inhibitor 20801893 Covalent ligand that targets 3 EN4 cysteine 171 (C171) of MYC PMID: to Inhibit MYC 32966806 transcriptional activity 4 Mycro 3 MAX dimerization inhibitor. PMID: Inhibits MYC DNA binding 25332683 5MYCMI-MYC-MAX dimerization PMID: 6 inhibitor 29968736 6 KJ Pyr 9 MYC-MAX dimerization PMID: inhibitor 25114221 Attorney Docket No.702581.02595 (NU2023-153-02) Inhibitor of MYC / MAX 7 KSI-376 binding to target gene PMID: promoters 23872029 The formulae of the compounds and molecules disclosed herein should be interpreted as encompassing all possible stereoisomers, enantiomers, or epimers of the compounds and molecules unless the formulae indicates a specific stereoisomer, enantiomer, or epimer. The formulae of the compounds and molecules disclosed herein should be interpreted as encompassing salts, esters, amides, or solvates thereof of the compounds and molecules. The compounds may exhibit one or more biological activities. The compounds may inhibit binding of the Myc / Max complex to DNA (e.g., in a DNA gel shifting assay). In some embodiments, the compounds inhibit binding of the Myc / Max complex to DNA by at least 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% at a concentration of less than about 100 μM, 50 μM, 10 μM, 1 μM, 0.1 μM, 0.05 μM, 0.01 μM, 0.005 μM, 0.001 μM, or less. The compounds may not produce significant DNA damage (e.g., in an rH2AX staining assay at a concentration greater than about 0.001 μM, 0.005 μM , 0.01 μM, 0.1 μM, 1.0 μM, 10 μM, 100 μM, or higher). The compounds may inhibit the growth of cells that express c-Myc (preferably by at least 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% at a concentration of less than about 100 μM, 50 μM, 10 μM, 1 μM, 0.1 μM, 0.05 μM, 0.01 μM, 0.005 μM, 0.001 μM, or less). The compounds may not inhibit the growth of cells that do not express c-Myc (preferably by not more than 50%, 40%, 30%, 20%, 10%, 5%, 4%, 3%, 2% or less at a concentration of greater than about 0.001 μM, 0.005 μM, 0.01 μM, 0.5 μM, 0.1 μM, 1.0 μM, 10 μM, and 100 μM or higher). Concentration ranges also are contemplated herein, for example, a concentration range bounded by end-point concentrations selected from 0.001 μM, 0.005 μM, 0.01 μM, 0.5 μM, 0.1 μM, 1.0 μM, 10 μM, and 100 μM. The compounds may be effective in inhibiting cell proliferation of cancer cells, including cancer cells that express c-MYC and whose proliferation is inhibiting by inhibiting the biological activity of c-MYC. The compounds may be effective in inhibiting cell proliferation of one or more types of cancer cells including: multiple myeloma cells, such as MM.1S cells; leukemia cells, such as CCRF-CEM, HL-60(TB), MOLT-4, RPMI-8226 and SR; non-small lung cancer cells, such as A549 / ATCC, EKVX, HOP-62, HOP-92, NCI-H226, NCI-H23, NCI-H322M, NCI-H460 and NCI-H522; colon cancer cells, such as COLO 205, HCC-2998, HCT-116, HCT-15, HT29, KM12 and SW-620; CNS: SF-268, SF-295, SF-539, Attorney Docket No.702581.02595 (NU2023-153-02) SNB-19, SNB-75 and U251; melanoma cancer cells, such as LOX IMVI, MALME-3M, M14, MDA-MB-435, SK-MEL-2, SK-MEL-28, SK-MEL-5, UACC-257 and UACC-62; ovarian cancer cells, such as IGR-OV1, OVCAR-3, OVCAR-4, OVCAR-5, OVCAR-8, NCI / ADR-RES and SK-OV-3; renal cancer cells, such as 786-0, A498, ACHN, CAKI-1, RXF 393, SN12C, TK-10 and UO-31; prostate cancer cells, such as DU-145 and PC-3; and breast cancer cells, such as MCF7, MDA-MB-231 / ATCC, MDA-MB-468, HS 578T, BT-549 and T-47D. Cell proliferation and inhibition thereof by the presently compounds may be assessed by cell viability methods disclosed in the art including colorimetric assays that utilize dyes such as MTT, XTT, and MTS to assess cell viability. Preferably, the compounds have an IC50of less than about 10 μM, 5 μM, 1 μM, 0.5 μM, 0.01 μM, 0.005 μM, 0.001 μM or lower in the selected assay. The compounds may activate ATF4 or induce ATF4 expression. Activation of ATF4 or induction of ATF4 expression may be determined by methods known in the art, such as Western blot analysis. The compounds may be formulated as anti-cancer therapeutics, including hematologic malignancies, breast, lung, myeloma, pancreas and prostate malignancies. The compounds also may be formulated as anti-inflammation therapeutics. The compounds utilized in the methods disclosed herein may be formulated as pharmaceutical compositions that include: (a) a therapeutically effective amount of one or more compounds as disclosed herein; and (b) one or more pharmaceutically acceptable carriers, excipients, or diluents. The pharmaceutical composition may include the compound in a range of about 0.1 to 2000 mg (preferably about 0.5 to 500 mg, and more preferably about 1 to 100 mg). The pharmaceutical composition may be administered to provide the compound at a daily dose of about 0.1 to 100 mg / kg body weight (preferably about 0.5 to 20 mg / kg body weight, more preferably about 0.1 to 10 mg / kg body weight). In some embodiments, after the pharmaceutical composition is administered to a subject (e.g., after about 1, 2, 3, 4, 5, or 6 hours post-administration), the concentration of the compound at the site of action may be within a concentration range bounded by end-points selected from 0.001 μM, 0.005 μM, 0.01 μM, 0.5 μM, 0.1 μM, 1.0 μM, 10 μM, and 100 μM (e.g., 0.1 μM - 1.0 μM). The compounds and pharmaceutical compositions comprising the compounds may be administered in methods of treating a subject in need thereof. For example, in the methods of treatment a subject in need thereof may include a subject having a cell proliferative disease, Attorney Docket No.702581.02595 (NU2023-153-02) disorder, or condition such as cancer (e.g., cancers such as multiple myeloma, leukemia, lung cancer, colon cancer, cancer of the central nervous system, melanoma, ovarian cancer, renal cancer, prostate cancer, and breast cancer). In some embodiments of the disclosed treatment methods, the subject may be administered a dose of a compound as low as 1.25 mg, 2.5 mg, 5 mg, 7.5 mg, 10 mg, 12.5 mg, 15 mg, 17.5 mg, 20 mg, 22.5 mg, 25 mg, 27.5 mg, 30 mg, 32.5 mg, 35 mg, 37.5 mg, 40 mg, 42.5 mg, 45 mg, 47.5 mg, 50 mg, 52.5 mg, 55 mg, 57.5 mg, 60 mg, 62.5 mg, 65 mg, 67.5 mg, 70 mg, 72.5 mg, 75 mg, 77.5 mg, 80 mg, 82.5 mg, 85 mg, 87.5 mg, 90 mg, 100 mg, 200 mg, 500 mg, 1000 mg, or 2000 mg once daily, twice daily, three times daily, four times daily, once weekly, twice weekly, or three times per week in order to treat the disease or disorder in the subject. In some embodiments, the subject may be administered a dose of a compound as high as 1.25 mg, 2.5 mg, 5 mg, 7.5 mg, 10 mg, 12.5 mg, 15 mg, 17.5 mg, 20 mg, 22.5 mg, 25 mg, 27.5 mg, 30 mg, 32.5 mg, 35 mg, 37.5 mg, 40 mg, 42.5 mg, 45 mg, 47.5 mg, 50 mg, 52.5 mg, 55 mg, 57.5 mg, 60 mg, 62.5 mg, 65 mg, 67.5 mg, 70 mg, 72.5 mg, 75 mg, 77.5 mg, 80 mg, 82.5 mg, 85 mg, 87.5 mg, 90 mg, 100 mg, 200 mg, 500 mg, 1000 mg, or 2000 mg, once daily, twice daily, three times daily, four times daily, once weekly, twice weekly, or three times per week in order to treat the disease or disorder in the subject. Minimal and / or maximal doses of the compounds may include doses falling within dose ranges having as end-points any of these disclosed doses (e.g., 2.5 mg – 200 mg). In some embodiments, a minimal dose level of a compound for achieving therapy in the disclosed methods of treatment may be at least about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1200, 1400, 1600, 1800, 1900, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 15000, or 20000 ng / kg body weight of the subject. In some embodiments, a maximal dose level of a compound for achieving therapy in the disclosed methods of treatment may not exceed about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1200, 1400, 1600, 1800, 1900, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 15000, or 20000 ng / kg body weight of the subject. Minimal and / or maximal dose levels of the compounds for achieving therapy in the disclosed methods of treatment may include dose levels falling within ranges having as end- points any of these disclosed dose levels (e.g., 500 – 2000 ng / kg body weight of the subject). The compounds utilized in the methods disclosed herein may be formulated as a pharmaceutical composition in solid dosage form, although any pharmaceutically acceptable dosage form can be utilized. Exemplary solid dosage forms include, but are not limited to, Attorney Docket No.702581.02595 (NU2023-153-02) tablets, capsules, sachets, lozenges, powders, pills, or granules, and the solid dosage form can be, for example, a fast melt dosage form, controlled release dosage form, lyophilized dosage form, delayed release dosage form, extended release dosage form, pulsatile release dosage form, mixed immediate release and controlled release dosage form, or a combination thereof. The compounds utilized in the methods disclosed herein may be formulated as a pharmaceutical composition that includes a carrier. For example, the carrier may be selected from the group consisting of proteins, carbohydrates, sugar, talc, magnesium stearate, cellulose, calcium carbonate, and starch-gelatin paste. The compounds utilized in the methods disclosed herein may be formulated as a pharmaceutical composition that includes one or more binding agents, filling agents, lubricating agents, suspending agents, sweeteners, flavoring agents, preservatives, buffers, wetting agents, disintegrants, and effervescent agents. Filling agents may include lactose monohydrate, lactose anhydrous, and various starches; examples of binding agents are various celluloses and cross-linked polyvinylpyrrolidone, microcrystalline cellulose, such as Avicel® PH101 and Avicel® PH102, microcrystalline cellulose, and silicified microcrystalline cellulose (ProSolv SMCC™). Suitable lubricants, including agents that act on the flowability of the powder to be compressed, may include colloidal silicon dioxide, such as Aerosil®200, talc, stearic acid, magnesium stearate, calcium stearate, and silica gel. Examples of sweeteners may include any natural or artificial sweetener, such as sucrose, xylitol, sodium saccharin, cyclamate, aspartame, and acsulfame. Examples of flavoring agents are Magnasweet® (trademark of MAFCO), bubble gum flavor, and fruit flavors, and the like. Examples of preservatives may include potassium sorbate, methylparaben, propylparaben, benzoic acid and its salts, other esters of parahydroxybenzoic acid such as butylparaben, alcohols such as ethyl or benzyl alcohol, phenolic compounds such as phenol, or quaternary compounds such as benzalkonium chloride. Suitable diluents may include pharmaceutically acceptable inert fillers, such as microcrystalline cellulose, lactose, dibasic calcium phosphate, saccharides, and mixtures of any of the foregoing. Examples of diluents include microcrystalline cellulose, such as Avicel® PH101 and Avicel® PH102; lactose such as lactose monohydrate, lactose anhydrous, and Pharmatose® DCL21; dibasic calcium phosphate such as Emcompress®; mannitol; starch; sorbitol; sucrose; and glucose. Suitable disintegrants include lightly crosslinked polyvinyl pyrrolidone, corn starch, potato starch, maize starch, and modified starches, croscarmellose sodium, cross-povidone, sodium starch glycolate, and mixtures thereof. Attorney Docket No.702581.02595 (NU2023-153-02) Examples of effervescent agents are effervescent couples such as an organic acid and a carbonate or bicarbonate. Suitable organic acids include, for example, citric, tartaric, malic, fumaric, adipic, succinic, and alginic acids and anhydrides and acid salts. Suitable carbonates and bicarbonates include, for example, sodium carbonate, sodium bicarbonate, potassium carbonate, potassium bicarbonate, magnesium carbonate, sodium glycine carbonate, L-lysine carbonate, and arginine carbonate. Alternatively, only the sodium bicarbonate component of the effervescent couple may be present. The compounds utilized in the methods disclosed herein may be formulated as a pharmaceutical composition for delivery via any suitable route. For example, the pharmaceutical composition may be administered via oral, intravenous, intramuscular, subcutaneous, topical, and pulmonary route. Examples of pharmaceutical compositions for oral administration include capsules, syrups, concentrates, powders and granules. The compounds utilized in the methods disclosed herein may be administered in conventional dosage forms prepared by combining the active ingredient with standard pharmaceutical carriers or diluents according to conventional procedures well known in the art. These procedures may involve mixing, granulating and compressing or dissolving the ingredients as appropriate to the desired preparation. Pharmaceutical compositions comprising the compounds may be adapted for administration by any appropriate route, for example by the oral (including buccal or sublingual), rectal, nasal, topical (including buccal, sublingual or transdermal), vaginal or parenteral (including subcutaneous, intramuscular, intravenous or intradermal) route. Such formulations may be prepared by any method known in the art of pharmacy, for example by bringing into association the active ingredient with the carrier(s) or excipient(s). Pharmaceutical compositions adapted for oral administration may be presented as discrete units such as capsules or tablets; powders or granules; solutions or suspensions in aqueous or non-aqueous liquids; edible foams or whips; or oil-in-water liquid emulsions or water-in-oil liquid emulsions. Pharmaceutical compositions adapted for transdermal administration may be presented as discrete patches intended to remain in intimate contact with the epidermis of the recipient for a prolonged period of time. For example, the active ingredient may be delivered from the patch by iontophoresis. Pharmaceutical compositions adapted for topical administration may be formulated as ointments, creams, suspensions, lotions, powders, solutions, pastes, gels, impregnated Attorney Docket No.702581.02595 (NU2023-153-02) dressings, sprays, aerosols or oils and may contain appropriate conventional additives such as preservatives, solvents to assist drug penetration and emollients in ointments and creams. For applications to the eye or other external tissues, for example the mouth and skin, the pharmaceutical compositions are preferably applied as a topical ointment or cream. When formulated in an ointment, the compound may be employed with either a paraffinic or a water-miscible ointment base. Alternatively, the compound may be formulated in a cream with an oil-in-water cream base or a water-in-oil base. Pharmaceutical compositions adapted for topical administration to the eye include eye drops where the active ingredient is dissolved or suspended in a suitable carrier, especially an aqueous solvent. Pharmaceutical compositions adapted for nasal administration where the carrier is a solid include a coarse powder having a particle size (e.g., in the range 20 to 500 microns) which is administered in the manner in which snuff is taken (i.e., by rapid inhalation through the nasal passage from a container of the powder held close up to the nose). Suitable formulations where the carrier is a liquid, for administration as a nasal spray or as nasal drops, include aqueous or oil solutions of the active ingredient. Pharmaceutical compositions adapted for parenteral administration include aqueous and non-aqueous sterile injection solutions which may contain anti-oxidants, buffers, bacteriostats and solutes which render the formulation isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions which may include suspending agents and thickening agents. The formulations may be presented in unit-dose or multi-dose containers, for example sealed ampoules and vials, and may be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid carrier, for example water for injections, immediately prior to use. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tablets. Tablets and capsules for oral administration may be in unit dose presentation form, and may contain conventional excipients such as binding agents, for example syrup, acacia, gelatin, sorbitol, tragacanth, or polyvinylpyrrolidone; fillers, for example lactose, sugar, maize-starch, calcium phosphate, sorbitol or glycine; tabletting lubricants, for example magnesium stearate, talc, polyethylene glycol or silica; disintegrants, for example potato starch; or acceptable wetting agents such as sodium lauryl sulphate. The tablets may be coated according to methods well known in normal pharmaceutical practice. Oral liquid preparations may be in the form of, for example, aqueous or oily suspensions, solutions, emulsions, syrups or elixirs, or may be presented as a dry product for reconstitution with water or other suitable vehicle before use. Such liquid preparations may contain conventional Attorney Docket No.702581.02595 (NU2023-153-02) additives, such as suspending agents, for example sorbitol, methyl cellulose, glucose syrup, gelatin, hydroxyethyl cellulose, carboxymethyl cellulose, aluminium stearate gel or hydrogenated edible fats, emulsifying agents, for example lecithin, sorbitan monooleate, or acacia; non-aqueous vehicles (which may include edible oils), for example almond oil, oily esters such as glycerine, propylene glycol, or ethyl alcohol; preservatives, for example methyl or propyl p-hydroxybenzoate or sorbic acid, and, if desired, conventional flavoring or coloring agents. The compounds or pharmaceutical compositions comprising the compounds may be administered in methods of treatment. For example, the compounds or pharmaceutical compositions comprising the compounds may be administered in methods of treating cell proliferative diseases and disorders. Cell proliferative diseases and disorders treated by the disclosed methods may include, but are not limited to, cancers selected from the group consisting of multiple myeloma, leukemia, non-small cell lung cancer, colon cancer, cancer of the central nervous system, melanoma, ovarian cancer, renal cancer, prostate cancer, and breast cancer. Optionally, the compounds or pharmaceutical compositions comprising the compounds may be administered with additional therapeutic agents, optionally in combination, in order to treat cell proliferative diseases and disorders. In some embodiments of the disclosed methods, one or more additional therapeutic agents are administered with the compounds or with pharmaceutical compositions comprising the compounds, where the additional therapeutic agent is administered prior to, concurrently with, or after administering the compounds or the pharmaceutical compositions comprising the compounds. In some embodiments, the disclosed pharmaceutical composition is formulated to comprise the compounds and further to comprise one or more additional therapeutic agents, for example, one or more additional therapeutic agents for treating cell proliferative diseases and disorders. In some embodiments, additional therapeutic agents may include, but are not limited to, therapeutic agents for treating leukemias and lymphomas, such as acute myeloid leukemia (AML), acute lymphocytic leukemia (ALL), chronic myelogenous leukemia (CML), and non-Hodgkin’s lymphoma. In some embodiments, additional therapeutic agents may include, but are not limited to, antimetabolite antineoplastic agents that inhibit the synthesis of DNA. Suitable antimetabolite antineoplastic agents that inhibit the synthesis of DNA may include, but are not limited to, nucleoside and / or nucleotide derivatives. Suitable nucleoside and / or nucleotide Attorney Docket No.702581.02595 (NU2023-153-02) derivatives may include, but are not limited to, cytosine arabinoside (ara-C), otherwise called cytarabine. Unless otherwise specified or indicated by context, the terms “a”, “an”, and “the” mean “one or more.” For example, “a molecule” should be interpreted to mean “one or more molecules.” As used herein, “about”, “approximately,” “substantially,” and “significantly” will be understood by persons of ordinary skill in the art and will vary to some extent on the context in which they are used. If there are uses of the term which are not clear to persons of ordinary skill in the art given the context in which it is used, “about” and “approximately” will mean plus or minus ≤10% of the particular term and “substantially” and “significantly” will mean plus or minus >10% of the particular term. As used herein, the terms “include” and “including” have the same meaning as the terms “comprise” and “comprising.” The terms “comprise” and “comprising” should be interpreted as being “open” transitional terms that permit the inclusion of additional components further to those components recited in the claims. The terms “consist” and “consisting of” should be interpreted as being “closed” transitional terms that do not permit the inclusion additional components other than the components recited in the claims. The term “consisting essentially of” should be interpreted to be partially closed and allowing the inclusion only of additional components that do not fundamentally alter the nature of the claimed subject matter. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., “such as”) provided herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention. All references, including publications, patent applications, and patents, cited herein are hereby incorporated by reference to the same extent as if each reference were individually and specifically indicated to be incorporated by reference and were set forth in its entirety herein. Preferred aspects of this invention are described herein, including the best mode known to the inventors for carrying out the invention. Variations of those preferred aspects may become apparent to those of ordinary skill in the art upon reading the foregoing description. The inventors expect a person having ordinary skill in the art to employ such Attorney Docket No.702581.02595 (NU2023-153-02) variations as appropriate, and the inventors intend for the invention to be practiced otherwise than as specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context. EXAMPLES The following Examples are illustrative and are not intended to limit the scope of the claimed subject matter. Example 1. MYC inhibitors activate the ATF4-ISR pathway To obtain a comprehensive view of the molecular effects of MYC inhibitors, we examined the gene expression and chromatin accessibility profile changes induced by treatment with MYCi975. We examined RNAseq data from PC3 prostate cancer cells with MYC knockdown by siRNA, P493-6 lymphoma cells after tetracycline-mediated suppression of a MYC transgene, or MYCi975 treatment of both cell models. We identified enrichment of the Unfolded Protein Response (UPR) pathway in MYCi975-treated cells upon gene set enrichment analysis (Figure 2, panels a-c) The UPR overlaps with the integrated stress response (ISR), an evolutionarily conserved signaling pathway whose induction is coupled to UPR activation and decreased global protein synthesis (5). We queried the RNA-seq datasets with an “ISR gene signature” obtained from mice with inactivating mutation in eIF2B, which leads to persistent ISR (5, 6) and observed robust activation of the ISR signature in MYCi975-treated cells (Figure 2, panel d). Analysis of chromatin accessibility by ATAC-seq in MYCi975-treated prostate and breast cancer cell lines showed significant increase in ATAC-seq signals in regions enriched for consensus motifs for the stress response master transcription factor ATF4 and its binding partner CEBPG (Figure 1, panel a; Figure 2, panel e). As ATF4 is a central node in the ISR pathway, these convergent observations suggest that MYCi975 may activate the ATF4-ISR pathway. Indeed, ATF4 protein levels increased within 2 to 4 hours of MYCi975 treatment of multiple c-MYC expressing cell lines (Figure 1, panel c; Figure 2, panel f). MYCi975 also induced ATF4 in the N-MYC-expressing neuroblastoma cell line SK-N-BE(2) (Figure 2, panel g). As a negative control, we examined the ability of a novel regio-isomer of MYCi975 called “Compound 733” to induce ATF4. While MYCi975 bound MYC in a biolayer interferometry (BLI) assay (Kd=272nM), compound 733 failed to bind (Figure 1, panels d,e). Attorney Docket No.702581.02595 (NU2023-153-02) Notably, 733 failed to induce ATF4 protein expression (Figure 1, panel f), suggesting a link between MYC binding / inhibition and ATF4 induction. In support of this notion, analysis of MYC degradation and ATF4 induction by 41 compounds generated as part of a medicinal chemistry campaign to generate new MYCi analogs revealed a correlation between MYC degradation and ATF4 induction (Figure 2, panel h). This is strikingly exemplified by NUCC-226435 (“435”) and its stereoisomer NUCC-226436 (“436”). While “435” showed minimal ATF4 induction, MYC degradation or E-box reporter inhibition, “436” was active in all three assays (Figure 2, panels i-k). These observations prompted us to further examine whether other structurally diverse small molecule MYC binders reported in the literature could also activate ATF4. We assessed the ability of 7 chemically distinct small-molecule MYC inhibitors to activate ATF4. These include 5 MYC / MAX dimerization inhibitors (10058-F4, 10074-G5, Mycro 3, KJ-Pyr-9, MYCMI-6); an inhibitor of MYC / MAX / DNA complex formation (KS-3716); and a covalent inhibitor that targets cysteine 171 (C171) of MYC (EN4) (7-13). We found that all these compounds can induce ATF4 protein expression with variable potencies in MycCaP and PC3 prostate cancer cells. Thus, ATF4 activation is a common feature of chemically diverse MYC binding small molecules. By contrast, the dominant negative MYC peptide OMOMYC did not induce ATF4 expression (Figure 2, panel m). Example 2. MYCi induces ATF4 in a partially MYC-dependent manner To determine the role of MYC in ATF4 induction by MYCi, we assessed ATF4 induction in P493-6 cells in which MYC was suppressed by doxycycline treatment. MYC suppression attenuated induction of ATF4 by MYCi975 as well as by the ER tress inducer thapsigargin (Figure 4, panel a). Furthermore, ATF4 induction by MYCi975 was attenuated in PC3 cells after MYC depletion by siRNA, as well as in MYC-null rat fibroblasts HO.15 (Figure 4, panels b,c). Thus, MYCi induction of ATF4 is only partially dependent on MYC expression. As MYC transcriptionally regulates ATF4, reduced levels of ATF4 mRNA in MYC-deficient cells may result in suboptimal ATF4 induction by MYCi in these cells. To explore further the mechanism of ATF4 induction by MYCi, we examined phosphorylation of eukaryotic initiation factor 2α (EIF2α) kinase, the central node that integrates multiple stress pathways to activate ATF4 translation and shut down global protein synthesis. MYCi treatment induced EIF2α phosphorylation and suppressed global protein synthesis coincident with ATF4 protein expression (Figure 4, panel d). EIF2α phosphorylation is required for ATF4 induction by MYCi975, as MYCi975 failed to induce ATF4 in mouse embryo fibroblasts (MEFs) from genetically engineered mice with knock-in Attorney Docket No.702581.02595 (NU2023-153-02) of non-phosphorylatable serine (S) at position 51 to alanine (A) (14) (Figure 3, panel a). Next, we assessed the potential role of kinases upstream of EIF2α, including PERK and GCN2. MYCi-induced ATF4 activation was attenuated by treatment with GCN2 inhibitor in PC3 and MycCaP with a more modest effect observed for PERK inhibitor (Figure 3, panel b; Figure 4, panel e). Example 3. MYCi targets a shared intrinsically disordered region between MYC and GCN1 To investigate the possibility that MYCi might affect the ATF4 pathway through additional targets, we examined proteomics data of proteins binding to MYCi975 and a close analog MYCi361 (1). Of the XXX proteins bound by both compounds in two different cell lines, GCN1, a GCN2-binding protein, was the second top hit. GCN1 is essential for GCN2 function in the amino acid stress response. Furthermore, a PHI-BLAST pattern search with the MycHot motif [KR]-[KR]-x(3)-K-R-x(5,7)-[KR]-[DE]-Q-I-x-E-L-E-x(2)-E (SEQ ID NOs: 12-13) returned GCN1 as the only hit (Figure 3, panels c,d). We confirmed MYCi binding to GCN1 in biotinylated-MYCi975 pulldown studies (Figure 3, panel e). Thus, GCN1 contains an IDR with sequence similarity to the MycHot region in MYC that binds MYCi. Notably, GCN1 shows a striking sequence similarity to MYC in this region which is even better than N-MYC and L-MYC in the C-terminal portion FALRDQIPELENNE (SEQ ID NO: 14) (Figure 3, panel d), and several amino acids conserved between MYC and GCN1 in this region are divergent in MAX, the MYC homodimerizing partner which does not bind MYCi (1). The intrinsically disordered region of GCN1 has 42.9% identity with 14 residues overlap, a score of 30.0, and gap frequency 0.0%. This region of GCN1 has been shown to be involved in binding to ribosomes. We therefore examined whether MYCi975 affects ribosome profiles. Treatment of PC3 cells with MYCi975 led to disruption of 60S / 80S ribosomal and polysomal profiles within 2hrs (Figure 3, panel f), consistent with a direct effect of MYCi975 on ribosomes. Additionally, GCN1 deletion by CRISPR / Cas9 attenuated ATF4 induction by MYCi975 (Figure 3, panel g). Example 4. “SAR-by-Proxy”: The shared IDR in MYC and GCN1 provides insights into MYCi binding To explore the structural basis of MYCi binding to GCN1, we modeled the MycHot IDR and the surrounding amino acids of the GCN1 protein using alpha-fold. This revealed a potential pocket bounded by the IDR and two helices (Figure 5, panel a). We next used molecular docking guided by MYC-inhibitory / ATF4-actiavtion function of MYCi975 and several analogs to determine a favorable binding pose (Figure 5, panel b). Attorney Docket No.702581.02595 (NU2023-153-02) A comparison of this putative MYCi-binding pocket in GCN1 to the MycHot region in the MYC bHLH / LZ domain alpha-fold structure revealed the lack of a similar pocket. Based on the observation that the KD for MYCi975 binding to full-length MYC of 272 nM is ~8-fold lower than for binding to the MycHot-containing bHLH domain at 2.23 μM in the BLI assay (Figure 5, panel g), we reasoned that another part of the MYC protein may contribute to MYCi binding. Using a series of MYC deletion mutants in a MYC pulldown assay with biotinylated-MYCi975, we found that in addition to the deletion overlapping the MycHot region (ΔG), deletion of a distal N-terminal region of MYC (ΔC), also impaired MYCi binding (Figure 5, panel c). In a BLI assay, a truncated MYC (aa1-353) containing the ΔC region showed association with MYCi975 with a fast dissociation (Figure 5, panel f). Next, using a recombinant mutant protein in which the ΔC region was connected with a flexible glycine linger to the ΔG region (C-linker-G MYC), we show that binding both regions improves MYCi binding. The binding affinity for this construct was ~20-fold better than for the MYC bHLH / LZ domain alone (Figure 5, panel g). We next generated alpha-fold models of full-length MYC and of C-linker-G MYC (Figure 5, panel d). The ΔC regions has potential helices that could come into close proximity to the ΔG region to form a pocket. These results indicate that structural-activity-relationship (SAR) insights of small molecule binding to a shared IDR in a more structured protein such as GCN1, could inform SAR for small molecule binding to a related IDR in a more disordered protein, such as MYC. We call this approach “SAR-by-Proxy”. Example 5. The functional role of MYCi-induced ATF4 pathway in cancer cells To define the functional role of ATF4 pathway activation by MYCi, we generated MycCaP cells expressing a doxycycline-inducible shRNA targeting ATF4 (Dox / shATF4 MycCaP cells). In these cells, doxycycline treatment suppressed MYCi-induced ATF4 expression (Figure 6, panel a) and attenuated MYCi cytotoxicity (Figure 7, panel a). Similar results were obtained in 22RV1 cells expressing doxycycline-inducible shATF4 (Figure 7, panels b,c). We then used RNAseq to define the genes modulated by MYCi975 treatment in an ATF4-dependent manner. ATF4 suppression by doxycycline treatment impaired the expression of a subset of MYCi-induced genes consisting of classical ATF4-regulated genes of the ISR pathway, including DDIT4, CHAC1 and VEGF (Figure 6, panel b; Figure 7, panel d). Notably, under the conditions of this experiment, some ATF4 targets were more dramatically affected by ATF4 knockdown than others. This was not merely a consequence of the expression level of these genes in the MYCi-treated cells. For example, ASNS and DDIT4 / CHOP were both expressed at similar absolute levels after MYCi975 treatment. Yet, Attorney Docket No.702581.02595 (NU2023-153-02) DDIT4 was significantly suppressed upon ATF4 depletion while ASNS was largely unaffected. Next, we examined the functional significance of ATF4 induction to the anti-tumor efficacy of MYCi975 in vivo in immunocompetent mice. We established Dox / shATF4 MycCaP allograft tumors in FVB / N mice, followed by treatment of the animals with vehicle, Dox, MYCi975 or Dox+MYCi975. Analysis of tumor size over time showed that ATF4 knockdown by Dox treatment significantly attenuated MYCi975 efficacy (Figure 6, panel c; Figure 7, panel e). All the treatments were well tolerated and did not affect mouse body weight (Figure 7, panel f). Analysis of tumors at endpoint showed that MYCi975 increased the apoptotic marker cleaved-caspase 3 (CC3) and suppressed the proliferation marker Ki-67 (Figure 6, panels d,e). Both changes were largely reversed upon ATF4 depletion in the Dox+MYCi975 group. MYCi-induced changes in the tumor immune microenvironment including infiltration of CD3+ T cells, Natural Killer (NK) cells, FoxP3+ Treg cells, and upregulation of tumor PD-L1 expression were also all largely reversed by ATF4 depletion (Figure 6, panels d,e). Overall, these results indicate that activation of ATF4 by MYCi975 contributes significantly to MYCi-induced remodeling of the tumor immune microenvironment and anti-tumor efficacy. Table 2 shows a result table for the linear mixed effects model employed for statistical analysis of the in vivo experiments shown in Figure 6, panel c. Table 2. Models for Figure 6, panel c Compound N Obs Num DF Den DF F Statistic p-value MYCi vs. Vehicle 220 2 194 26.51 <.0001 MYCi + Dox vs. Vehicle 220 2 194 5.79 0.0036 MYCi + Dox vs. Dox 220 2 194 3.78 0.0246 MYCi + Dox vs. MYCi 220 2 194 8.28 0.0004 Example 6. MYC inhibitor Response Signature (MiRS) score Motivated by our observations that MYCi efficacy is dependent on both selective suppression of MYC target genes and activation of the ATF4 pathway, we sought to use an unbiased approach to develop a MYCi response signature (MiRS) score that could facilitate selection of tumor types that may best respond to MYCi treatment. To this end, we curated the common MYCi regulated in multiple cell lines (22Rv1, PC3 and MycCaP cells) by RNAseq. By applying a log2 fold change (FC) cutoff = 2, we identified 32 MYCi responsive Attorney Docket No.702581.02595 (NU2023-153-02) genes (25 upregulated, 7 downregulated). This gene list was used in gene set variance analysis (GSVA), a gene set enrichment method that estimates variation of pathway activity over a sample population in an unsupervised manner, to obtain MiRS scores for biological samples of interest (Figure 8, panels a,b). To assess the relationship between tumor cell line MiRS score and MYCi975 efficacy, we used data from a panel of 6 xenograft / allograft models treated with 100mg / kg once a day p.o. These include MDA-MB231triple-negative breast cancer (TNBC), PLC / PR15 hepatocellular carcinoma, KMS11 multiple myeloma, A2780 ovarian cancer, and A549 non-small cell lung cancer xenografts as well as CT26 mouse colon cancer allograft. A subset of these models (MDA-MB-231, KMS11 and A549) also received 100mg / kg twice a day p.o. showing a dose-dependent effect on tumor growth inhibition (Figure 9, panel a). We then examined the correlation between the MiRS score and tumor growth inhibition (TGI) by MYCi975 given at 100mg / kg / d and observed a significant positive correlation (Figure 8, panel c). The samples segregated into two clusters based on relative sensitivity to MYCi with a MiRS score >0.2 showing better response (Figure 8, panel d). Notably, neither a classical “MYC Hallmarks” signature nor “UPR / ATF4” signatures correlated to MYCi975 efficacy (Figure 9, panel b). Table 3 shows a result table for the linear mixed effects model employed for statistical analysis of the in vivo experiments shown in Figure 9. Table 3. Models for Figure 9 G2 Compound vs. G1 Vehicle N Obs Num DF Den DF F Statistic p-value QDA2780 140 2 118 0.31 0.7309 A549 240 2 218 2.02 0.1346 KMS11 220 2 198 3.01 0.0513 MDA-MB-231 280 2 258 2.01 0.1356 CT26 176 2 152 0.02 0.9815 PLC-PRF-5 240 3 216 3.36 0.0197 BIDA549 240 2 218 3.34 0.0372 KMS-11 200 2 178 9.62 0.0001 MDA-MB-231 240 2 218 13.80 <.0001 Example 7. GCN1 activates or induces expression of ATF4 As demonstrated in Figures 10 and 11, GCN1 is required for full ATF4 induction. These results, along with, the in vivo experiment showing that ATF4 is necessary for fully Attorney Docket No.702581.02595 (NU2023-153-02) sensitizing the tumors to MYCi treatment (Figure 6) establish that GCN1 ligands that activate or induce expression of ATF4 potentiate tumor sensitivity. ATF4 induction was assessed in PC12 cells following GCN1 knockout (KO) using pLentiCRISPR v2. Cells were treated with MYCi-975 (20μM) for 24 and 48 hours. The results demonstrate an attenuation of ATF4 induction in GCN1 KO cells compared to Cas9 control, indicating the role of GCN1 in mediating the cellular response to MYCi-975 (Figure 10). ATF4 induction was evaluated in 22Rv1 cells with simultaneous GCN1 KO and MYC knockdown (KD), treated with 10μM MYCi-975 using western blot analysis. The results demonstrate a significant attenuation of ATF4 compared to control cells, highlighting the combined impact of GCN1 KO and MYC KD on MYCi-975-mediated ATF4 induction (Figure 11). Example 8. Summary The two distinct sites in the MYC protein (SEQ ID. NO: 23), one in the C-terminal bHLH domain and the other overlapping the N-terminal MBII (MYC Box II) domain that participate in the binding of small molecule MYC inhibitors, have been further characterized. The N-terminal region overlapping MBII (which can also be called eMBII for extended MYC Box II) region contains 2 lysines, K148 / 149 and K157 / 158 (human / mouse), that are known to be acetylated by P300. Acetylation of MYC at these residues leads to increased MYC oncogenic activity in vivo. It was found that MYC acetylated at these lysine residues showed greater affinity for MYCi binding. Prostate tumors express elevated levels of K148-acetylated MYC, compared to normal prostate tissues. The fact that MYCi preferentially binds acetylated MYC which is more oncogenic may contribute to the therapeutic index of MYC inhibitors as well as path for developing a potential predictive biomarker. Results MYC acetylation on K148 / 149 (human / mouse) and K157 / 158 (human / mouse) enhances MYCi975 biding The acetylation of lysines K148 / 149 (human / mouse) and K157 / 158 (human / mouse) in the eMBII region by P300 is important for in vivo tumorigenicity and the regulation of specific MYC target gene programs (15). To assess how K148 / K157 acetylation might affect MYCi binding, the impact of acetylation on the predicted structure of this region in alphafold-3 was first evaluated (16). The models showed that K148 and K157 acetylation increased the stability of the helix containing these lysine residues (Figure 12, panels a-c). Attorney Docket No.702581.02595 (NU2023-153-02) Although glutamine does not fully mimic acetyl-lysine due to lack of its recapitulation of steric changes that come with acetylation, lysine-to-glutamine substitution mimics the loss of positive charge in acetyl-lysine. MYC mutants were next generated in which K148 / K157 were mutated to glutamines and tested for binding to MYCi975 in the BLI assay (Figure 12, panel d). The MYC lysine-to-arginine mutant was used as a control. MYCi975 bound to the R148 / R157 mutant with a similar affinity to wild type MYC (KD= 230 + 6.1 nM vs 262 + 3.8 nM). By contrast, the Q148 / Q157 mutant showed higher affinity binding (KD= 130 + 1.5 nM), which is similar to the affinity of the eMBII-linker-bHLH-LZ mutant (KD= 120 + 1.5 nM). These results suggest that acetylated, oncogenic MYC, may bind better to MYCi975. Next, it was examined whether MYCi975 preferentially binds to acetylated MYC in cells. To this end, biotinylated-MYCi975 (17) was used to pull down MYC from cancer cells and examine the K148 acetylation status of the recovered protein by probing with specific K148-Ac MYC antibodies (18). The results indicate that Biotin-MYCi975 recovers higher levels of K148-Ac MYC relative to total MYC, suggesting that in cells, MYCi975 may have a higher affinity for acetylated MYC than non-acetylated MYC (Figure 13). Next, it was showed that human prostate cancer tissues express higher levels of K148-Ac MYC than normal prostate tissues (Figure 14). Materials and Methods Cell Culture 22Rv1, PC-3, PC-12, SK-N-BE (2) were obtained from ATCC; P493-6 B cells were a generous gift from Prof. Chi Van Dang (University of Pennsylvania) and mouse MycCaP cells were the kind gift of Charles Sawyers (Memorial Sloan-Kettering Cancer Centre). MycCaP Pten-KO cells are generated from the parental MycCaP cells as previously described (1). Mouse Embryonic fibroblast (MEF) cells – MEF S / S and MEF A / A cells were provided by Prof. Randal J. Kaufman (Sanford Burnham Prebys Medical Discovery Institute). TGR-1 and HO.15.19 rat fibroblast cells were a gift from Prof. John Sedivy (Brown University). All cells were authenticated and tested as mycoplasma-free several times throughout the studies. 22Rv1, PC-3, MycCaP and P-493-6 cells were grown in RPMI1 1640 (Gibco #11875119) medium; SK-N-BE (2) cells in F-12K medium (ATCC #30-2004) and MEF S / S and MEF A / A cells in DMEM / F12(Ham) 1:1 (Thermo Scientific #1132033) supplemented with non- essential amino acids (Thermo Scientific #11140050), all medium supplemented with 10% heat-inactivated fetal bovine serum (FBS, Gibco # 10437028). PC-12 cells in F-12K medium (ATCC #30-2004) with 2% heat-inactivated FBS and 12.5% horse serum (Thermo Scientific #16050122). TGR-1 and HO.15.19 cells were cultured in DMEM (Gibco #11965118) with Attorney Docket No.702581.02595 (NU2023-153-02) 10% calf serum (Fisher Scientific, #SH3007303). All cells were cultured in 1% Penicillin- Streptomycin (10,000U / ml, Life Technologies) and 5% CO2 in humidified incubator at 370C. Generation of inducible shATF4 cell lines For ATF4 knockdown, we constructed 22Rv1 and MycCaP Tet-on shATF4 cells by infecting wild type cells with pLKO-Tet-On-shATF4 virus followed by continuous puromycin selection. The virus was generated as follows: Lentiviral shRNA plasmids were constructed by inserting target oligonucleotides into Tet-pLKO-puro (Addgene, #21915) plasmid. Plasmid DNA was extracted using a DNA extraction kit (Vazyme, DC112–01). The lentivirus was packaged by transfecting the plasmids with packaging vectors (psPAX and pMD2.G) and Lipofectamine 2000 (Invitrogen, #11668-019) in Opti-MEM media (Gibco) into HEK293T cells. Afterward, the virus supernatant was collected, filtered with a 0.45 μm strainer, concentrated with PEG6000 (Sigma-Aldrich, #81253), resolved in PBS and then aliquoted and stored for subsequent transfection. Cells were infected with viruses and selected for 72 hours with puromycin (2 μg / mL, Sigma-Aldrich, #P8833). The following oligonucleotide sequences were used: GCCAAGCACTTCAAACCTCAT (SEQ ID NO: 15) for the shATF4 sequence employed for the human cell line 22Rv1 and CGGACAAAGATACCTTCGAGT (SEQ ID NO: 16) for the mouse cell line MycCaP. Cell proliferation assays MycCaP shATF4 cells were plated into 96-well plates at approximately 1500 cells / well in triplicate. Cells were plated and allowed to adhere for 6 hrs and then treated with the indicated concentration of DMSO, MYCi975, Doxycycline, or both drug treatments (n=3). After 72 hrs of drug treatment, medium was removed completely and viability was estimated by Cell Counting Kit-8 (Dojindo Molecular Technologies) and CellTiter 96 Aqueous One Solution Cell Proliferation Assay- MTS (Promega) as described (2). After removing the complete medium, a reagent was added, and absorbance was measured using a plate reader (Perkin Elmer Victor 3V) Recombinant protein production of MYC Human c-MYC (NM_002467.6 ORF clone) was introduced into backbone vector pET-28a(+)-TEV (GenScript) with an N-terminal hexa-histidine (His 6) tag separated by a TEV (Tobacco Etch Virus) protease digestion site and expressed in bacteria BL21-Gold (DE3) strain (Agilent #230132). For bacterial transformation, 100 μl of bacterial stock was added to pre-chilled 15-mL Falcon tubes, to which 50ng of plasmid (50ng / μL concentration in nuclease-free water) was added and incubated on ice for 30 minutes. Following the incubation period, the cells were placed in 42 ℃ water bath for 30 seconds, after which were Attorney Docket No.702581.02595 (NU2023-153-02) further incubated on ice for 2 minutes. Then 0.9 mL of pre-heated Luria Broth (LB) medium was added to each transformation reaction and incubated at 37 ℃ for 1 hour with shaking at 225-250 rpm. The falcon tubes were quickly spun down at 1000g for 4 minutes and the pellet concentrated using 100 μL of the supernatant. The solution was then spread on LB agar plates with 50 μg / mL working Kanamycin concentration and incubated at 37 ℃ overnight. Single colonies were picked from the plates and grown overnight in 5 mL of LB medium with 50 μg / mL of Kanamycin at 37 ℃ and shaken at 200 rpm. Next day, culture volume was scaled up 6x (30mL total volume) with fresh LB containing 50 μg / mL of Kanamycin and also left overnight under same conditions. Finally, 10 mL of this culture was expanded 30x with fresh LB broth media without antibiotics for a further 3 to 4 hour culture to reach an A600-0.6 to 0.8, and then expression induced by adding 0.5 mM isopropyl-L- thio-B-D-galactopyranoside (IPTG, Sigma-Aldrich cat#I1284) for 4 hours at 37 ℃. Cell pellet from the culture was harvested by centrifugation for 10 min at room temperature at 3000 g and lysed in a buffer containing 8 M urea, 100 mM NaH2PO4, and 10 mM Tris-HCl (pH 8.0), using 12 mL of buffer for the pooled cell pellets from the 300 mL culture. Lysis was allowed to proceed for 1 hour (shaken at 260rpm) at room temperature, followed by centrifugation for 30 minutes at 10,000 g at room temperature. The supernatant was collected and mixed with Ni-NTA agarose slurry (Qiagen #30210), using 1 mL slurry per 4 mL of cleared supernatant, for a total of 3 mL Ni-NTA slurry. The supernatant-slurry mixture was placed back on rocker at 260 rpm for 1 more hour at room temperature, after which the protein was purified using 5 mL polypropylene columns (Qiagen #34964) with a pH gradient elution as instructed in QIAexpressionist (protocols 17, page 90). For removal of urea, Zeba Desalt spin columns (Thermo Fisher #89892, 5mL capacity) were used, exchanging per the manufacturer protocols into a pH 8.050 mM NaH2PO4, 150 mM NaCl, 10 mM Tris base buffer. Biolayer interferometry assay (BLI) Binding assays for small molecule MYCi to full-length recombinant MYC were conducted using an Octet K2 instrument (ForteBio) in a solid black 96-well micro-plate at 37 °C. His-tagged Myc protein (439 amino acids long, UniProt accession number P01106-1) was immobilized on a Ni-NTA sensor by incubating the sensor in 200 μl of peptide solution (1 μg / mL) for five minutes. Kinetics assay consisted of the following steps: equilibration in buffer (50 mM HEPES-KOH, pH 7.4, 150 mM NaCl) for three minutes to establish a baseline signal, association with different concentrations of MYCi for six minutes, followed Attorney Docket No.702581.02595 (NU2023-153-02) by dissociation in the same buffer as the baseline. Data analysis was done using ForteBio Data Analysis 7.0. Western Blot analysis Western blot was performed as described (3). Following transfer of proteins from SDS-PAGE to membrane, the membranes were blocked for 1 h at room temperature with 5% blotting-grade blocker non-fat dry milk (Bio-Rad) or 2% BSA, followed by overnight 4 °C incubation with the appropriate primary antibody and 1 h room temperature incubation with an anti-rabbit or anti-mouse IgG (H + L)-HRP conjugate (Bio-Rad) secondary antibody. Blots were imaged using Supersignal West Femto Maximum Sensitivity Substrate detection system (Thermo Scientific #34096) and the ChemiDoc Imaging System (Bio-Rad #12003153). The following primary antibodies were used: c-Myc (Y69) (Abcam #ab32072, 1:1000), MYCN (Proteintech #10159-2-AP, 1:1000), eIF2α S51 (Cell Signaling #9721, 1:1000), eiF2α (Cell Signaling #9722, 1:1000), CHOP (Cell Signaling #2895, 1:1000), GAPDH (Cell Signaling #3683, 1:5000) and Actin (Cell Signaling #5125, 1:5000). Quantification analyses were performed by Biorad ChemiDoc Imager and Bio-Rad Image Lab software. Small molecule inhibitors Small molecule inhibitors were purchased from MedChem express. The inhibitors used are: 10058-F4 (#HY-12702), 10074-G5 (#HY-100996), Mycro 3 (#HY-100669), KJ- Pyr-9 (#HY-19735), MYCMI-6 (#HY-124675), KSI-3716 (#HY-12703), EN4 (#HY- 134761), PERKi / GSK2656157 (#HY-13820) and GCN2i / GCN2-IN-1 (#HY-100877). ICD- ATP secretion assay MycCaP shATF4 cells were plated into 6-well plates at approximately 1500 cells / well in triplicate. Cells were plated and allowed to adhere for 6 hours and then treated with the indicated concentration of DMSO, MYCi975, Doxycycline, or both drug treatments. After 24 hours of drug treatment, supernatants were collected and cell counts performed for quantifying secreted ATP as per manufacturer’s protocol (Bioluminescent Assay Kit, Sigma- Aldrich # FLAA). Puromycin incorporation assay PC3 cells were seeded on 6-well plates in growth media. On the next day, cells were treated with 10 μM MYCi975 for 1, 2, 4, 8, 16, or 24 hours. For puromycin labeling, puromycin (10 μg / ml, Sigma-Aldrich #P9620) was added during the last 30 min before harvest. For Western Blotting, anti-puromycin antibody (Sigma-Aldrich, MABE343) at 1:2000 dilution was used. Flow cytometry of immune cells Attorney Docket No.702581.02595 (NU2023-153-02) Single-cell suspensions from spleen and lymph nodes were stained at 4°C using predetermined optimal concentrations of Abs for 30 min. Cells with the forward and side scatter properties of lymphocytes were analyzed using Fortessa flow cytometer (BD Bioscience, San Jose, CA). Background staining was assessed using isotype matched control (Ctrl) Abs. The following Abs were used: FITC-conjugated anti-mouse CD3, pacific blue- conjugated anti-mouse F4 / 80 (BioLegend, San Diego, CA) , FITC-conjugated anti-mouse CD45, pacific blue-conjugated anti-mouse CD4, PE-conjugated anti-mouse CD8a, APC- conjugated anti-mouse CD69, PE or APC-conjugated anti-mouse CD11b, APC-conjugated anti-mouse Gr-1 (BD Biosciences, San Jose, CA), PE-conjugated anti-mouse iNOS, APC- conjugated anti-mouse Arginase 1 (eBioscience, San Diego, CA). Splenic CD1dhiCD5+ B cells were determined using V450-conjugated antimouse CD19, PE-conjugated anti-CD5, Alexa fluor 647-conjugated CD1d (Biolegend, San Diego, CA). For the detection of Tregs, splenocytes were stained with FITC-conjugated anti-mouse CD4 and APC-conjugated anti- mouse CD25 Abs, fixed, permeabilized, and subsequently stained with PE-conjugated anti- mouse Foxp3 Ab (eBioscience, San Diego, CA). Isolation and generation of bone marrow-derived macrophages Mouse femur and tibia from vehicle-treated or MYCi975-treated mice were flushed (2-3 times) using a syringe with ice-cold DMEM supplemented with 10% FBS until the bones are completely white. Collected cells were cultured in DMEM with 10% FBS and 20 ng / ml M-CSF (PeproTech) media in a 37oC incubator with 5% CO2. On day 7, BMDMs were treated with conditioned media in indicated experiments. Migration assay BMDMs (5x104) in media containing 2% FBS were seeded on the top chamber of Transwell with a pore size of 8 μm (Corning cat# 353097). The conditioned media of Myc- CaP cells were added into the bottom chamber. After 24 hours, the chambers were fixed with 70% ethanol for 5 minutes and stained with crystal violet. Cells on the upper surface of the transwell membrane were wiped with a cotton swab. Cell migration was quantified by counting the migrated cells in four random fields under a microscope. ATAC seq Cells were plated in 10 cm2plates and treated with 10 μM MYCi975 or DMSO (0.2%) for 48 hrs. ATAC-seq libraries were generated as previously described with slight modifications (4, 5). Treated and control cells were trypsinized and 1 million cells were washed in ice-cold PBS. Cells were pelleted at 500g for 5 min at 4°C and resuspended in 1 ml of lysis reaction mix (0.1% Tween-20, 0.1% IGEPAL (Sigma-Aldrich, cat#: I8896), 0.01 Attorney Docket No.702581.02595 (NU2023-153-02) % Digitonin (Promega, cat#: G9441) in ATAC resuspension buffer (10 mM Tris-HCl, pH 8.0, 10 mM NaCl, 3 mM MgCl2). The lysis reaction was carried out on ice for 3 min, then 4 ml of wash buffer (0.1% Tween-20 in ATAC resuspension buffer) was added and mixed end- over-end. Nuclei were pelleted at 500g for 10 min at 4°C. The pellet was resuspended in 125 μl of ice-cold PBS and the nuclei were counted and inspected for quality. In total, 12,500 nuclei were aliquoted into the transposase tagmentation mix (2.5 μl TDE1 enzyme and 25 μl of TD Buffer (Illumina, FC-121-1030), 16.5 μl of nuclei in ice-cold PBS, 0.5 μl of 1% Digitonin, 0.5 μl of 10% Tween-20 and 5 μl molecular biology-grade water). Tagmentation was carried out at 37°C for 30 min in a Thermomixer (Eppendorf) at 300 rpm. DNA was isolated using the Zymo DNA clean and concentrator (cat#: D4013) and amplified for 9 cycles with New England Biosystems High Fidelity 2X PCR Master Mix (NEB, cat#: M0541S) as described (4). PCR amplified ATAC-seq libraries were purified using the Zymo kit (cat#: D4013) and Ampure XP beads (Cat#A63880) were used for 0.6X-1.8X size selection. Library distribution was analyzed with an Agilent 2100 Bioanalyzer. Paired-end sequencing was performed (2x42bp) using an Illumina NextSeq 500. RNA-sequencing PC-3, 22Rv1 and MycCaP cells were treated with 8μM (PC3) or 10 μM MYCi975 (22Rv1 and MycCaP), and P493-6 cells were treated with 100ng / ml of tetracycline or 6μM of MYCi975 for 24 hrs. PC-3 cells were also treated with 10 μM NUCC-226435 (“435”) and 10 μM NUCC-226436 (“436”) for 24 hours. Cells were collected using trypsinization and washed with ice-cold PB and later centrifuge at 1500 rpm for 5 minutes. Total RNA was extracted from cell pellet using Qiagen RNeasy Plus kit. The stranded total RNA-seq was conducted in the Northwestern University NUSeq Core Facility. Briefly, total RNA examples were checked for quality on Agilent Bioanalyzer 2100 and quantified with Qubit fluorometer. The NEBNext Ultra II RNA Library Prep Kit for Illumina was used to prepare sequencing libraries. The Kit procedure was performed without modifications. This procedure includes rRNA depletion, remaining RNA purification and fragmentation, cDNA synthesis, end preparation, Illumina adapter ligation, library PCR amplification and validation. lllumina HiSeq 4000 Sequencer was used to sequence the libraries with the production of paired-end, 50 bp reads. Immunofluorescence and Immunohistochemistry Tumor sections were fixed and processed for immunostaining as described (6, 7). Primary antibodies used are: CD-3 (Roche Ventana #790-4341), c-Myc (Y69) (Abcam #ab32072), Ki-67 (Abcam #ab15580 & BD Biosciences #550609), Cleaved Caspase-3 (Cell Attorney Docket No.702581.02595 (NU2023-153-02) Signaling #9661), PD-L1 (Cell Signaling #13684), NKp46 (Biolegend 137601), FoxoP3 (Abcam #ab20034), HMGB1 (Abcam #ab18256) and Calreticulin (Abcam # ab2907). For Immuno-histochemistry, slides were incubated with ImmPRESS HRP anti-mouse (Vector #MP-7402) or ImmPRESS HRP anti-rabbit (Vector#MP-7401). Protein expression was visualized by using AEC peroxidase substrate (Vector #SK-4200). Slides were incubated with Hematoxylin (Vector #3404) and mounted with Glycergel Mounting Medium (Dako #C056330-2). For immuno-fluorescence, slides were incubated with secondary antibodies labelled with Alexa Fluor 488 anti-rabbit (Thermo Scientific # A11008), Alexa 594 anti- rabbit (Thermo Scientific #A-21207), Alexa 594 anti-mouse (Molecular probes #A11005) and Alexa Fluor 568 Goat anti-Mouse IgG (H+L) (Thermo Scientific #A-11004). Slides were counterstained with DAPI (Sigma-Aldrich #D-9542) and mounted with ProLong Gold Antifade reagent (Invitrogen / Molecular Probes #P36961). Immunofluorescence images were visualized using fluorescent microscope or Leica A1R spectral confocal microscope. ChIP-seq 22Rv1 cells were plated at ~5x106cells in 15cm2dishes. Two days later, cell culture media was replenished with 30 mL of fresh media containing either 10 M MYCi975 or vehicle control (0.1% DMSO). Following 16-hours of treatment, formaldehyde crosslinking was performed by the addition of 1 / 15thvolume (2mls) of 16% methanol-free paraformaldehyde (Electron Microscopy Sciences #15710) for 10 minutes at room temperature. Formaldehyde crosslinking was terminated by the addition of glycine to 119 mM (1.6ml of 2.5M glycine) for 5 minutes at room temperature. Cells were washed three times with ice-cold PBS, scraped into ice-cold PBS, and recovered by centrifugation at1500g, for 10 minutes at 4 C. Formaldehyde-crosslinked cell pellets were snap frozen inliquid nitrogen and stored at -80 C.Soluble chromatin was prepared by sonication and chromatin immunoprecipitation, library preparation, and sequencing were performed as described previously with minor modifications (8). Briefly, frozen cell pellets were quick-thawed and resuspended in 4mls of Lysis Buffer 1 (50 mM HEPES-KOH, pH 7.6, 140 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 10% Glycerol, 0.5% IGEPAL-CA630, 0.25% Triton X-100 supplemented with 1x Complete Protease Inhibitor cocktail [Roche]) and incubated with end-over-end mixing for 10 minutesat 4 C. Chromatin was recovered by centrifugation (2000g, 10 min, 4 C), resuspended in4mls of Lysis Buffer 2 (10 mM Tris-HCl, pH 8.0, 200 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 1x Complete Protease Inhibitor cocktail) and extracted with end-over-end mixing for Attorney Docket No.702581.02595 (NU2023-153-02)10 minutes at 4 C. The insoluble chromatin was recovered by centrifugation (2000g, 10 min,4 C), resuspended in 0.9 ml of Lysis Buffer 3 (10 mM Tris-HCl, pH 8.0, 200 mM NaCl, 1mM EDTA, 1 mM EGTA, 0.1% Sodium Deoxycholate, 0.5% Sarkosyl, 1x Complete Protease Inhibitor cocktail) and transferred to pre-chilled 1.5ml microcentrifuge tubes on ice. Chromatin was sheared by sonication in an ice-water bath using a Misonix micro-tip equipped sonicator at setting 5 (~5W RMS output power) for 13 cycles of 15 seconds sonication followed by a 45 second cooling interval. The sonicated chromatin was adjusted to 1% Triton X-100 from a 10% stock solution and debris removed by centrifugation at 20,000g at 4°C for 20 minutes. The protein concentration of solubilized chromatin was determined by BCA assay. Approximately 200 g of chromatin was used for H3K27ac ChIP and 400 g of chromatin was used for ATF4 and MYC ChIP. Immunoprecipitations were performed overnight at 4°C with the indicated antibodies and amounts: H3K27ac (Active Motif #39133, 3 g), ATF4 (Cell Signaling #11815, 0.35 g), MYC[Y69] (Abcam #ab32072, 2 g). Chromatin immunoprecipitation complexes were recovered by the addition of 30 l ofProtein G Dynabeads followed by incubation at 4 C with end-over-end mixing for 3 hours.Magnetic beads were collected using a Dyna-Mag2 magnetic stand and beads were washed four times with 1 ml of ChIP-RIPA wash buffer (50 mM HEPES-KOH, pH 7.6, 500 mM LiCl, 1 mM EDTA, 1.0% IGEPAL-CA630, 0.7% Na- Deoxycholate) and once with TE containing 50 mM NaCl. Bead-bound immunoprecipitated chromatin was eluted by the addition of 50 l of 0.1M NaHCO3, 1% SDS followed by incubation at 65°C with orbital mixing (900rpm) in an Eppendorf Thermomixer R for 15 minutes. Eluates were transferred to clean 1.5ml Eppendorf LoBind tubes. The elution step was repeated, and eluates were combined. Input chromatin was thawed and adjusted to contain 0.1 M NaHCO3, 1% SDS in a final volume of 100 l. All samples were adjusted to contain 190 mM NaCl and reverse crosslinking was performed by incubating at 65°C for 12 hours in an Eppendorf Thermomixer R. Reverse-crosslinked chromatin was sequentially treated with RNAse A and proteinase K, and DNA was purified using Qiagen MinElute spin columns. Libraries were prepared from less than 5ng of ChIP-enriched or input DNA using KAPA Hyper Prep Kit (Roche #KK8502) according to manufacturer’s instructions except adapter ligations were extended from 15 to 60 minutes at room temperature. A post-adapter ligation, pre- amplification double-sided size selection (0.7-0.9X) step using Ampure XP beads (Beckman- Coulter #A63880) was included. Library concentration was determined by fluorometry using Qubit high-sensitivity dsDNA assay kit (Thermo Fisher Scientific #Q32851) and library size Attorney Docket No.702581.02595 (NU2023-153-02) was determined using an Agilent 2100 Bioanalyzer. Individual libraries were pooled, and the library pool concentration determined using KAPA Library Quantification Kit (Roche #KK4835). Libraries were sequenced as 100bp single-end reads using an Illumina NextSeq 2000 (Division of Endocrinology, Department of Medicine, Northwestern University). Bioinformatics and Data analysis ChIP-seq Single-end ChIP-seq reads were aligned using bowtie2 (v2.2.6, settings –end-to-end) and a pre-built human reference genome index (GRCh38 no alt analysis set) retrieved from http: / / bowtie-bio.sourceforge.net / manual.shtml. ChIP-seq peaks were identified using HOMER (v4.10) with settings -tbp 1 and either -style factor (for ATF4 / MYC ChIP-seq) or - style histone (H3K27ac ChIP-seq). Regions of differential ATF4 / MYC chromatin binding or H3K27ac deposition were determined using the R / Bioconductor package DiffBind (v3.4.11). Problematic genomic regions of known artifactual signal / noise were excluded from differential affinity analysis using the built-in DiffBind function dba.blacklist. For each ChIP- seq (e.g., ATF4, MYC, H3K27ac), consensus peaksets were determined for DMSO and MYCi975 treatments separately and only peaks present in every biological replicate were included. The union of these treatment-level consensus peaks was then determined to give the final peak sets for DiffBind analysis. Read counts at peak summits were then extracted using the DiffBind function dba.count with option bRemoveDuplicates = FALSE and these counts were normalized to sequencing depth using the function dba.normalize. Regions of differential signal were identified using DiffBind’s dba.analyze function with default parameters. Sites with an adjusted false discovery rate of less than 0.01 (FDR < 0.01) were considered differentially enriched. Volcano plots of ChIP-seq signal were generated using the R / Bioconductor package EnhancedVolcano (v1.12.0). Peak set overlaps were determined using bedtools intersect (v2.30.0). UpSet plots were created using the R / Bioconductor package ComplexHeatmap (v2.10.0). ATAC-seq For paired-end ATAC-seq, adaptors were removed with NGmerge (v0.2_dev) (11) and reads were aligned with bowtie2 (v2.2.6, settings: --very-sensitive -X 800). Mitochondrial reads were removed from alignment files using the Unix utility ‘sed’. Peak calling was performed using MACS2 (v2.2.6) (9) with the DNase-seq enriched cut site method (--nomodel --shift -75 --extsize 150 --nolambda -p 0.01). Peaks from each ATAC-seq dataset (DMSO vs. 48-hr MYCi975) were used as input for differential transcription factor activity analysis (DAStk) using default settings (12). Attorney Docket No.702581.02595 (NU2023-153-02) RNA-seq For paired-end RNA-seq, reads were aligned to either human (hg38) or mouse (mm10) reference genomes using the STAR aligner (9) (v2.7.5, settings: --alignIntronMin 20 --alignIntronMax 500000). Count matrices were generated using featureCounts from the Subread package (v1.6.1, settings: -g gene_id -t gene -p -s 2) with either human (hg38.ncbiRefSeq.gtf, http: / / hgdownload.soe.ucsc.edu / goldenPath / hg38 / bigZips / genes / ) or mouse (mm10.ncbiRefSeq.gtf, http: / / hgdownload.soe.ucsc.edu / goldenPath / mm10 / bigZips / genes / ) annotation files. Differential RNA levels in 24-hour MYCi975 treated samples versus DMSO controls were determined using DESeq2 (10) with default settings. For GSEA (11) analysis of RNA-seq, each gene was assigned a rank metric [-log10(FDR)Xlog2(foldchange)] and this pre-ranked list was used as input for GSEA hallmarks v7.4 database. GSVA scoring and MiRS generation Cell line baseline transcriptomic data were obtained from the Cancer Cell Line Encyclopedia (CCLE) hosted on the Cancer Dependency Portal (DepMap) (12) for human cancer cell lines as raw counts and from the Tumor Immune Syngeneic Mouse (TISMO) (13) portal for murine lines as TPM normalized counts. The matrix of normalized counts for a given sample was scored using candidate gene sets (either canonical, for example HALLMARK gene sets, or gene set candidates obtained in house) via Gene Set Variation Analysis using the gsva() function in R and the following settings: kcdf = Gaussian, mx.diff = FALSE. For generating geneset candidates for GSVA analysis, we used RNAseq experiments performed in 3 different cell lines (MycCaP, PC3 and 22Rv1), treated with 8 μM (PC3) or 10 μM (MycCaP, 22Rv1) MYCi975 for 24 hours. Using DESeq2, differences in RNA levels were determined by comparing MYCi vs DMSO treated conditions to obtain significantly differentially expressed genes. The genes that are significantly differentially expressed and in the same direction (upregulated / downregulated) in all three cell lines are termed the MYCi response genes and serve as the baseline pool of genes from which candidate genesets were obtained for GSVA analysis. The candidate genesets were derived from the baseline set of 1881 genes by applying log2 fold change filters. The GSVA rank sum algorithm outputs scores which depend on the matrix of normalized counts as input, as well as the gene set this matrix is scored against. We obtained different sets of scores for the xenograft tumor cell lines by employing different candidate genesets and each set of different scores was subsequently assessed against MYCi in vivo Attorney Docket No.702581.02595 (NU2023-153-02) experimental data, using tumor growth inhibition percentages as quantifiable markers of MYCi efficacy. The candidate gene set which showed the best correlation of GSVA scores with TGI percentages from in vivo experimental data was labeled as the MYCi Response Signature (MiRS). Modeling Unacetylated and Acetylated MYC From the AlphaFold 3 Server, the top five predicted models of unacetylated MYC; MYC with N6-acetylation of lysine at residues 148 and 157; MYC with arginine residues substituted for lysine residues at positions 148 and 157; and MYC with glutamine residues substituted for lysine residues at positions 148 and 157 were downloaded. Predicted Local Distance Difference Test (pLDDT) scores for each atom of an amino acid were averaged to generate average pLDDT scores for each amino acid in the 439-amino acid protein; these pLDDT scores were plotted across the protein in each model. Predicted aligned error (PAE) values for each amino acid pair in each model were also extracted. In Vitro Pull Down Assay Nuclear extracts from 22RV1 cells were pre-cleared with streptavidin beads (Thermo, 88817) for 1 hr at 4C°.100 μg nuclear extract was applied to each sample and incubated with 10 μM of Biotin975, 10 μM of D-Biotin or DMSO on a rotator over night at 4C°. Next day, 60 μl of streptavidin beads was added to each sample and further rotated for 1 hr at 4C°. Beads were washed, then eluted with 2x sample buffer and boiled at 95C° for 5 min. The supernatant was subjected to Western Blot. General Chemical Methods All chemical reagents were obtained from commercial suppliers and used without further purification, unless otherwise stated. Reactions were run without taking precautions to exclude air or moisture, unless otherwise noted. Normal phase column chromatography was performed using silica gel columns and ACS grade solvents. Analytical TLC was performed on EM Reagent 0.25 mm silica gel 60 F254 plates and visualized by UV light. Compound identities were confirmed by1H and F (NMR) spectroscopy which were recorded on a Bruker 400 MHz spectrometer using the corresponding residual slovent peak (CDCl3,1H δ = 7.27; CD3OD, 1H δ = 3.31; DMSO-d6, 1H δ = 2.50) as an internal standard. The chemical shifts for1H-NMR is reported to the second decimal place. Proton coupling constants are expressed in hertz (Hz). Standard abbreviations were used to denote spin multiplicity for1H NMR data. Statistical Analysis Attorney Docket No.702581.02595 (NU2023-153-02) All statistical analyses were performed in GraphPad Prism 9 software and R packages. For in vitro assays, unpaired two-sided t test was used for data that followed a normal distribution. For data that did not follow a normal distribution, Mann-Whitney test was used. For in vivo xenografts, tumor growth was compared between groups using linear mixed effects models with mouse as the random effect and first-order autoregressive (AR(1)) correlation structure between repeated tumor size measurements. The number of technical replicates, biological replicates, and independent experiments performed are indicated in the figure legends. Data are presented as mean ± standard error of the mean (S.E.M.) and statistical significance was defined by P < 0.05 (two-tailed). Pearson’s correlation coefficient and corresponding P value were used to measure the extent of correlation between MYCi response signature scores and MYCi in vivo tumor growth inhibition percentages. For bioinformatics data, false discovery rates (FDRs) were calculated using the Benjamini- Hochberg method and cut off at 5%. In figures, “*” indicates P < 0.05, “**” indicates P < 0.01, “***” indicates P < 0.001, “****” indicates P < 0.0001, and not significant “ns” indicates P ≥ 0.05 for the indicated pairwise comparison. Error bars in all figures indicate SEM unless stated otherwise. Additional information can be found in Supplementary Materials and Methods. Scheme 1. CF3O 4'-chloro-6-((4-chlorobenzyl)oxy)-3-(1-methyl-5-(trifluoromethyl)-1H-pyrazol-3-yl)- 3'-(trifluoromethyl)-[1,1'-biphenyl]-2-ol (NUCC-0200733). To a solution of 8-(4-chloro-3- (trifluoromethyl)phenyl)-7-((4-chlorobenzyl)oxy)-2-(trifluoromethyl)-4H-chromen-4-one (14)(1, 400 mg, 750.12 mol, 1 eq) in EtOH (8 mL) was added CH3NHNH2(259.19 mg, 2.25 mmol, 40% purity, 3 eq), the reaction was stirred at 80°C for 3 hrs. LCMS showed the starting material was consumed and two new peaks with desired product mass were detected. Attorney Docket No.702581.02595 (NU2023-153-02) The reaction was concentrated to give crude product, which was purified by Prep-HPLC (column: Waters Xbridge Prep OBD C18 150*40 mm*10 m; mobile phase: [water (NH4HCO3)-ACN]; B%: 70%-95%, 8 mins) to give NUCC-0200733 (51.9 mg, yield 12.3%) as a white solid.1H-NMR (400 MHz, CDCl3): δ 4.01 (s, 3H), 5.05 (s, 2H), 6.65 (d, J=8.76 Hz, 1H), 6.93 (s, 1H), 7.17 (d, J=8.25 Hz, 2H), 7.30 (d, J=8.38 Hz, 2H), 7.51 (d, J=8.63 Hz, 1H), 7.53-7.58 (m, 1H), 7.58-7.64 (m, 1H), 7.85 (d, J=1.63 Hz, 1H), 10.67 (s, 1H).19F-NMR (376 MHz, CDCl3): δ 62.37, 60.57. MS (ESI+): m / z 561.0 [M + H]+. Scheme 2. O O O O CF COONa I , pyridineCF g, 65.73 mmol, 8.47 mL, 1 eq) in TFAA (37 mL) was placed in a high-pressure tube (100 mL). Sodium trifluoroacetate (19.67 g, 144.60 mmol, 2.2 eq) was added and the system was capped and stirred at 130oC for 24 h. A total of 30 batches (10 g each) were set in parallel. LCMS showed all starting materials consumed. The reaction were allowed to cool to 25oC and combined and then diluted with EtOAc (1 L). The mixture was neutralized by saturated Attorney Docket No.702581.02595 (NU2023-153-02) aqueous K2CO3solution until no more bubbling was observed. The organic layer was separated and the aqueous portion was extracted with EtOAc (3 × 500 mL). The organic layer was washed by brine, dried over anhydrous Na2SO4, concentrated to 1 / 3 volume of EtOAc and the flask was allowed to stand at 25oC for 8 hrs. Compound 2 (180 g, 793.0 mmol, 40.0% yield) was collected as a white solid.1H-NMR (400 MHz, CD3OD) δ ppm 6.75 (s, 1 H) 6.93 (d, J=2.20 Hz, 1 H) 7.01 (dd, J=8.86, 2.26 Hz, 1 H) 8.02 (d, J=8.80 Hz, 1 H). LCMS (ESI+): m / z = 231.1 (M+H)+. O O I2, pyridine 7-hydroxy-8-iodo-2-(trifluoromethyl)-4H-chromen-4-one (3). A solution of Compound 2 (180 g, 782.13 mmol, 1 eq), iodine (794.05 g, 3.13 mol, 630.20 mL, 4 eq), pyridine (247.47 g, 3.13 mol, 252.52 mL, 4 eq) in chloroform (1 L) was stirred at 25oC for 8 h. LCMS showed the reaction completed. The mixture was poured into water (500 mL) and triturated with petroleum ether: ethyl acetate (10:1, 800 mL) to give 3 (210 g, 589.83 mmol, 75.4% yield) as yellowish solid.1H-NMR (400 MHz, CD3OD) δ ppm 6.79 (s, 1 H) 7.02 (d, J=8.82 Hz, 1 H) 7.95 - 7.99 (m, 1 H). LCMS (ESI+): m / z = 356.9 (M+H)+. O O Br 7-(benzyloxy)-8-iodo-2-(trifluoromethyl)-4H-chromen-4-one (4). A solution of Compound 3 (130 g, 365.13 mmol, 1 eq), bromomethylbenzene (74.94 g, 438.16 mmol, 52.04 mL, 1.2 eq), bromomethylbenzene (74.94 g, 438.16 mmol, 52.04 mL, 1.2 eq) in acetone (1 L) was added potassium carbonate (100.93 g, 730.26 mmol, 2 eq). The mixture was stirred at 80°C for 8 h. TLC showed the reaction completed. The mixture was poured into water (500 mL) and extracted with ethyl acetate (3 × 500 mL). The organic layer was dried over Na2SO4and concentrated to give crude product, which was purified by chromatography on silica, eluted with petroleum ether:ethyl acetate= 10:1 to 5:1 to give Attorney Docket No.702581.02595 (NU2023-153-02) desired product 4 (120 g, 268.96 mmol, 73.7% yield) as yellowish solid.1H-NMR (400 MHz, CDCl3) δ ppm 5.27 (s, 2 H) 6.63 (s, 1 H) 6.96 (d, J=8.93 Hz, 1 H) 7.10 - 7.39 (m, 8 H) 7.40 - 7.46 (m, 2 H) 8.08 (d, J=8.93 Hz, 1 H). Cl O O OH F C B CF3 - - - chromen-4-one (5). A solution of 4 (100 g, 22.41 mmol, 1 eq), [4-chloro-3- (trifluoromethyl)phenyl]boronic acid (5.03 g, 22.41 mmol, 1 eq), cesium carbonate (14.61 g, 44.83 mmol, 2 eq) in toluene (2 L) and ethanol (400 mL) and water (80 mL) was added Pd(dppf)Cl2(1.64 g, 2.24 mmol, 0.1 eq) under N2atmosphere. The mixture was stirred at 80°C for 8 h. TLC showed the reaction completed. The mixture was poured into water (500 mL) and extracted with ethyl acetate (3×500 mL). The organic layer was dried over Na2SO4and concentrated to give crude product which was triturated with ethyl acetate: petroleum ether (1:10, 500 mL) to give desired product 5 (93 g, 18.64 mmol, 83.2% yield) as yellowish solid.1H-NMR (400 MHz, CDCl3) δ ppm 5.26 (s, 2 H) 6.71 (s, 1 H) 7.27 (d, J=2.93 Hz, 2 H) 7.34 - 7.41 (m, 3 H) 7.55 - 7.66 (m, 2 H) 7.85 (s, 1 H) 8.26 (d, J=8.93 Hz, 1 H). CFO3N 6-(benzyloxy)-4'-chloro-3-(1-methyl-3-(trifluoromethyl)-1H-pyrazol-5-yl)-3'- (trifluoromethyl)-[1,1'-biphenyl]-2-ol (6). A suspension of 5 (100 g, 200.48 mmol, 1 eq) , methylhydrazine (27.71 g, 601.44 mmol, 31.67 mL, 3 eq) in ethanol (500 mL) was bubbled with N2 gas for 10 minutes. The vial was then heated at 80oC for 16 h. LCMS showed all Attorney Docket No.702581.02595 (NU2023-153-02) starting materials consumed. The resulting mixture was concentrated to give crude residue which was triturated by toluene to obtain 6 (75 g, 142.35 mmol, 71.0% yield) as brown solid. LCMS (ESI+): m / z = 527.2 (M+H)+. CF3CF3N N 6-(benzyloxy)-4'-chloro-3-(1-methyl-3-(trifluoromethyl)-1H-pyrazol-5-yl)-3'- (trifluoromethyl)-[1,1'-biphenyl]-2-yl acetate (7). A solution of 6 (42 g, 79.72 mmol, 1 eq) in DCM (500 mL) was added acetyl chloride (7.51 g, 95.66 mmol, 6.83 mL, 1.2 eq) and TEA (9.68 g, 95.66 mmol, 13.31 mL, 1.2 eq). The mixture was stirred at 25oC for 8 hrs. TLC showed all starting materials consumed. Once completion, the mixture was poured into water and extracted with DCM (2×100mL). The organic layer was dried over Na2SO4, concentrated to give 7 (45 g, 79.10 mmol, 99.2% yield) as white solid which was used directly in next step.1H-NMR (400 MHz, CDCl3) δ 1.72-1.81 (m, 3H), 3.75-3.84 (m, 3H), 5.10-5.16 (m, 2H), 6.45-6.52 (m, 1H), 7.07 (d, J=8.60 Hz, 1H), 7.21-7.27 (m, 3H), 7.38 (s, 2H), 7.44-7.49 (m, 1H), 7.51-7.57 (m, 1H), 7.72-7.76 (m, 1H). CF CF33N 4'-chloro-6-hydroxy-3-(1-methyl-3-(trifluoromethyl)-1H-pyrazol-5-yl)-3'- (trifluoromethyl)-[1,1'-biphenyl]-2-yl acetate (8). A solution of 7 (50 g, 87.89 mmol, 1 eq) in Attorney Docket No.702581.02595 (NU2023-153-02) DCM (200 mL) was cooled to 0oC. BCl3(1 M, 175.78 mL, 2 eq) was added to the mixture dropwise and maintain the temperature below 0oC. The mixture was stirred at 0oC for 4 hs. TLC showed all starting materials consumed. On completion, ice (100 mL) was added to the mixture and the organic layer was separated. The aqueous was extracted with EtOAc (3 × 100 mL). The organic layer was dried over Mg2SO4 and concentrated to give a product which was purified by chromatography on silica, eluted with PE:EA=10:1 to 1:1 give 8 (25 g, 52.22 mmol, 59.4% yield) as white solid.1H-NMR (400 MHz, CDCl3) δ 1.78 (s, 3H), 3.76- 3.84 (m, 3H), 5.95 (s, 1H), 6.48-6.53 (m, 1H), 6.96-7.02 (m, 1H),7.23-7.28 (m, 1H), 7.48- 7.53 (m, 1H), 7.62 (d, J=8.16 Hz, 1H), 7.70-7.74 (m, 1H). - 3'-(trifluoromethyl)-[1,1'-biphenyl]-2-ol (NUCC-0226435) and (R)-4'-chloro-3-(1-methyl-3- (trifluoromethyl)-1H-pyrazol-5-yl)-6-(1-(p-tolyl)ethoxy)-3'-(trifluoromethyl)-[1,1'-biphenyl]- 2-ol (NUCC-0226436). A mixture of 8 (200 mg, 417.74 mol), Cpd 2 (85.34 mg, 626.6 mol), diisopropyl azodicarboxylate (145.75 mg, 835.48 mol), triphenylphosphine (220.14 mg, 835.48 mol) in tetrahydrofuran (4 mL) was degassed and purged with N2for 3 times, and then the reaction was stirred at 25°C for 12 h. TLC showed the reaction was completed. (Rt=0.4, PE:EA=3:1) After that K2CO3 (115.46 mg, 835.48 mol) was added to the reaction, the mixture was stirred at 25°C for 1 h. LCMS showed the reaction was completed. The reaction was poured into water (3 mL) and extracted with ethyl acetate (5 mL). The organic layer was concentrated to give a residue which was purified by Prep-HPLC (column: WatersXbridge BEH C18 100*30mm*10 m;mobile phase: [water(10mM NH4HCO3)-ACN]; B%:70%-95%,10min) to give 9 (42 mg, 18%). There was partial racemization during the Mitsunobu reaction and compound 9 was a 65:35 mixture of S- and R-isomers (30% e.e.).The 42 mg of 9 was separated by SFC (DAICEL CHIRALPAK AD (250mm*30mm,10 m);mobile phase: [0.1%NH3H2O ETOH]; B%: 21%-21%,6min. Two fractions were concentrated to give NUCC-0226435 (12 mg, yield 29%) and NUCC-0226436 (5 mg, yield 12%). Attorney Docket No.702581.02595 (NU2023-153-02) NUCC-0226435.1H-NMR (400 MHz, CD3OD) δ 1.42 (d, J = 6.39 Hz, 3H), 2.29 (s, 3H), 3.75 (s, 3H), 5.37 (d, J = 6.17 Hz, 1H), 6.54 (s, 1H), 6.65 (d, J = 8.60 Hz, 1H), 7.02-7.15 (m, 5H), 7.59-7.70 (m, 2H), 7.76 (s, 1H). LCMS (ESI+) 555.1 (M+H)+. NUCC-0226436.1H-NMR (400 MHz, CD3OD) δ 1.43 (d, J = 6.62 Hz, 3H), 2.30 (s, 3H), 3.76 (s, 3H), 5.32-5.43 (m, 1H), 6.54 (s, 1H), 6.65 (d, J = 8.82 Hz, 1H), 7.04-7.13 (m, 5H), 7.59-7.71 (m, 2H), 7.76 (s, 1H). LCMS (ESI+) 555.2 (M+H)+. References in the Examples 1. Anker JF, Naseem AF, Mok H, Schaeffer AJ, Abdulkadir SA, Thumbikat P. Multi- faceted immunomodulatory and tissue-tropic clinical bacterial isolate potentiates prostate cancer immunotherapy. Nat Commun.2018;9(1):1591. 2. Sagar V, Vatapalli R, Lysy B, Pamarthy S, Anker JF, Rodriguez Y, et al. EPHB4 inhibition activates ER stress to promote immunogenic cell death of prostate cancer cells. Cell Death Dis.2019;10(11):801. 3. Carneiro BA, Pamarthy S, Shah AN, Sagar V, Unno K, Han H, et al. Anaplastic Lymphoma Kinase Mutation (ALK F1174C) in Small Cell Carcinoma of the Prostate and Molecular Response to Alectinib. Clin Cancer Res.2018;24(12):2732-9. 4. Buenrostro JD, Wu B, Chang HY, Greenleaf WJ. ATAC-seq: A Method for Assaying Chromatin Accessibility Genome-Wide. Curr Protoc Mol Biol.2015;109:2191- 99. 5. Corces MR, Trevino AE, Hamilton EG, Greenside PG, Sinnott-Armstrong NA, Vesuna S, et al. An improved ATAC-seq protocol reduces background and enables interrogation of frozen tissues. Nat Methods.2017;14(10):959-62. 6. Vatapalli R, Sagar V, Rodriguez Y, Zhao JC, Unno K, Pamarthy S, et al. Histone methyltransferase DOT1L coordinates AR and MYC stability in prostate cancer. Nat Commun.2020;11(1):4153. 7. Desai AS, Sagar V, Lysy B, Weiner AB, Ko OS, Driscoll C, et al. Inflammatory bowel disease induces inflammatory and pre-neoplastic changes in the prostate. Prostate Cancer Prostatic Dis.2021. 8. Holmes AG, Parker JB, Sagar V, Truica MI, Soni PN, Han H, et al. A MYC inhibitor selectively alters the MYC and MAX cistromes and modulates the epigenomic landscape to regulate target gene expression. Sci Adv.2022;8(17):eabh3635. 9. Dobin A, Davis CA, Schlesinger F, Drenkow J, Zaleski C, Jha S, et al. STAR: ultrafast universal RNA-seq aligner. Bioinformatics.2013;29(1):15-21. Attorney Docket No.702581.02595 (NU2023-153-02) 10. Love MI, Huber W, Anders S. Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome Biol.2014;15(12):550. 11. Subramanian A, Tamayo P, Mootha VK, Mukherjee S, Ebert BL, Gillette MA, et al. Gene set enrichment analysis: a knowledge-based approach for interpreting genome-wide expression profiles. Proc Natl Acad Sci U S A.2005;102(43):15545-50. 12. Ghandi M, Huang FW, Jane-Valbuena J, Kryukov GV, Lo CC, McDonald ER, 3rd, et al. Next-generation characterization of the Cancer Cell Line Encyclopedia. Nature. 2019;569(7757):503-8. 13. Zeng Z, Wong CJ, Yang L, Ouardaoui N, Li D, Zhang W, et al. TISMO: syngeneic mouse tumor database to model tumor immunity and immunotherapy response. Nucleic Acids Res.2022;50(D1):D1391-D7. 14. Han H, Jain AD, Truica MI, Izquierdo-Ferrer J, Anker JF, Lysy B, et al. Small- Molecule MYC Inhibitors Suppress Tumor Growth and Enhance Immunotherapy. Cancer Cell.2019;36(5):483-97 e15. SEQUENCES MYC WT Amino acid sequence, region underlined is C region, region double underlined is MYCHot. In italics is G region,removed in MYC G (SEQ ID NO:1).MDFFRVVENQQPPATMPLNVSFTNRNYDLDYDSVQPYFYCDEEENFYQQQQQSELQPPAPSE DIWKKFELLPTPPLSPSRRSGLCSPSYVAVTPFSLRGDNDGGGGSFSTADQLEMVTELLGGD MVNQSFICDPDDETFIKNIIIQDCMWSGFSAAAKLVSEKLASYQAARKDSGSPNPARGHSVC STSSLYLQDLSAAASECIDPSVVFPYPLNDSSSPKSCASQDSSAFSPSSDSLLSSTESSPQG SPEPLVLHEETPPTTSSDSEEEQEDEEEIDVVSVEKRQAPGKRSESGSPSAGGHSKPPHSPL VLKRCHVSTHQHNYAAPPSTRKDYPAAKRVKLDSVRVLRQISNNRKCTSPRSSDTEENVKRR THNVLERQRRNELKRSFFALRDQIPELENNEKAPKVVILKKATAYILSVQAEEQKLISEEDL LRKRREQLKHKLEQLRNSCA MYCHot region (SEQ ID NO:2): NELKRSFFALRDQIPELENNEKAMYC C (SEQ ID NO:3):MDFFRVVENQQPPATMPLNVSFTNRNYDLDYDSVQPYFYCDEEENFYQQQQQSELQPPAPSE DIWKKFELLPTPPLSPSRRSGLCSPSYVAVTPFSLRGDNDGGGGSFSTADQLEMVTELLGGD MVNQSFICDPDDETFIKDPSVVFPYPLNDSSSPKSCASQDSSAFSPSSDSLLSSTESSPQGS Attorney Docket No.702581.02595 (NU2023-153-02) PEPLVLHEETPPTTSSDSEEEQEDEEEIDVVSVEKRQAPGKRSESGSPSAGGHSKPPHSPLV LKRCHVSTHQHNYAAPPSTRKDYPAAKRVKLDSVRVLRQISNNRKCTSPRSSDTEENVKRRT HNVLERQRRNELKRSFFALRDQIPELENNEKAPKVVILKKATAYILSVQAEEQKLISEEDLL RKRREQLKHKLEQLRNSCAMYC G (SEQ ID NO:4):MDFFRVVENQQPPATMPLNVSFTNRNYDLDYDSVQPYFYCDEEENFYQQQQQSELQPPAPSE DIWKKFELLPTPPLSPSRRSGLCSPSYVAVTPFSLRGDNDGGGGSFSTADQLEMVTELLGGD MVNQSFICDPDDETFIKNIIIQDCMWSGFSAAAKLVSEKLASYQAARKDSGSPNPARGHSVC STSSLYLQDLSAAASECIDPSVVFPYPLNDSSSPKSCASQDSSAFSPSSDSLLSSTESSPQG SPEPLVLHEETPPTTSSDSEEEQEDEEEIDVVSVEKRQAPGKRSESGSPSAGGHSKPPHSPL VLKRCHVSTHQHNYAAPPSTRKDYPAAKRVKLDSVRVLRQISNNRKCTSPRSSDTEENVKRR THNVLERQRRNELKRSFFALRD MYC 1-353 (SEQ ID NO:5): MDFFRVVENQQPPATMPLNVSFTNRNYDLDYDSVQPYFYCDEEENFYQQQQQSELQPPAPSE DIWKKFELLPTPPLSPSRRSGLCSPSYVAVTPFSLRGDNDGGGGSFSTADQLEMVTELLGGD MVNQSFICDPDDETFIKNIIIQDCMWSGFSAAAKLVSEKLASYQAARKDSGSPNPARGHSVC STSSLYLQDLSAAASECIDPSVVFPYPLNDSSSPKSCASQDSSAFSPSSDSLLSSTESSPQG SPEPLVLHEETPPTTSSDSEEEQEDEEEIDVVSVEKRQAPGKRSESGSPSAGGHSKPPHSPL VLKRCHVSTHQHNYAAPPSTRKDYPAAKRVKLDSVRVLRQISNNRKCTSPRSSDTEE C-linker-G (i.e., C-linker-bHLH-LZ) (SEQ ID NO:6): NIIIQDCMWSGFSAAAKLVSEKLASYQAARKDSGSPNPARGHSVCSTSSLYLQDLSAAASEC IDGSGSGSGSGSGSGSGSGSGSGSGSGSGSGSGSGSGSGSGSNVKRRTHNVLERQRRNELKR SFFALRDQIPELENNEKAPKVVILKKATAYILSVQAEEQKLISEEDLLRKRREQLKHKLEQL RNSCA Note: all of the above are the sequences for the protein construct (inserted into vector) based on UNIPROT. However, our final expressed constructs for the proteins which we have purified for biophysical studies all have the following amino acids attached to the N-terminus = His-tag + a TEV cleavable site (SEQ ID NO:7):MGSSHHHHHHSSGENLYFQG Attorney Docket No.702581.02595 (NU2023-153-02) GCN1: includes only amino acids 700-900, with the above His tag and TEV site sequence N-terminus, so: Sequence GCN1 700-900 (SEQ ID NO:8): FITRHLDQIIPRMTTQSPLNQSSMNAMGSLSVLSPDRVLPQLISTITASVQNPALRLVTREE FAIMQTPAGELYDKSIIQSAQQDSIKKANMKRENKAYSFKEQIIELELKEEIKKKKGIKEEV QLTSKQKEMLQAQLDREAQVRRRLQELDGELEAALGLLDIILAKNPSGLTQYIPVLVDSFLP LLKSPLAAPRIKNP Final expressed protein sequence (SEQ ID NO:9): MGSSHHHHHHSSGENLYFQGFITRHLDQIIPRMTTQSPLNQSSMNAMGSLSVLSPDRVLPQL ISTITASVQNPALRLVTREEFAIMQTPAGELYDKSIIQSAQQDSIKKANMKRENKAYSFKEQ IIELELKEEIKKKKGIKEEVQLTSKQKEMLQAQLDREAQVRRRLQELDGELEAALGLLDIIL AKNPSGLTQYIPVLVDSFLPLLKSPLAAPRIKNP MYCHot in GCN1 = GCN1Hot (SEQ ID NO:10): KKANMKRENKAYSFKEQIIELELKE PHI-BLAST pattern Q-I-x-E-L-E-x(2)-E (SEQ ID NO: 11) GCN1 is only hit, with an E value of 0.003, search strategy is: Query: P01106 (MYC Uniprot code) Database: UniProtKB / Swiss-Prot(swissprot) Organism: Homo Sapiens (taxid:9606) Short queries: deselected, under “Algorithm parameters”; Expect (E-value) threshold = 0.05 Matrix: BLOSUM62 Gap costs: Existence: 11, Extension: 1 c-MYC (SEQ ID NO: 23). Amino acids 120-191 are the extended MYC Box II (bolded). Lysines K148 and K157 are italicized. Attorney Docket No.702581.02595 (NU2023-153-02) MPLNVSFTNRNYDLDYDSVQPYFYCDEEENFYQQQQQSELQPPAPSEDIWKKFELLPTPPLS PSRRSGLCSPSYVAVTPFSLRGDNDGGGGSFSTADQLEMVTELLGGDMVNQSFICDPDDETF IKNIIIQDCMWSGFSAAAKLVSEKLASYQAARKDSGSPNPARGHSVCSTSSLYLQDLSAAAS ECIDPSVVFPYPLNDSSSPKSCASQDSSAFSPSSDSLLSSTESSPQGSPEPLVLHEETPPTT APPSTRKDYPAAKRVKLDSVRVLRQISNNRKCTSPRSSDTEENVKRRTHNVLERQRRNELKR SFFALRDQIPELENNEKAPKVVILKKATAYILSVQAEEQKLISEEDLLRKRREQLKHKLEQL RNSCA REFERENCES H. Han et al., Small-Molecule MYC Inhibitors Suppress Tumor Growth and Enhance Immunotherapy. Cancer cell 36, 483-497 e415 (2019). M. I. Truica, M. C. Burns, H. Han, S. A. Abdulkadir, Turning Up the Heat on MYC: Progress in Small-Molecule Inhibitors. Cancer Res 81, 248-253 (2021). F. X. Schaub et al., Pan-cancer Alterations of the MYC Oncogene and Its Proximal Network across the Cancer Genome Atlas. Cell Syst 6, 282-300 e282 (2018). M. Kalkat et al., MYC Deregulation in Primary Human Cancers. Genes (Basel) 8, (2017). M. Costa-Mattioli, P. Walter, The integrated stress response: From mechanism to disease. Science 368, (2020). Y. L. Wong et al., eIF2B activator prevents neurological defects caused by a chronic integrated stress response. eLife 8, (2019). M. J. Huang, Y. C. Cheng, C. R. Liu, S. Lin, H. E. Liu, A small-molecule c-Myc inhibitor, 10058-F4, induces cell-cycle arrest, apoptosis, and myeloid differentiation of human acute myeloid leukemia. Experimental hematology 34, 1480-1489 (2006). D. M. Clausen et al., In vitro cytotoxicity and in vivo efficacy, pharmacokinetics, and metabolism of 10074- G5, a novel small-molecule inhibitor of c-Myc / Max dimerization. J Pharmacol Exp Ther 335, 715-727 (2010). B. J. Chen, Y. L. Wu, Y. Tanaka, W. Zhang, Small molecules targeting c-Myc oncogene: promising anti- cancer therapeutics. Int J Biol Sci 10, 1084-1096 (2014). J. R. Hart et al., Inhibitor of MYC identified in a Krohnke pyridine library. Proc Natl Acad Sci U S A 111, 12556-12561 (2014). A. Castell et al., A selective high affinity MYC-binding compound inhibits MYC:MAX interaction and MYC- dependent tumor cell proliferation. Sci Rep 8, 10064 Attorney Docket No.702581.02595 (NU2023-153-02) (2018). 12. K. C. Jeong et al., Intravesical instillation of c-MYC inhibitor KSI-3716 suppresses orthotopic bladder tumor growth. J Urol 191, 510-518 (2014). 13. L. Boike et al., Discovery of a Functional Covalent Ligand Targeting an Intrinsically 5 Disordered Cysteine within MYC. Cell Chem Biol 28, 4-13 e17 (2021). 14. M. J. Davies et al., Neuroserpin polymers activate NF-kappaB by a calcium signaling pathway that is independent of the unfolded protein response. J Biol Chem 284, 18202-18209 (2009). 15. M. Hurd et al., MYC acetylated lysine residues drive oncogenic cell transformation 10 and regulate select genetic programs for cell adhesion-independent growth and survival. Genes Dev.2023 Oct 1;37(19-20):865-882. doi: 10.1101 / gad.350736.123. Epub 2023 Oct 18. PMID: 37852796; PMCID: PMC10691474. 16. J. Abramson et al., Accurate structure prediction of biomolecular interactions with AlphaFold 3. Nature.2024 Jun;630(8016):493-500. doi: 10.1038 / s41586-024-07487-w. Epub 15 2024 May 8. PMID: 38718835; PMCID: PMC11168924. 17. A. G. Holmes et al., MYC inhibitor selectively alters the MYC and MAX cistromes and modulates the epigenomic landscape to regulate target gene expression. Sci Adv. 2022 Apr 29;8(17):eabh3635. doi: 10.1126 / sciadv.abh3635. Epub 2022 Apr 27. PMID: 35476451; PMCID: PMC9045724. 1280. M. Hurd M et al., MYC acetylated lysine residues drive oncogenic cell transformation and regulate select genetic programs for cell adhesion-independent growth and survival. Genes Dev.2023 Oct 1;37(19-20):865-882. doi: 10.1101 / gad.350736.123. Epub 2023 Oct 18. PMID: 37852796; PMCID: PMC10691474.

Claims

Attorney Docket No.702581.02595 (NU2023-153-02) CLAIMS We claim:

1. A method for treating a subject for a cancer responsive to MYC inhibition, the method comprising administering a MYC inhibitor to the subject.

2. The method of claim 1, wherein the MYC inhibitor Response Signature (MiRS) score for the cancer is greater than 0.

3. The method of claim 2 further comprising determining the MiRS score for the cancer.

4. The method of claim 3, wherein the MiRS score is determined by gene set variance analysis of a MYC inhibition responsive gene.

5. The method of claim 4, wherein the MYC inhibition responsive gene is selected from ADM2, AIG1, AKNA, ANK2, ARHGEF2, ATF3, BBC3, BMF, CCNE2, CHAC1, DDIT3, DDIT4, ERN1, FYN, GADD45A, GDF15, GINS2, GTPBP2, HMOX1, KDM7A, MCM4, NUPR1, PPP1R15A, RELN, RRM2, TCP11L2, THSD7A, TK1, TR1B3, UHRF1, UNG, YPEL2or any combination thereof.

6. The method of claim 5, wherein the MYC inhibition responsive gene is a MYC target or a ATF4 target.

7. The method of claim 1, wherein the cancer has acetylated MYC.

8. The method of claim 7 further comprising screening the cancer for acetylated MYC.

9. The method of claim 7, wherein the acetylated MYC has an acetylated lysine in an extended MYC Box II domain.

10. The method of claim 9, wherein K148 or K157 is acetylated.Attorney Docket No.702581.02595 (NU2023-153-02) 11. The method of any one of claims 1-10, wherein the MYC inhibitor binds to an intrinsically disordered region of MYC and one or both of a distal N-terminal region of MYC (ΔC) and an intrinsically disordered region of GCN1.

12. The method of claim 11, wherein the intrinsically disordered region of MYC comprises a MycHot motif.

13. The method of any one of claims 1-10, wherein the MYC inhibitor activates ATF4 or induces ATF4 expression.

14. The method of any one of claims 1-10, wherein the cancer is a breast cancer, a myeloma, a lung cancer, or a prostate cancer.

15. The method of any one of claims 1-10, wherein the MYC inhibitor is .

16. A method for treating a subject for a cancer, the method comprising determining the binding of an MYC inhibitor to an intrinsically disordered region of MYC and one or both of a distal N-terminal region of MYC (ΔC) and an intrinsically disordered region of GCN1 and administering the MYC inhibitor if the MYC inhibitor binds to the intrinsically disordered region of MYC and one or both of the distal N-terminal region of MYC (ΔC) and the intrinsically disordered region of GCN1.

17. The method of claim 16, wherein the intrinsically disordered region of MYC comprises a MycHot motif.Attorney Docket No.702581.02595 (NU2023-153-02) 18. The method of any one of claims 16-17, wherein MYC is acetylated.

19. The method of claim 18, wherein the acetylated MYC has an acetylated lysine in an extended MYC Box II domain.

20. The method of claim 19, wherein K148 or K157 is acetylated.

21. The method of any one of claims 16-17, wherein the MYC inhibitor activates ATF4 or induces ATF4 expression.

22. The method of any one of claims 16-17, wherein the MYC inhibitor Response Signature (MiRS) score for the cancer is greater than 0.

23. The method of any one of claims 16-17, wherein the cancer is a breast cancer, a myeloma, a lung cancer, or a prostate cancer.

24. The method of any one of claims 16-17, wherein the MYC inhibitor is .

25. A method for screening a compound for biological activity toward an intrinsically disordered protein (IDP), the method comprising determining a binding affinity of the compound to a proxy protein; and evaluating the biological activity of the compound toward the IDP based on the binding affinity of the compound to the proxy protein, wherein the proxy protein is less disordered than the IDP and comprises a binding domain with at least 80% sequence identity to an intrinsically disordered region (IDR) of the IDP;Attorney Docket No.702581.02595 (NU2023-153-02) wherein the compound binds to the binding domain of the proxy protein.

26. The method of claim 25, wherein the IDR comprises a basic helix-loop-helix (bHLH) domain.

27. The method of any one of claims 25-26, wherein the IDP is a MYC protein.

28. The method of claim 27, wherein the compound is an inhibitor of the MYC protein.

29. The method of claim 27, wherein the proxy protein is a GCN1 protein.

30. A method of identifying a cancer responsive to MYC inhibition, the method comprising determining a MYC inhibitor Response Signature (MiRS) score for the cancer.

31. The method of claim 30, wherein the MiRS score is determined by gene set variance analysis of a MYC inhibition responsive gene.

32. The method of claim 31, wherein the MYC inhibition responsive gene is selected from ADM2, AIG1, AKNA, ANK2, ARHGEF2, ATF3, BBC3, BMF, CCNE2, CHAC1, DDIT3, DDIT4, ERN1, FYN, GADD45A, GDF15, GINS2, GTPBP2, HMOX1, KDM7A, MCM4, NUPR1, PPP1R15A, RELN, RRM2, TCP11L2, THSD7A, TK1, TR1B3, UHRF1, UNG, YPEL2 or any combination thereof.

33. The method of claim 32, wherein the MYC inhibition responsive gene is a MYC target or a ATF4 target.

34. A method for treating a subject for a cancer responsive to ATF4 activation or induction, the method comprising administering a GCN1 ligand that activates or induces expression of ATF4.

35. The method of claim 34, wherein the GCN1 ligand binds an intrinsically disordered region of GCN1Attorney Docket No.702581.02595 (NU2023-153-02) 36. A method for sensitizing a subject having cancer to MYC inhibition treatment, the method comprising activating or inducing expression of ATF4 in the subject.

37. The method of claim 36, wherein activating or inducing expression of ATF4 in the subject comprises administering to the subject a GCN1 ligand that activates or induces expression of ATF4.

38. The method of claim 37, wherein the GCN1 ligand binds an intrinsically disordered region of GCN1.

39. The method of any one of claims 34-38, wherein the GCN1 ligand is a MYC inhibitor.

40. The method of claim 39, wherein the MYC inhibitor is .

41. The method of any one of claims 34-40, wherein the cancer is a breast cancer, a myeloma, a lung cancer, or a prostate cancer.

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