EP4734993A2 - Methods for sensitizing drug-resistant cancer cells - Google Patents

Abstract

The present invention is directed to methods of sensitizing tyrosine kinase inhibitor (TKI)-resistant cancer cells in a subject using a SWI/SNF complex modulator.

Description

METHODS FOR SENSITIZING DRUG-RESISTANT CANCER CELLS

[0001] This application claims priority to U.S. Provisional Application No. 63/524,563, filed on June 30, 2023, and U.S. Provisional Application No. 63/526,676, filed on July 13, 2023, the entire contents of each of which are incorporated herein by reference.

[0002] All patents, patent applications and publications cited herein are hereby incorporated by reference in their entirety. The disclosures of these publications in their entireties are hereby incorporated by reference into this application in order to more fully describe the state of the art as known to those skilled therein as of the date of the invention described and claimed herein.

[0003] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0004] This patent disclosure contains material that is subject to copyright protection. The copyright owner has no objection to the facsimile reproduction by anyone of the patent document or the patent disclosure as it appears in the U.S. Patent and Trademark Office patent file or records, but otherwise reserves any and all copyright rights.

GOVERNMENT INTERESTS

[0005] This invention was made with government support under R35 CA220497 awarded by the National Institutes of Health. The government has certain rights in the invention.

FIELD OF THE INVENTION

[0006] This invention is directed to compositions and methods of sensitizing tyrosine kinase inhibitor (TKI)-resistant cancer cells in a subject. For example, aspects of the invention are drawn to methods of administering to the subject a therapeutically effective amount of a SWI/SNF complex modulator, thereby sensitizing the cells to the ty rosine kinase inhibitor.

BACKGROUND OF THE INVENTION

[0007] Over the past two decades, targeted therapies such as tyrosine kinase inhibitors (TKIs) directed against mutant or hyperactive ty rosine kinases, have transformed clinical management across a range of cancer types, bringing precision medicine to the forefront of modem oncology. More than 70 different targeted therapies have been approved by the FDA (Food and Drug Administration) with utility across a broad range of cancers, from lung cancers, chronic myelogenous leukemia (CML), melanoma and others. Indeed, decreases in lung cancer mortality observed in recent years are, in part, attributed to the use of targeted therapies used to treat specific subsets of oncogene-driven lung adenocarcinomas such as epidermal growth factor receptor (EGFR) -mutant lung adenocarcinomas. Mutations in exons encoding the ty rosine kinase domain of EGFR account for -15% of lung adenocarcinomas in the US, -40- 50% in East Asians and 14-51% in Latin America. Tumors harboring most EGFR mutations respond to TKIs which have been approved for the first-line treatment of the disease and have significantly improved outcomes for patients.

SUMMARY OF THE INVENTION

[0008] Aspects of the invention are directed to methods of sensitizing tyrosine kinase inhibitor (TKI)-resistant cancer cells in a subject. In embodiments, the method comprises administering to the subject a therapeutically effective amount of a SWI/SNF complex modulator, thereby sensitizing the cells to the tyrosine kinase inhibitor.

[0009] In embodiments, the tyrosine kinase inhibitor comprises osimertinib. gefitinib, or trametinib.

[0010] In embodiments, the cancer comprises lung cancer. For example, the lung cancer comprises non-small cell lung cancer. For example, the lung cancer comprises EGFR-mutant lung cancer.

[0011] In embodiments, the SWI/SNF complex modulator comprises an inhibitor or a degrader.

[0012] In embodiments, the SWI/SNF complex modulator comprises a chromatin modifying agent.

[0013] In embodiments, the SWI/SNF complex modulator comprises a modulator of a cBAF subunit. For example, the modulator comprises ARID1A, ARID1B, DPF2, DPF3, BCL11 A, BCL1 IB, or any combination thereof.

[0014] In embodiments, the SWI/SNF complex modulator comprises an ATPase modulator. For example, the SWI/SNF complex modulator comprises a SWI/SNF ATPase modulator. In some embodiments, the SWI/SNF ATPase modulator can be a SMARCA4/SMARCA2 ATPase inhibitor, such as FHD-286 and Compound 14 described herein.

[0015] In embodiments, the SWI/SNF ATPase comprises SMARCA2, SMARCA4, or a combination of the two ATPases.

[0016] In embodiments, the SWI/SNF complex modulator comprises a nucleic acid molecule, a small molecule, a peptide, or a polypeptide. [0017] In embodiments, wherein the nucleic acid molecule comprises an RNA interfering agent or an antisense oligonucleotide.

[0018] In embodiments, the RNA interfering agent comprises small interfering RNA (siRNA), CRISPR RNA (crRNA), microRNA (miRNA), small hairpin RNA (shRNA), antisense RNA, guide RNA (gRNA), single guide RNA (sgRNA), piwi-interacting RNA (piRNA), modified forms thereof, or any combination thereof.

[0019] In embodiments, the small molecule comprises a structure according to:

Formula I, or a derivative or analog thereof. In embodiments, Ri can be Cl, or F.

[0020] In embodiments, the compound comprises:

or a derivative or analog thereof.

[0021] In embodiments, the polypeptide comprises an antibody or antigen-binding fragment thereof. For example, the antibody is murine, chimeric, humanized, mosaic, composite, or human.

[0022] In embodiments, the SWI/SNF complex modulator comprises a degrader directed to the SWI/SNF complex, a nucleic acid molecule targeting the SWI/SNF complex, a compound or prodrug thereof that binds to the SWI/SNF complex, or a pharmaceutically acceptable salt or ester of said compound or prodrug.

[0023] In embodiments, the SWI/SNF complex comprises canonical BAF (cBAF), polybromo-associated BAF (PBAF) or non-canonical BAF (ncBAF).

[0024] In embodiments, sensitization is indicated by reducing cancer cell proliferation, inducing cancer cell killing, inhibiting epithelial-to-mesenchymal transition, inhibiting epithelial cell differentiation, and/or modulating NRF2 signaling which exceeds that observed in cancer cells not treated with the SWI/SNF complex modulator.

[0025] Aspects of the invention are further drawn to methods of treating a subject afflicted with a tyrosine kinase inhibitor (TKI)-resistant cancer. In embodiments, the method comprises administering to the subject a SWI/SNF complex modulator described herein to sensitize cells of the TKI-resistant cancer to a tyrosine kinase inhibitor. In embodiments, the method further comprises administering to the subj ect a tyrosine kinase inhibitor.

[0026] In embodiments, sensitization is indicated by the amount and/or activity of at least one sample biomarker listed in Figure 1 or Figure 3.

[0027] Aspects of the invention are further drawn a method of identifying a subject afflicted with a cancer or at risk for developing a cancer that can benefit from increasing sensitivity of the cancer to a tyrosine kinase inhibitor. In embodiments, the method comprises determining in a cancerous or pre-cancerous subject sample the amount and/or activity of at least one biomarker listed in Figure 1 or Figure 3. In embodiments, the amount and/or activity of at least one biomarker listed in Figure 1 or Figure 3 is indicative of a subject afflicted with cancer or at risk for developing cancer that can benefit from increasing sensitivity⁷ of the cancer to a tyrosine kinase inhibitor. In embodiments, the method further comprises obtaining the cancerous or pre-cancerous sample from the subject. In embodiments, the method further comprises determining in a control sample the amount and/or activity of at least one control biomarker; and comparing the amount and/or activity of the at least one biomarker from the cancerous or pre-cancerous subject sample with the amount and/or activity of least one control biomarker from the control sample.

[0028] In embodiments, the presence of or a significant change in the amount and/or activity of the at least one biomarker from the cancerous or pre-cancerous subject sample relative to the amount and/or activity of the at least one control biomarker from the control sample is indicative of the subject having or at risk of developing a cancer that can benefit from increasing sensitivity of the cancer cells to a tyrosine kinase inhibitor.

[0029] In embodiments, the method further comprises recommending, prescribing, or administering an agent that modulates the at least one biomarker listed in Figure 1 or Figure 3. For example, the agent comprises a SWI/SNF complex modulator.

[0030] In embodiments, the method further comprises administering at least one additional cancer therapeutic agent or regimen. For example, the at least one additional cancer therapeutic agent or regimen comprises a targeted therapy, chemotherapy, radiation therapy, surgery, immunotherapy, and/or hormonal therapy.

[0031] In embodiments, the at least one additional cancer therapeutic agent or regimen comprises a tyrosine kinase inhibitor. For example, the tyrosine kinase inhibitor comprises osimertinib, gefitinib, or trametinib.

[0032] In embodiments, the amount and/or activity of at least one control biomarker is determined from a cancerous, pre-cancerous, or non-cancerous sample from the subject or a member of the same species to which the subject belongs. In embodiments, the cancer is a tyrosine kinase inhibitor (TKI)-resistant cancer or is at risk of developing resistance to a tyrosine kinase inhibitor. In embodiments, the cancer is lung cancer. For example, the lung cancer comprises non-small cell lung cancer. For example, the lung cancer comprises EGFR- mutant lung cancer.

[0033] Still further, aspects of the invention are drawn towards methods for predicting the clinical outcome of a subject afflicted with a cancer or at risk for developing cancer. In embodiments, the method comprises determining in a cancerous or pre-cancerous subject sample the amount and/or activity of at least one biomarker listed in Figure 1 or Figure 3. In embodiments, the method further comprises determining the amount and/or activity of at least one control biomarker having a good clinical outcome. In embodiments, the method further comprises comparing the amount and/or activity' of the at least one sample biomarker and the at least one control biomarker, wherein the presence of or a significant change in the amount and/or activity of the at least biomarker from the subject sample relative to the at least one control biomarker indicates that the subject afflicted with the cancer or at risk for developing the cancer has a poor clinical outcome.

[0034] Still further, aspects of the invention are drawn towards methods for monitoring the progression of a cancer in a subject. In embodiments, the method comprises detecting in a subject sample at a first point in time the amount and/or activity⁷ of at least one sample biomarker listed in Figure 1 or Figure 3. In embodiments, the method further comprises repeating the detecting step at one or more subsequent points in time. In embodiments, the method further comprises comparing the amount and/or activity of the at least one sample biomarker from the subsequent points in time to monitor the progression of the cancer in the subject.

[0035] Still further, aspects of the invention are drawn towards methods of assessing the efficacy of an agent for treating a cancer in a subject. In embodiments, the method comprises detecting in a subject sample at a first point in time the amount and/or activity of at least one biomarker listed in Figure 1 or Figure 3. In embodiments, the method further comprises repeating the detecting step at one or more subsequent points in time after administration of the agent. In embodiments, the method further comprises comparing the amount and/or activity of the at least one biomarker from the subsequent points in time, wherein the presence of or a significant change in the amount and/or activity of the at least one biomarker indicates that the agent treats the cancer in the subject.

[0036] In embodiments, between the first point in time and the subsequent point in time, the subject has undergone treatment, completed treatment, and/or is in remission for the cancer.

[0037] In embodiments, the first and/or at least one subsequent sample is selected from the group consisting of ex vivo and in vivo samples.

[0038] In embodiments, the first and/or at least one subsequent sample is obtained from an animal model of cancer.

[0039] In embodiments, the first and/or at least one subsequent sample is a portion of a single sample or pooled samples obtained from the subject.

[0040] In embodiments, the sample comprises cells, serum, peritumoral tissue, and/or intratumoral tissue obtained from the subject.

[0041] Still further, aspects of the invention are drawn towards a cell-based assay for screening for agents that sensitize a cancer cell to a tyrosine kinase inhibitor. In embodiments, the cell-based assay comprises contacting the cancer cell with a test agent. In embodiments, the cell-based assay further comprises determining the ability of the test agent to change the amount and/or activity of at least one biomarker listed in Figure 1 or Figure 3.

[0042] In embodiments, the step of contacting occurs in vivo, ex vivo, or in vitro.

[0043] In embodiments, the cell-based assay further comprises determining cancer cell proliferation, cancer cell killing, epithelial-to-mesenchymal transition, epithelial cell differentiation, and/or NRF2 signaling.

[0044] Other objects and advantages of this invention will become readily apparent from the ensuing description.

BRIEF DESCRIPTION OF THE FIGURES

[0045] The patent or application fde contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0046] FIG. 1 shows chromatin accessibility and gene regulatory underpinnings of osimertinib resistance in EGF/?-mutant lung cancer cell lines. Panel A provides a schematic representation of the generation of the osimertinib- and gefitinib- resistant cell lines. Panel B provides osimertinib dose-response curves for the parental cell lines PC9, H1975, HCC827, PC9*, HCC4006, and HCC827* and their osimertinib- or gefitinib-resistant counterparts (n=3 experimental replicates; mean ± SEM is shown). Panel C provides bar graphs (mean ± SEM) of EC50 values for parental and osimertinib-resistant isogenic cell line pairs. The fold-change in EC50 values between resistant and parental cells is indicated. Panel D provides a clustered heatmap performed on n=2-4 RNA-seq profiles (raw⁷ RPKM) from parental and resistant cell line pairs profiled. Significant gene examples are labeled. RPKM signals were z-scored separately within each cell line pair and combined horizontally to highlight differences between parental and resistant states. Panel E provides GSEA pathway analysis for pathway enrichment using DEGs from parental and resistant cell line pairs. Panel F provides a heatmap representation of ATAC-seq peaks in PC9*, HCC4006, HCC827* and PC9 parental cell lines and their resistant counterparts sorted by RPKM values over accessible genomic sites. Note: ATAC-seq studies in PC9-OR were performed on cells with washout of osimertinib; washout was not applied to other cell lines. Panel G provides a pie chart representation of proportion of DEGs (resistant cell line vs parental) near concordantly changed ATAC-seq peaks in PC9*, HCC4006, HCC827* and PC9 cell line pairs. Panel H provides ATAC-seq tracks over the MAPK1, CRKL loci in PC9 cell line pairs and JAG1 in HCC4006 cell line pair. Gene expression RPKM values are shown in bar graphs. Error bars represent the 95% confidence interval around the mean expression level for each cell line.

[0047] FIG. 2 shows mammalian SWI/SNF (BAF) complexes as critical regulators of resistance-associated gene loci. Panel A provides Ingenuity Pathway Analysis (IP A) performed on differentially-regulated genes in parental versus resistant cell line pairs. Top 20 most significant transcriptional upstream regulators are shown; Circle size indicates the number of genes regulated by each factor in each cell line. Panel B provides a heatmap for SMARC A4, SMARCC1, and H3K27ac occupancy (CUT&RUN) levels and ATAC-seq chromatin accessibility in HCC4006/HCC4006-OR cell lines across merged SMARCA4 sites. Panel C provides a heatmap displaying SMARCA4 occupancy levels and ATAC-seq chromatin accessibility in PC9*/PC9-OR*, HCC4006/HCC4006-GR and HCC827*/HCC827GR6 cell lines, across merged differential ATAC-seq sites. Panel D provides hockey-stick plots representing the normalized rank and signals of RNA-seq in PC9-OR*, HCC4006-OR and HCC827GR6 cell lines. Representative SMARCA4/ATAC gained-associated genes that are upregulated are in red and representative BAF/ATAC lost-associated genes that are downregulated are in blue. Panel E provides example SMARC A4 and ATAC-seq tracks at the ETV1 (in PC9*/OR*) and TWIST1 (in HCC4006/OR) loci. RNA-seq expression signal (RPKM) is shown for each; error bars represent the 95% confidence interval around the mean expression level.

[0048] FIG. 3 shows pharmacologic targeting of mSWI/SNF complex ATPase activity reverses the TKI resistance program in a subset of EG'F/?-mutant cancer cell lines. Panel A provides drug synergy plots in PC9* and PC9-OR* cells as assayed by Combenefit software. Bliss synergy scores were calculated for each drug combination, Osimertinib (osi) and Trametinib (Tram) in the absence or presence of Compounds (CmpI4) after 72 hours. One representative experiment out of N=3 independent experiments is shown. Panel B provides Caspase-3/7 activity assays performed in PC9* and PC9-OR* cells across 3 days of drug treatment. A low and high concentration of Osi and Tram were used in these assays to highlight enhanced sensitization effects. Graphs represent fluorescent signals normalized to cellular confluency at each timepoint. One representative experiment out of N=5 independent experiments is shown. Data presented as Mean ± SEM with significance calculated at the last timepoint using an unpaired t test ***P<0.0005, **P<0.005. Panel C provides a schematic of experimental design for ATAC-seq and RNA-seq performed in PC9* and PC9-OR* cells following 24 hours of each treatment condition. Panel D provides a RNA-seq clustered heatmap of Cmpl4 synergy genes in PC9-OR* cells. Biological replicates are represented for DMSO, OT and OT+Cmpl4 treatment conditions. Expression signals were z-scored across the samples. Genes were k-means clustered (k=4) and clusters were reordered. The greatest coordinated ATAC-seq changes (in logFCs) between OT+CMP14 and OT in PC9-OR* for each gene is shown as a yellow/purple heatmap. Select genes are labeled. Panel E provides metascape analysis of genes from each cluster of Cmpl4 synergy genes separated by correlation to ATAC-seq signal. Primary analysis represents DEGs which have a closest associated change in ATAC peak while secondary analysis represents DEGs without an associated ATAC peak change. Cluster specific or common terms are highlighted. Panel F provides bar graphs of key deregulated Cmpl4 synergy genes from each RNA-seq cluster from (D) showing average RPKM values for each condition with one SEM for the error bars. Panel G provides a Venn diagram representation of SMARCA4 occupied sites in PC9-OR* cells which overlap with lost ATAC sites at Cmpl4 synergy⁷ DARs (upper). A subset of these sites overlap with upregulated DEGs which characterize the resistant state (PC9-OR* vs PC9*) and are subsequently downregulated by Cmpl4 synergy treatment (lower). Panel H provides IGV tracks of SMARCA4 occupancy (Cut&Run) and accessibility⁷ (ATAC-seq) at the CES1 locus. Panel I provides a RNA-seq heatmap of RPKM values of Cmpl4 Synergy⁷ DEGs at resistantstate associated genes. Values are shown for PC9* and PC9-OR* cells under DMSO treatment as compared to PC9-OR* cells under OT and OT+Cmpl4 treatments. Panel J provides a RNA- seq heatmap of RPKM values of Cmpl4 Synergy DEGs in PC9* cells under DMSO and OT treatments and in PC9-OR* cells under OT and OT+Cmpl4 treatments.

[0049] FIG. 4 shows resensitization of osimertinib-resistant NSCLC cell lines reveals the attenuation of reactive oxygen species by SMARCA4. Panel A provides a schematic overview of cell lines which are responsive (resensitized: purple) and non-responsive (green) to osimertinib treatment upon inhibition or knock down of SMARCA4. Panel B provides GSEA pathway enrichment analysis of differentially expressed genes (DEGs) between each parental and osimertinib-resistant cell line pair as well as between responsive and non- responsive DEGs. Panel C provides quadrant plots of differentially expressed genes specific to the resistant PC9-OR* state as compared to HCC4006-OR and specific to the resistant PC9- OR state as compared to H1975-OR and HCC827-OR. Specific upregulated genes are in red and specific downregulated genes are in blue and gene examples are labelled. Panel D provides bar graphs of key gene examples that are specifically upregulated and downregulated common to both PC9-OR* and PC9-OR showing RPKM values across cell lines. Panels E-F provide motif analysis of lost ATAC sites attributed to Cmpl4+OT treatment in PC9-OR* cells (Panel E) and atributed to SMARCA4 knock down in PC9-OR and YU-005C cells (Panel F). Panel G provides immunofluorescence images (IF) of cells stained using CellROX™ to quantify ROS in PC9 and PC9-OR cells in the presence and absence of 750 nM osimertinib. Hoechst staining was used to detect nuclear DNA (left panel). CellROX™ IF quantification of three independent replicates is shown (right panel). A.U., arbitrary units. Panel H provides a schematic model of ROS levels, NRF2 pathway activity and SMARCA4 regulation in osimertinib sensitivity and resistance based on the RNA-seq data of NRF2 targets. Upon osimertinib treatment NRF2 targets are downregulated, and ROS levels increase in PC9 cells (1 and 2). In treated PC9-OR cells NRF2 targets are activated, and ROS levels are high (3 and 4); upon SMARCA4 knockdown, NRF2 targets are downregulated and ROS levels further increase causing toxicity in the cells. Panels I-J provide flow cytometry results using CellROX^1M to measure ROS in PC9-OR cells in the presence and absence of 750 nM osimertinib and with or without SMARCA4 knockdown (Panel I) and in YU-005C tumors in the presence and absence of osimertinib and with or without SMARCA4 knockout (Panel J). (-) Controls are from PC9-OR and YU-005C unstained cells respectively and (+) CellROX™ Deep Red control in (Panel J) is from stained YU-005C cells. CellROX™ MFI was assessed in RFP+/shRNA-containing cells (Panel I) and GFP+/sgRNA-containing cells (Panel J). Representative MFI profile of CellROX™+ cells (Panel I: left panel, Panel J: left panel). Quantification of three independent replicates (Panel I: right panel) and four tumors (Panel J: right panel). Panel K provides a western blot of PC9-OR cells transduced with three NRF2 shRNAs as indicated (upper panel). Osimertinib dose-response curves for PC9-OR cells after NRF2 knock-down (botom left panel). Bar graph of EC50 values (botom right panel). Panel L provides IHC staining for SMARCA4 and NRF2 in three representative cores of a TMA containing /A/FA-mutant TKI-treated tumors (upper panels). Correlation plot of NRF2 and SMARCA4 H-Scores for the tumors (lower panel). Significance was calculated using the Pearson r correlation test. Scr.: Scramble shRNA, sh #1 : SMARCA4 shRNA #1; sh #2: SMARCA4 shRNA #2. Significance was calculated using a paired t test and the Mean ± SEM is shown in Panels D, E and H. Significance was calculated using a Mann- Whitney test and the Median ± IQR is shown in F. **P<0.01, *P<0.05.

[0050] FIG. 5 shows pharmacological inhibition of mSWI/SNF ATPase activity sensitizes a patient-derived tumor to osimertinib. Panel A provides osimertinib, compound- 14, and combination (titrated osimertinib + IpM of Compound-14) dose-response curves for YU-005C cells. Panel B provides osimertinib. FHD-286, and combination (titrated osimertinib + lOOnM of FHD-286) dose-response curves for YU-005C cells. (A,B) N=4. The mean ± standard deviation is shown. Panel C provides a bar graph of IC50 values for YU-005C cells treated with osimertinib (alone), compound- 14 combination, or FHD-286 combination. The mean ± SEM is shown. Significance was calculated using the one-way repeated measures ANOVA test and Tukey’s multiple comparisons test. ***P<0.001, **P<0.01, *P<0.05. Panel D provides an outline of experimental methods. YU-005C cells were injected subcutaneously in mice that were treated with vehicle, osimertinib. FHD-286 or the combination of both. Treatment began 16-days after injection once tumor volume reached ~50mm³ and was continued for 18-days. Tumor volume w as measured twice a w eek and total body weight was measured once a week. Panel E provides normalized tumor volume of YU-005C cells treated with vehicle, osimertinib, FHD-286 or the combination of both. Individual tumor volumes reflect the change in volume from treatment baseline. Tumor volume mean and ± standard error of the mean is shown. Significance was calculated using the two-way repeated measures ANOVA test and Dunnetf s multiple comparisons test, with a single pooled variance. ***P<0.001, **P<0.01, *P<0.05. Panel F provides a schematic representation of the mechanistic model by which SMARCA4 promotes osimertinib resistance. Sensitive tumors rely on EGFR signaling pathway. Osimertinib blocks EGFR and generates ROS killing the cells. Resistant tumors rely on SMARCA4 to keep proliferating and neutralizing the accumulated ROS. Blocking SMARCA4 activity⁷ generates too much stress killing the cells. Created with Biorender.com.

[0051] FIG. 6 shows genetic and genomic profiles of parental and tyrosine kinase inhibitor- resistant cell line pairs. Panel A provides Sanger sequencing traces of the mutation identified in H1975-OR cells causing a premature stop codon in the CIC gene. Panel B provides immunofluorescence images of HCC827 and HCC827-OR cells stained with the epithelial marker E-cadherin (CDH1) and the mesenchymal marker Vimentin (VIM). Hoechst staining was used for nuclear DNA. Panel C provides a western blot showing upregulation of Axl and downregulation of E-cadherin in HCC4006-OR as compared to parental upon 7 days treatment of 1 OOnM osi. Panel D provides a bar graph of RAFI copy -number in normal human DNA (control), PC9 and PC9-OR cells measured using a TaqMan quantitative PCR assay. Panel E provides a western blot showing the levels of for RAFI in PC9 and PC9-OR cells confirming its overexpression and efficient reduction using siRNAs. Scramble (Scr.); RAFI (#1 and #2). Panel F provides proliferation curves of PC9 and PC-OR cells 72 hours after siRNA transfection and osimertinib treatment at different concentrations. Data from three independent replicates are shown. Panel G provides representative colony formation assays in PC9 and PC9-OR cells after transfection of RAFI siRNAs and 750 nM osimertinib treatment. Panel H provides dose-response curves of osimertinib, the MEK inhibitor trametinib and the combination of both inhibitors for PC9 and PC9-OR cells (left panels). Bar graph of EC50 values (right panels), n=3. Significance calculated with a paired t test and the Mean ± SEM is shown. Panel I provides western blots showing components of the EGFR/RAS/MAPK signaling pathway in PC9 and PC9-OR cells upon treatment with osimertinib and/or trametinib as indicated. Panel J shows the presence of BRAF G469A in PC9-OR* was confirmed by Sanger sequencing but was not observed in the parental PC9* cell line or after 4 days osi treatment (Left). CTG assay demonstrating that while 100 nM Osi + 30 nM Tram nearly eliminates the entire PC9* population, PC9-OR* cells remain significantly more resistant. Data presented as Mean ± SEM with significance calculated using an unpaired t test ***P<0.0005. Panel K provides volcano plots representing differentially expressed genes in PC9-OR, H1975-OR, HCC827-OR, PC9-OR*, HCC4006-OR, and HCC827GR6 cells. Selected top up- and down- regulated genes are labeled. Panel L (Top) provides a bar graph of DEGs specifically upregulated or downregulated in each cell line pair as well as across increasing numbers of cell line pairs; (Bottom) provides a grey bar chart showing the total number of DEGs per cell line pair. Panel M provides ATAC-seq tracks and gene expression (mean RPKM-/+ 95% confidence interval) at the GATA3 locus. Panel N provides metascape analysis reflecting up- and down- regulated pathway terms corresponding to DEGs with nearby changes in accessibility in PC9-OR*, HCC4006-OR, HCC827GR6 and PC9-OR cell lines. Selected specific and common terms are highlighted.

[0052] FIG. 7 shows chromatin occupancy of mSWI/SNF complexes at resistance- associated gene loci. Panel A provides mRNA expression (RPKM) of SMARCA4 in parental and osimertinib-resistant cell lines. Mean ± SEM is shown. Panel B provides a western blot of SMARCA4 and SMARCC1 protein levels in parental and resistant cells. GAPDH is shown as a loading control. Panel C provides Venn diagrams showing overlap between SMARCA4 and SMARCC1 peaks (CUT&RUN). Panel D provides a heatmap for SMARCA4, SMARCC1, and H3K27ac occupancy levels (CUT&RUN) and ATAC-seq chromatin accessibility' in PC9*/PC9-OR* and HCC827*/HCC827GR6 cell lines across merged SMARCA4 sites. Panel E provides Principal component analysis (PC A) of ATAC-seq experiments in PC9*/OR, HCC4006/OR, HCC827*/GR6 cell lines. Panel F provides Venn diagrams representing overlap betw een changing (lost and gained) SMARCA4 and ATAC sites in PC9-OR* vs PC9*, HCC827GR6 vs HCC827* and HCC4006-OR vs HCC4006 cells. Panel G provides a distance- to-TSS stacked bar graph for SMARCA4 gained (G) and lost (L) sites in PC9*/PC9-OR*, HCC4006/HCC4006-OR and HCC827*/HCC827GR6 cell lines. Panel H provides motifs enriched (red) and lost (blue) under SMARCA4-occupied, accessible sites in the resistance versus parental cell lines. Panel I provides Principal component analysis (PCA) of RNA-seq experiments performed across the cell lines. Panel J provides metascape terms for differentially-expressed SMARCA4/accessible target genes for the resistance versus parental state in PC9-OR*, HCC4006-OR, and HCC827GR6 cell line pairs.

[0053] FIG. 8 shows SMARCA4/2 ATPase inhibition in Osimertinib- and gefitinib- resistant cell lines reverses chromatin accessibility and gene expression signatures. Panel A provides cell viability assays for Cmpl4 treatment in PC9*, HCC4006, HCC827* cell lines and their resistant counterparts, n=3 replicates. Panel B provides a Western blot of pEGFR and pERKl/2 levels across time course treatment of 100 nM Osimertinib (osi) or osimertinib and 1000 nM Cmpl4 combo (OC) in PC9* and PC9-OR* cells. Insufficient protein collected for day 7 OC treatment in PC9* cells owing to overt cell toxicity. Panel C provides a western blot of pEGFR and pERKl/2 levels across time course treatment of lOOnM osimertinib and 30nM Trametinib (OT) or OT and lOOOnM Cmpl4 combo (OTC) in PC9* and PC9-OR* cells. Insufficient protein collected for day 4 OT and OTC treatment in PC9* cells owing to overt cell toxicity. Panel D provides a western blot for pEGFR and pERKl/2 levels across time course treatment of lOOnM osimertinib (osi) or osimertinib and lOOOnM Cmpl4 combo (OC) in HCC827* and HCC827GR6 cells. Panel E provides drug synergy’ plots in HCC4006/HCC4006-OR and HCC827*/HCC827GR6 cells as measured using Combenefit software. Bliss synergy scores were calculated for each drug combination, Osimertinib (Osi) and Trametinib (Tram) or Osi alone in the absence or presence of Compounds (Compl4) after 72 hours. Synergy is observed in HCC827GR6 cells when treated with Osi and Compl4. One representative experiment out of N=3 independent experiments is shown. Panel F provides caspase assays performed in HCC827* and HCC827GR6 cells following across 100 hours of drug treatment. A combination of lOOnM Osi with or without 30nM Tram was used were used in these assays. Cmpl4 was used at lOOOnM for the assays. Graphs represent fluorescent signals normalized to cellular confluency at each timepoint. Panel G provides caspase assay images for PC9*/PC9-OR* cells at low and high OT concentrations (FIG. 5, panel B). While 100 nM Osi + 30 nM Tram induce apoptosis in PC9-OR* cells, many healthy surviving cells can still be seen in the wells. This drug tolerant population is effectively eliminated upon addition of 1000 nM Comp 14 to OT. Panel H provides an immunoblot for SMARCA4, SMARCC1, GAPDH in PC9* /PC9-OR* upon 24hr OT and OT+Cmpl4 treatments. Panel I provides volcano plots reflecting differentially expressed genes across conditions indicated with significant genes up (red) and down (blue) indicated. Panel J provides Venn diagrams reflecting upregulated (left) and downregulated (right) genes overlapping between OT + Cmpl4 vs OT compared to OT+Compl4 vs DMSO. Panel K provides a distance-to-TSS plot for AT AC gains (G) and losses (L) at resistance-associated accessible sites upon Cmpl4 and Cmpl4+0T treatment (vs DMSO). Panel L provides a Venn diagram reflecting the overlap between downregulated genes in Cmpl4 vs DMSO, 0T+Cmpl4 vs DMSO, and 0T+Cmpl4 vs OT to identify synergy-specific genes. Panels M-N provide metascape analyses performed on up- and down-regulated genes from Cmpl4 synergy’ and resistance reversal gene sets and Cmpl4 synergy and sensitizing gene sets.

[0054] FIG. 9 shows SMARCA4 attenuates reactive oxygen species in osimertinib- resistant cells via NRF2 signaling. Panel A provides metascape analyses performed on up- and down-regulated genes from the differential gene analysis of sensitizing vs non-sensitizing cell lines (FIG. 5, panel C). Panel B provides a Venn diagram overlap of specific upregulated and downregulated genes amongst the three comparisons from FIG. 5, panel C. Panel C provides a metascape analysis from the overlap of common differentially expressed genes. Relevant pathways are highlighted. Panel D provides bar graphs of key gene examples that are specifically upregulated and downregulated common to both PC9-OR* and PC9-OR showing RPKM values across cell lines. Panels E-F provide motif analysis of gained ATAC sites attributed to Cmpl4+OT treatment in PC9-OR* cells (Panel E) and attributed to SMARCA4 knock down in PC9-OR and YU-005C cells (Panel F). Panel G provides flow cytometry results using CellROX™ to measure ROS in PC9 & PC9-OR cells in the presence and absence of 750 nM osimertinib. Controls are from unstained cells Representative MFI profile of in RFP+/shRNA-containing CellROX™+ cells (left panel). Quantification of three independent replicates (right panel). Paired t-test. Panel H provides a flow cytometry’ plot showing the controls and gating thresholds for GFP+ (Cas9/sgRNA+) and CellROX™ DeepRed+ cells used to analyze YU-005C tumors. The percentages show the fraction of cells from each sample in the quadrant. Panel I provides flow cytometry MFI profiles for individual YU-005C tumors upon SMARCA4 knock-out as indicated. (-) Controls are YU-005C unstained cells and (+) CellROX™ Deep Red control is from stained YU-005C cells. Panel J provides osimertinib dose-response curves for PC9, PC9-OR and YU-005C with or without 400 nM NAC (left panel). Bar graph of EC50 values. Mean ± SEM (right panel). ***P<0.001, **P<0.01, *P<0.05. [0055] FIG. 10 shows pharmacological inhibition of mSWI/SNF ATPase activity in a patient-derived model of EGFR-driven lung cancer. Panel A provides single agent dose response curves for osimertinib. compound-14, and FHD-286 for YU-005C cells. N=4. The mean ± standard deviation is shown. Panel B provides a bar graph of individual 1C50 values for YU-005C cells treated with osimertinib, compound-14, or FHD-286. The mean ± SEM is shown. Significance was calculated using the one-way repeated measures ANOVA test and Tukey’s multiple comparisons test. ***P<0.001, **P<0.01, *P<0.05. Panel C provides a waterfall plot showing the % tumor volume change from treatment baseline of YU-005C cells injected subcutaneously in mice that were treated with vehicle, osimertinib, FHD-286 or the combination of both. Females are indicated by a lighter shade and males are indicated by darker shaded boxes. Panel D provides an oncoprint of the lung adenocarcinomas from the GENIE Cohort vl l.O-public with EGFR and/or SMARCA4 alterations. The data and the mutual exclusivity analysis was obtained from cBioPortal.

[0056] FIG. 11 provides data showing mSWI/SNF complexes control a molecular signature of Osimertinib resistance in EGFR-mutant NSCLC.

[0057] FIG. 12 provides data showing reversal of resistance following combination SMARCA4/2 inhibition + Osimertinib.

[0058] FIG. 13 provides data showing Osimertinib + SMARCA4/2 dual ATPase inhibition (FHD-286) attenuates tumor growth.

DETAILED DESCRIPTION OF THE INVENTION

[0059] Detailed descriptions of one or more embodiments are provided herein. It is to be understood, however, that the invention can be embodied in various forms. Therefore, specific details disclosed herein are not to be interpreted as limiting, but rather as a basis for the claims and as a representative basis for teaching one skilled in the art to employ the invention in any appropriate manner.

[0060] The singular forms “a”, “an” and “the” include plural reference unless the context clearly dictates otherwise. The use of the word “a” or “an” when used in conjunction with the term “comprising” in the claims and/or the specification can mean “one,” but it is also consistent with the meaning of “one or more,” “at least one,” and “one or more than one.” [0061] Wherever any of the phrases “for example,” “such as,” “including” and the like are used herein, the phrase “and without limitation” is understood to follow unless explicitly stated otherwise. Similarly, “an example,” “exemplary” and the like are understood to be nonlimiting. [0062] The term “substantially” allows for deviations from the descriptor that do not negatively impact the intended purpose. Descriptive terms are understood to be modified by the term “substantially” even if the word “substantially” is not explicitly recited.

[0063] The terms “comprising” and “including” and “having” and “involving” (and similarly “comprises”, “includes,” “has,” and “involves”) and the like are used interchangeably and have the same meaning. Specifically, each of the terms is defined consistent with the common United States patent law definition of “comprising⁷’ and is therefore interpreted to be an open term meaning “at least the following,” and is also interpreted not to exclude additional features, limitations, aspects, etc. Thus, for example, “a process involving steps a, b, and c” can refer to the process including at least steps a, b and c. Wherever the terms “a” or “an” are used, “one or more” is understood, unless such interpretation is nonsensical in context.

[0064] The term “about” can refer to approximately, roughly, around, or in the region of. When the term “about” is used in conjunction with a numerical range, it modifies that range by extending the boundaries above and below the numerical values set forth. In general, the term “about” is used herein to modify a numerical value above and below the stated value by a variance of 20 percent up or down (higher or lower).

[0065] The term “zh vivo” can refer to an event that takes place in a subject's body.

[0066] The term in vitro can refer to an event that takes places outside of a subject's body. [0067] The term “ex vivo” can refer to outside a living subject. Examples of ex vivo cell populations include in vitro cell cultures and biological samples such as fluid or tissue samples from humans or animals. Such samples can be obtained by methods well known in the art. Exemplary biological fluid samples include blood, cerebrospinal fluid, urine, saliva. Exemplary tissue samples include tumors and biopsies thereof. In this context, the compounds can be in numerous applications, both therapeutic and experimental.

[0068] Aspects of the invention are drawn towards methods of sensitizing tyrosine kinase inhibitor (TKI)-resistant cancer cells in a subject. For example, embodiments comprise sensitizing tyrosine-kinase inhibitor (TKI)-resistant cancer cells in a subject to a tyrosinekinase inhibitor. For example, embodiments as described herein comprise administering to a subject a therapeutically effective amount of a SWI/SNF complex modulator, thereby sensitizing the cells to the tyrosine kinase inhibitor.

[0069] As used herein, the terms “sensitize” or “sensitizing” can refer to altering cancer cells or tumor cells in a way that allows for more effective treatment of the associated cancer with a cancer therapy (e.g, anti-immune checkpoint, immunotherapy, chemotherapeutic, and/or radiation therapy). In embodiments, normal cells are not affected to an extent that causes the normal cells to be unduly injured by the therapies. An increased sensitivity or a reduced sensitivity⁷ to a therapeutic treatment can be measured according to a known method in the art for the treatment and methods described herein, including, but not limited to, cell proliferative assays (Tanigawa N. Kern D H, Kikasa Y. Morton D L. Cancer Res 1982; 42: 2159-2164) and cell death assays (Weisenthal L M, Shoemaker R H, Marsden J A, Dill P L, Baker J A, Moran E M, Cancer Res 1984; 94: 161-173; Weisenthal L M, Lippman M E. Cancer Treat Rep 1985; 69: 615-632; Weisenthal L M. In: Kaspers G J L, Pieters R. Twentyman P R. Weisenthal L M, Veerman A J P, eds. Drug Resistance in Leukemia and Lymphoma. Langhorne, P A: Harwood Academic Publishers, 1993: 415-432; Weisenthal L M, Contrib Gynecol Obstet 1994; 19: 82- 90). The sensitivity or resistance can also be measured in an animal by measuring the tumor size reduction over a period of time, for example, 6 month for human and 4-6 weeks for mouse. A composition or a method can sensitize response to a therapeutic treatment if the increase in treatment sensitivity or the reduction in resistance is about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, or more, such as about 2-fold. 3-fold, 4-fold, 5-fold, 10-fold. 15-fold, 20-fold or more, or any range in between, inclusive, compared to treatment sensitivity or resistance in the absence of such composition or method. The determination of sensitivity or resistance to a therapeutic treatment is routine in the art and within the skill of an ordinarily skilled clinician. Any method described herein for enhancing the efficacy of a cancer therapy can be equally applied to methods for sensitizing hyperproliferative or otherwise cancerous cells (e.g., resistant cells) to the cancer therapy. In embodiments, sensitization of tyrosine kinase inhibitor (TKI)-resistant cancer cells in a subject can be indicated by reducing cancer cell proliferation, inducing cancer cell killing, inhibiting epithelial-to-mesenchymal transition, inhibiting epithelial cell differentiation, and/or modulating NRF2 signaling which exceeds that observed in cancer cells not treated with the SWI/SNF complex modulator. In embodiments, TKI resistance can be indicated by molecular biomarkers which correlate with reversal of the resistance state, such as those listed in FIG. 1. For example, cancer cell proliferation is reduced with administration of Osimertinib. For example, cancer cell differentiation/cell state changes is increased and thus permissive of Osimertinib efficacy.

[0070] Tyrosine kinases are important mediators of this signal transduction process, leading to cell proliferation, differentiation, migration, metabolism and programmed cell death. Tyrosine kinases are a family of enzymes, which catalyzes phosphorylation of select tyrosine residues in target proteins, using ATP. Tyrosine kinases are implicated in several steps of neoplastic development and progression. As the role of tyrosine kinases in cancer molecular pathogenesis is immense, tyrosine kinases are potential anti-cancer targets for drugs such as tyrosine kinase inhibitors. The terms “tyrosine kinase inhibitor” or “TKI” can refer to an anti-cancer agent, such as a small molecule or protein, that inhibits the activity of one or more tyrosine kinase. In one embodiment, the tyrosine kinase inhibitor inhibits the tyrosine kinase by binding directly to it and inhibiting its kinase activity. For example, the tyrosine kinase inhibitor can comprise osimertinib, gefitinib, or trametinib. In embodiments, the tyrosine kinase inhibitor can comprise osimertinib or gefitinib. In embodiments, reduction in cell proliferation or increased cell differentiation and/or cell state change following treatment with osimertinib can be permissive of osimertinib efficacy.

[0071] Aspects of the invention are also drawn to methods of treating a subject afflicted with a tyrosine kinase inhibitor-resistant cancer or preventing the development of or progression of a tyrosine kinase inhibitor-resistant cancer. The terms “treat,” “treatment,” and “treating” can refer to the management and care of a subject for the purpose of combating a condition, disease or disorder, such as a cancer, in any manner in which one or more of the symptoms of a disease or disorder are ameliorated or otherwise beneficially altered. The term can include the full spectrum of treatments for a given condition from which the patient is suffering, such as administration of the active compound for the purpose of: alleviating or relieving symptoms or complications; delaying the progression of the condition, disease or disorder; curing or eliminating the condition, disease or disorder; and/or preventing the condition, disease or disorder. "Preventing" or "prevention" can refer to the management and care of a patient for the purpose of hindering the development of the condition, disease or disorder, and includes the administration of the active compounds to prevent or reduce the risk of the onset of symptoms or complications.

[0072] The terms "cancer” or “tumor” or “hyperproliferative disorder” can refer to the presence of cells possessing characteristics typical of cancer-causing cells, such as uncontrolled proliferation, immortality, metastatic potential, rapid growth and proliferation rate, and certain characteristic morphological features. Cancer is associated with uncontrolled cell growth, invasion of such cells to adjacent tissues, and the spread of such cells to other organs of the body by vascular and lymphatic means. Cancer invasion occurs when cancer cells intrude on and cross the normal boundaries of adjacent tissue, which can be measured by assaying cancer cell migration, enzymatic destruction of basement membranes by cancer cells, and the like. In some embodiments, a certain stage of cancer is relevant and such stages can include the time period before and/or after angiogenesis, cellular invasion, and/or metastasis. Cancer cells are often in the form of a solid tumor, but such cells can exist alone within an animal, or can be a non-tumorigenic cancer cell, such as a leukemia cell.

[0073] In embodiments, the cancer can be a drug resistant cancer, such as a tyrosine kinase inhibitor resistant cancer. %The term "drug resistance" can refer to the circumstance when a disease, such as cancer, does not respond to a drug or treatment drugs. Drug resistance can be intrinsic, which means that the disease, such as cancer, has never responded to the drug or drugs, or it can be acquired, which means that the disease, such as cancer, stops responding to a drug or drugs to which (they) had previously responded to the disease. Aspects of the invention can comprise methods of sensitizing drug-resistant cancer cells or tumors in a subject by administering to the subject a therapeutically effective amount of a SWI/SNF complex modulator, thereby sensitizing the cells to the drug.

[0074] In embodiments, the cancer can be resistant to a tyrosine kinase inhibitor. Tyrosine kinase inhibitors treat several kinds of cancers, including HER2-positive breast cancer, chronic lymphocytic leukemia, gastrointestinal stromal tumors, kidney cancer, Waldenstrom macroglobulinemia, non-small cell lung cancer, and melanoma. Accordingly, resistance to tyrosine kinase inhibitors can be intrinsic, or it can be acquired. As such, aspects of the invention can comprise methods of sensitizing tyrosine kinase inhibitor (TKl)-resistant cancer cells or tumors in a subject by administering to the subject a therapeutically effective amount of a SWI/SNF complex modulator, thereby sensitizing the cells to the ty rosine kinase inhibitor. [0075] In embodiments, aspects of the invention are drawn to methods to treat, prevent, or ameliorate the symptoms of a subject afflicted with a lung tumor, including but not limited to abnormally proliferative or aberrantly proliferative lung cells and/or malignant lung tumor cells. The term “lung tumor” can refer to any lung tumors, including but not limited to primary lung tumors and/or metastatic lung tumors. For example, metastatic lung tumors can be those that have formed in a way that tumors at other positions are metastasized to the lung through various metastasis modes. The lung tumors can be benign (non-cancerous), preinvasive lesion (precancerous lesion), or malignant (cancerous) lung tumors, such as lung cancers. In embodiments, the lung cancer comprises non-small cell lung cancer. In embodiments, the lung cancer comprises EGFR-mutant lung cancer.

[0076] In some embodiments, the lung cancer comprises EGFR-mutant lung cancer. "EGFR" or "Epidermal growth factor receptor" or "EGFR" can refer to a tyrosine kinase cell surface receptor and is encoded by one of four alternative transcripts appearing as GenBank accession NM_005228.3. NM_201282.1, NM_201283.1 and NM_201284.1. Variants of EGFR include a deletion in exon 19, an insertion in exon 20, and amino acid substitutions T790M and L858R. [0077] The term “resistance” can refer to an acquired or natural resistance of a cancer sample or a mammal to a cancer therapy (z.e., being nonresponsive to or having reduced or limited response to the therapeutic treatment), such as having a reduced response to a therapeutic treatment by about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%. 75%. 80%. 85%. 90%. 95%. 100%, or more, such as about 2-fold, 3-fold, 4-fold. 5-fold. 10-fold, 15-fold, 20-fold or more, or any range in between, inclusive. The reduction in response can be measured by comparing with the same cancer sample or mammal before the resistance is acquired, or by comparing with a different cancer sample or a mammal that is known to have no resistance to the therapeutic treatment. In embodiments, an acquired resistance to chemotherapy is called “multi drug resistance.” The determination of resistance to a therapeutic treatment is routine in the art and within the skill of an ordinarily skilled clinician, for example, can be measured by cell proliferative assays and cell death assays as described herein as “sensitizing.” In embodiments, the term “reverses resistance” can refer to the use of a second agent (e.g., a SWI/SNF complex modulator) in combination with a primary cancer therapy (e.g., chemotherapeutic, immunotherapy , or radiation therapy; e.g. a tyrosine kinase inhibitor) is able to produce a significant decrease in tumor volume at a level of statistical significance (e.g.. p<0.05) when compared to tumor volume of untreated tumor in the circumstance where the primary cancer therapy (e.g., chemotherapeutic, immunotherapy or radiation therapy, e.g, a tyrosine kinase inhibitor) alone is unable to produce a statistically significant decrease in tumor volume compared to tumor volume of untreated tumor. This applies to tumor volume measurements made at a time when the untreated tumor is growing log rhythmically.

[0078] The terms “response” or “responsiveness” can refer to a cancer response, e.g. in the sense of reduction of tumor size or inhibiting tumor grow th. The terms can also refer to an improved prognosis, for example, as reflected by an increased time to recurrence, which is the period to first recurrence censoring for second primary cancer as a first event or death without evidence of recurrence, or an increased overall survival, which is the period from treatment to death from any cause. To respond or to have a response means there is a beneficial endpoint attained when exposed to a stimulus. Alternatively, a negative or detrimental symptom is minimized, mitigated or attenuated on exposure to a stimulus. It will be appreciated that evaluating the likelihood that a tumor or subject will exhibit a favorable response is equivalent to evaluating the likelihood that the tumor or subject will not exhibit favorable response (z.e., will exhibit a lack of response or be non-responsive).

[0079] The term “survival” includes the following: survival until mortality, also known as overall survival (wherein said mortality can be irrespective of cause or tumor related); “recurrence-free survival” (wherein the term recurrence shall include both localized and distant recurrence); metastasis free survival; disease free survival (wherein the term disease shall include cancer and diseases associated therewith). The length of said survival can be calculated by reference to a defined start point (e.g. time of diagnosis or start of treatment) and end point (e.g. death, recurrence or metastasis). In addition, criteria for efficacy of treatment can be expanded to include response to chemotherapy, probability' of survival, probability of metastasis within a given time period, and probability of tumor recurrence.

[0080] In accordance with embodiments of the invention, a subject in need thereof is administered a therapeutically effective amount of a SWI/SNF complex modulator, thereby sensitizing the cells to the tyrosine kinase inhibitor.

[0081] Embodiments as described herein can comprise administering to a subject a therapeutically effective amount of a mammalian SWI/SNF complex modulator. The term “SWI/SNF complex” can refer to SWItch/Sucrose Non-Fennentable, an evolutionarily conserv ed ATP-dependent complex found in both eukaryotes and prokaryotes that comprises multiple subunits. (Neigebom Carlson (1984) Genetics 108:845-858; Stem et al. (1984) J. Mol. Biol. 178:853-868). In embodiments, the subunits are assembled in to three main types or subcomplexes, termed canonical BAF (cBAF), polybromo-associated BAF (PBAF), and non- canonical BAF (ncBAF). mSWI/SNF ATPase inhibitors targeted against SMARCA4 and/or SMARCA2 can inhibit the three forms of these complexes. The SWI/SNF complex has a key role in chromatin remodeling and regulation of transcription by recruitment of transcription factors, coactivators and repressors and histone modifiers. These complexes consist of one of the two mutually exclusive catalytic ATPase subunits: SMARCA2 (Brahma or BRM) or SMARCA4 (BRG1), and other subunits such as SMARCB1, SMARCC1, and SMARCC2. PBAF complexes can be distinguished from BAF complexes because the former contains PBRM1 and ARID2 but lack ARID1A/B and DPF1/2/3. SWI/SNF complexes regulate chromatin access by controlling the processes of histone dimer ejection, nucleosome ejection, and repositioning of nucleosomes by sliding. ARID1A binds DNA and can regulate the chromatin remodeling activity of the SWI/SNF complex through recruitment and binding of transcriptional factors. ARID subunits help with binding of the ATPase subcomplex. PBRM1 is essential for the stability of the SWI/SNF chromatin remodeling complex SWI/SNF -B (PBAF). ACTL6A, an actin domain, SMARCE1, and DPF1/2/3 are accessory subunits common to both BAF and PBAF and are rarely mutated in cancers.

[0082] In embodiments, the modulator comprises a canonical BAF (cBAF) inhibitor or degrader. cBAF can refer to at least one type of mammalian SWI/SNF complex. Its nucleosome remodeling activity can be reconstituted with a set of four core subunits (BRG1/SMARCA4, SNF5/SMARCB1, BAF155/SMARCC1, and BAF170/SMARCC2), which have orthologs in the yeast complex. However, mammalian SWI/SNF contains several subunits not found in the yeast counterpart, which can provide interaction surfaces for chromatin (for example, acetyllysine recognition by bromodomains) or transcription factors and thus contribute to the genomic targeting of the complex. A key attribute of mammalian S WI/SNF is the heterogeneity of subunit configurations that can exist in different tissues and even in a single cell type (for example, as BAF, PBAF, neural progenitor BAF (npBAF), neuron BAF (nBAF), embryonic stem cell BAF (esBAF), etc.). In some embodiments, the BAF complex described herein refers to one type of mammalian SWI/SNF complexes, which is different from PBAF complexes. In one embodiment, the cBAF complex is a mammalian cBAF complex. In one embodiment, the cBAF complex is a human cBAF complex. The components of the cBAF complex can include, for example, SMARCC1/2, SMARCD1/2/3, SMARCB1, SMARCE1, ARID1A/B, DPF1/2/3, ACTL6A/B, beta- Actin, BCL7A/B/C, SMARCA2/4, and SS18/L1.

[0083] The term ‘"BAF complex” can refer to at least one type of mammalian SWI/SNF complexes. Its nucleosome remodeling activity can be reconstituted with a set of four core subunits (BRG1/SMARCA4, SNF5/SMARCB1, BAF155/SMARCC1, and BAF170/SMARCC2), which have orthologs in the yeast complex (Phelan et al. (1999) Mol Cell. 3:247-253). However, mammalian SWI/SNF contains several subunits not found in the yeast counterpart, which can provide interaction surfaces for chromatin (e.g. acetyl-lysine recognition by bromodomains) or transcription factors and thus contribute to the genomic targeting of the complex (Wang et al. (1996) EMBO J. 15:5370-5382; Wang et al. (1996) Genes Dev. 10:2117-2130; Nie et al. (2000) ). A key attribute of mammalian SWI/SNF is the heterogeneity of subunit configurations that can exist in different tissues and even in a single cell type (e.g., as BAF, PBAF, neural progenitor BAF (npBAF), neuron BAF (nBAF), embryonic stem cell BAF (esBAF), etc.). In some embodiments, the BAF complex described herein refers to one ty pe of mammalian SWI/SNF complexes, which is different from PBAF complexes.

[0084] In embodiments, the modulator can comprise a chromatin modifying agent. A "chromatin modifying agent" can refer to an agent that can modify genomic DNA, in the context of nuclear chromatin. In embodiments, genomic DNA can be modified in a detectable manner.

[0085] “Modulating” can refer to regulating or adjusting the degree of activity of a process or the degree of an effect. “Modulating” includes activation, inhibition, degradation, amplification, attenuation, and suppression, for example. For example, modulating the activity of the SWI/SNF complex can refer to altering the level or activity of the SWI/SNF complex, component thereof, or a related dow nstream effect. The activity level of a BAF complex can be measured using any method known in the art. [0086] A “modulator” can refer to an agent that agonizes (activates or enhances) or antagonizes (inhibits or reduces) the function of a biological target. In embodiments, the SWI/SNF complex modulator can comprise an inhibitor or a degrader. In embodiments, the SWI/SNF complex modulator can comprise a nucleic acid molecule, a small molecule, a peptide, or a polypeptide. [0087] An “inhibitor” can refer to any agent which reduces the level and/or activity of a protein or protein complex, such as the SWI/SNF complex. The term “inhibiting” can refer to decrease, limiting, and/or blocking a certain action, function, or interaction. Non-limiting examples of inhibitors include small molecule inhibitors, degraders, antibodies, enzymes, or polynucleotides (e.g., siRNA). In some embodiments, cancer is “inhibited” if at least one symptom of the cancer is alleviated, terminated, slowed, or prevented. As used herein, cancer is also “inhibited” if recurrence or metastasis of the cancer is reduced, slowed, delayed, or prevented.

[0088] A “degrader” can refer to a molecule, such as a compound, that interacts with a protein (e.g., a protein of the SWI/SNF complex) in a way which results in degradation of the protein. For example, binding of the degrader results in at least 5% reduction of the level of the protein, e.g., in a cell or subject. In embodiments, the degrader can comprise a degradation moiety, which can refer to a moiety whose binding results in degradation of a protein. For example, the degradation moiety can bind to a protease or a ubiquitin ligase that metabolizes the protein.

[0089] In embodiments, the modulator can comprise an ATPase modulator. An “ATPase modulator” can refer to a molecule which binds to ATPase and inhibits or reduces the ATP- hydrolyzing activity of ATPase. For example, the modulator can comprise a SWI/SNF ATPase inhibitor or degrader. For example, the SWI/SNF ATPase modulator can inhibit SMARCA2 and SMARCA4.

[0090] The term "SMARCA2" can refer to SWI/SNF related, matrix associated, actin dependent regulator of chromatin, subfamily a, member 2, a member of the SWI/SNF family of proteins and is highly similar to the brahma protein of Drosophila. Members of this family have helicase and ATPase activities and can regulate transcription of certain genes by altering the chromatin structure around those genes. The encoded protein is part of the large ATP- dependent chromatin remodeling complex SNF/SWL which is required for transcriptional activation of genes normally repressed by chromatin. SMARCA2 is a component of SWI/SNF chromatin remodeling complexes that carry out key enzymatic activities, changing chromatin structure by altering DNA-histone contacts within a nucleosome in an ATP-dependent manner. SMARCA2 binds DNA non-specifically (Euskichen et al. (2012) J Biol Chem 287:30987- 30905; Kadoch et al. (2015) Sci Adv l(5):e!500447). SMARCA2 belongs to the neural progenitors-specific chromatin remodeling complex (npBAF complex) and the neuron-specific chromatin remodeling complex (nBAF complex). During neural development a switch from a stem/progenitor to a postmitotic chromatin remodeling mechanism occurs as neurons exit the cell cycle and become committed to their adult state. The transition from proliferating neural stem/progenitor cells to postmitotic neurons requires a switch in subunit composition of the npBAF and nBAF complexes. As neural progenitors exit mitosis and differentiate into neurons, npBAF complexes which contain ACTL6A/BAF53Aand PHF10/BAF45A, are exchanged for homologous alternative ACTL6B/BAF53B and DPF1/BAF45B or DPF3/BAF45C subunits in neuron-specific complexes (nBAF). The npBAF complex is essential for the self- renewal/proliferative capacity of the multipotent neural stem cells. The nBAF complex along with CREST plays a role regulating the activity of genes essential for dendrite growth. Human SMARCA2 protein has 1590 amino acids and a molecular mass of 181279 Da. The known binding partners of SMARCA2 include, e.g., PHF10/BAF45A, CEBPB, TOPBP1, and CEBPA. [0091] The term "SMARCA4" can refer to SWI/SNF related, matrix associated, actin dependent regulator of chromatin, subfamily a, member 4, a member of the SWI/SNF family of proteins and is highly similar to the brahma protein of Drosophila. Members of this family have helicase and ATPase activities and can regulate transcription of certain genes by altering the chromatin structure around those genes. The encoded protein is part of the large ATP- dependent chromatin remodeling complex SNF/SWI, which is required for transcriptional activation of genes normally repressed by chromatin. In addition, this protein can bind BRCA1, as well as regulate the expression of the tumorigenic protein CD44. Mutations in this gene cause rhabdoid tumor predisposition syndrome ty pe 2. SMARCA4 is a component of SWI/SNF chromatin remodeling complexes that carry out key enzymatic activities, changing chromatin structure by altering DNA-histone contacts within a nucleosome in an ATP-dependent manner. SMARCA4 is a component of the CREST-BRG1 complex, a multiprotein complex that regulates promoter activation by orchestrating a calcium-dependent release of a repressor complex and a recruitment of an activator complex. In resting neurons, transcription of the c- FOS promoter is inhibited by BRG1 -dependent recruitment of a phospho-RBl-HDAC repressor complex. Upon calcium influx, RBI is dephosphorylated by calcineurin, which leads to release of the repressor complex. At the same time, there is increased recruitment of CREBBP to the promoter by a CREST-dependent mechanism, yvhich leads to transcriptional activation. The CREST-BRG1 complex also binds to the NR2B promoter, and activity dependent induction of NR2B expression involves a release of HDAC1 and recruitment of CREBBP. SMARCA4 belongs to the neural progenitors-specific chromatin remodeling complex (npBAF complex) and the neuron-specific chromatin remodeling complex (nBAF complex). During neural development a switch from a stem/progenitor to a postmitotic chromatin remodeling mechanism occurs as neurons exit the cell cycle and become committed to their adult state. The transition from proliferating neural stem/progenitor cells to postmitotic neurons requires a switch in subunit composition of the npBAF and nBAF complexes. As neural progenitors exit mitosis and differentiate into neurons, npBAF complexes which contain ACTL6A/BAF53A and PHF 10/BAF 45A, are exchanged for homologous alternative ACTL6B/BAF53B and DPF1/BAF45B or DPF3/BAF45C subunits in neuron-specific complexes (nBAF). The npBAF complex is essential for the sei f-renewal/proli ferati ve capacity of the multipotent neural stem cells. The nBAF complex along with CREST plays a role regulating the activity of genes essential for dendrite growth. SMARCA4/BAF190A promote neural stem cell self-renewal/proliferation by enhancing Notch-dependent proliferative signals, while concurrently making the neural stem cell insensitive to SHH-dependent differentiating cues. SMARCA4 acts as a corepressor of ZEB1 to regulate E-cadherin transcription and is required for induction of epithelial-mesenchymal transition (EMT) by ZEB1. Human SMARCA4 protein has 1647 amino acids and a molecular mass of 184646 Da. The known binding partners of SMARCA4 include, e.g., PHF10/ BAF45A, MYOG, IKFZ1, ZEB1, NR3C1, PGR, SMARD1, TOPBP1 and ZMIM2/ZIMP7.

[0092] The catalytic core of the SWI/SNF complex can be one of two closely related ATPases, SMARCA2 (BRM) or SMARCA4 (BRG1). The choice of alternative subunits can be a key determinant of specificity. Instead of impeding differentiation as was seen with SMARCA4 (BRG1) depletion, depletion of SMARCA2 (BRM) caused accelerated progression to the differentiation phenotype. SMARCA2 (BRM) was found to regulate genes different from those as SMARCA4 (BRG1) targets and can override SMARCA4 (BRG1) -dependent activation of the osteocalcin promoter, due to its interaction with different ARID family members (Flowers et al. (2009), supra).

[0093] The term "BAF250A" or "ARID1A" refers to AT-rich interactive domain-containing protein IA. a subunit of the SWI/SNF complex, which can be find in BAF but not PBAF complex. In humans there are two BAF250 isoforms, BAF250A/ARID 1A and BAF250B/ARID1B. They can be E3 ubiquitin ligases that target histone H2B (Li et al. (2010) Mai. Cell. Biol. 30: 1673-1688). ARID1A is highly expressed in the spleen, thymus, prostate, testes, ovaries, small intestine, colon and peripheral leukocytes. ARID 1 A is involved in transcriptional activation and repres-sion of select genes by chromatin remodeling. It is also involved in vitamin D-coupled transcription regulation by associating with the WINAC complex, a chromatin-remod-eling complex recruited by vitamin D receptor. ARID 1 A belongs to the neural progenitors-specific chromatin remod-eling (npBAF) and the neuron-specific chromatin remodel-ing (nBAF) complexes, which are involved in switching developing neurons from stem/progenitors to post-mitotic chromatin remodeling as they exit the cell cycle and become committed to their adult state. ARID 1 A also plays key roles in maintaining embryonic stem cell pluripotency and in cardiac development and function (Lei et al. (2012) J. Biol. Chem. 287:24255-24262; Gao et al. (2008) Proc. Natl. Acad. Sci. U.S.A. 105:6656- 6661). Loss of BAF250a expression was seen in 42% of the ovarian clear cell carcinoma samples and 21 % of the endometrioid carcinoma samples, compared with just 1 % of the highgrade serous carcinoma samples. ARID 1 A deficiency also impairs the DNA damage checkpoint and sensitizes cells to PARP inhibitors (Shen et al. (2015) Cancer Discov. 5:752- 767). Human ARID1 A protein has 2285 amino acids and a molecular mass of 242045 Da, with at least a DNA-binding domain that can specifically bind an AT-rich DNA sequence, recognized by a SWI/SNF complex at the beta-globin locus, and a C-terminus domain for glucocorticoid receptor-depen-dent transcriptional activation. ARID IA has been shown to interact with proteins such as SMARCB1/BAF47 (Kato et al. (2002) J. Biol. Chem. 277:5498- 505; Wang et al. (1996) EMBO J. 15:5370-5382) and SMARCA4/BRG1 (Wang et al. (1996), supra; Zhao et al. (1998) Cell 95:625-636), etc.

[0094] The term "BAF250B" or "ARID1B" can refer to AT-rich interactive domain-containing protein IB, a subunit of the SWI/SNF complex, which can be find in BAF but not PBAF complex. ARID IB and ARID IA are alternative and mutually exclusive ARID-subunits of the SWI/SNF com-plex. Germline mutations in ARID1B are associated with Coffm-Siris syndrome (Tsurusaki et al. (2012) Nat. Genet. 44:376-378; Santen et al. (2012) Nat. Genet. 44:379-380). Somatic mutations in ARID1B are associated with several cancer subtypes, indicating that it is a tumor suppressor gene (Shai and Pollack (2013) PLoS ONE 8:e55119; Sausen et al. (2013) Nat. Genet. 45:12-17; Shain et al. (2012) Proc. Natl. Acad. Sci. U.S.A. 109:E252-E259; Fujimoto et al. (2012) Nat. Genet. 44:760-764). Human ARID IA protein has 2236 amino acids and a molecular mass of 236123 Da, with at least a DNA-binding domain that can specifically bind an AT-rich DNA sequence, recognized by a SWI/SNF complex at the beta-globin locus, and a C-terminus domain for glucocorticoid receptor-dependent transcriptional activa-tion. ARID1B has been shown to interact with SMARCA4/ BRG1 (Hurlstone et al. (2002) Biochem. J. 364:255-264; Inoue et al. (2002) J. Biol. Chem. 277:41674-41685 and SMARCA2/BRM (Inoue et al. (2002). supra). [0095] A nucleic acid molecule can refer to DNA molecules and RNA molecules. A nucleic acid molecule can be single-stranded or double-stranded. In embodiments, the nucleic acid molecule is single stranded. In embodiments, the nucleic acid molecule is double-stranded DNA. As used herein, the term “isolated nucleic acid molecule” can refer to a nucleic acid molecule in which the nucleotide sequences are free of other nucleotide sequences, which other sequences can naturally flank the nucleic acid in human genomic DNA. Non-limiting examples of a nucleic acid molecule comprise a siRNA, miRNA, shRNA, antisense RNA, guide RNA (gRNA), single-guide RNA (sgRNA), modified forms thereof, or combination thereof. For example, the nucleic acid molecule can comprise an RNA interfering agent or an antisense oligonucleotide.

[0096] An “isolated” nucleic acid molecule is free of sequences (such as protein-encoding sequences) which naturally flank the nucleic acid (i.e., sequences located at the 5' and 3' ends of the nucleic acid) in the genomic DNA of the organism from which the nucleic acid is derived. For example, in various embodiments, the isolated nucleic acid molecule can contain less than about 5 kB, 4 kB, 3 kB, 2 kB, 1 kB, 0.5 kB or 0. 1 kB of nucleotide sequences which naturally flank the nucleic acid molecule in genomic DNA of the cell from which the nucleic acid is derived. Moreover, an “isolated” nucleic acid molecule, such as a cDNA molecule, can be substantially free of other cellular material or culture medium when produced by recombinant techniques, or substantially free of chemical precursors or other chemicals when chemically synthesized.

[0097] A biomarker nucleic acid molecule of the invention can be isolated using standard molecular biology techniques and the sequence information in the database records described herein. Using all or a portion of such nucleic acid sequences, nucleic acid molecules of the invention can be isolated using standard hybridization and cloning techniques (e.g.. as described in Sambrook et al., ed., Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989).

[0098] A nucleic acid molecule of the invention can be amplified using cDNA, mRNA, or genomic DNA as a template and appropriate oligonucleotide primers according to standard PCR amplification techniques. The nucleic acid molecules so amplified can be cloned into an appropriate vector and characterized by DNA sequence analysis. Furthermore, oligonucleotides corresponding to all or a portion of a nucleic acid molecule of the invention can be prepared by standard synthetic techniques, e.g., using an automated DNA synthesizer. [0099] An “RNA interfering agent” as used herein, is defined as any agent which interferes with or inhibits expression of a target biomarker gene by RNA interference (RNAi). Such RNA interfering agents include, but are not limited to, nucleic acid molecules including RNA molecules which are homologous to the target biomarker gene of the invention, or a fragment thereof, short interfering RNA (siRNA), and small molecules which interfere with or inhibit expression of a target biomarker nucleic acid by RNA interference (RNAi). Non-limiting examples of a RNA interfering agent comprise a small interfering RNA (siRNA), CRISPR RNA (crRNA), microRNA (miRNA), small hairpin RNA (shRNA), antisense RNA, guide RNA (gRNA), single guide RNA (sgRNA), or a pi wi -interacting RNA (piRNA).

[00100] '‘RNA interference (RNAi)” is an evolutionally conserved process whereby the expression or introduction of RNA of a sequence that is identical or highly similar to a target biomarker nucleic acid results in the sequence specific degradation or specific post- transcriptional gene silencing (PTGS) of messenger RNA (mRNA) transcribed from that targeted gene (see Cobum and Cullen (2002) J. Virol. 76:9225), thereby inhibiting expression of the target biomarker nucleic acid. In one embodiment, the RNA is double stranded RNA (dsRNA). This process has been described in plants, invertebrates, and mammalian cells. In nature, RNAi is initiated by the dsRNA-specific endonuclease Dicer, which promotes processive cleavage of long dsRNA into double-stranded fragments termed siRNAs. siRNAs are incorporated into a protein complex that recognizes and cleaves target mRNAs. RNAi can also be initiated by introducing nucleic acid molecules, e.g., synthetic siRNAs or RNA interfering agents, to inhibit or silence the expression of target biomarker nucleic acids. As used herein, “inhibition of target biomarker nucleic acid expression” or “inhibition of marker gene expression” includes any decrease in expression or protein activity or level of the target biomarker nucleic acid or protein encoded by the target biomarker nucleic acid. The decrease can be of at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 99% or more as compared to the expression of a target biomarker nucleic acid or the activity or level of the protein encoded by a target biomarker nucleic acid which has not been targeted by an RNA interfering agent.

[00101] “Short interfering RNA” (siRNA), also referred to herein as “small interfering RNA” is defined as an agent which functions to inhibit expression of a target biomarker nucleic acid, e.g., by RNAi. An siRNA can be chemically synthesized, can be produced by in vitro transcription, or can be produced within a host cell. In one embodiment, siRNA is a double stranded RNA (dsRNA) molecule of about 15 to about 40 nucleotides in length, about 15 to about 28 nucleotides, about 19 to about 25 nucleotides in length, and about 19, 20, 21, or 22 nucleotides in length, and can contain a 3‘ and/or 5^? overhang on each strand having a length of about 0, 1, 2, 3, 4, or 5 nucleotides. The length of the overhang is independent between the two strands, i.e., the length of the overhang on one strand is not dependent on the length of the overhang on the second strand. The siRNA can promote RNA interference through degradation or specific post-transcriptional gene silencing (PTGS) of the target messenger RNA (mRNA).

[00102] In another embodiment, an siRNA is a small hairpin (also called stem loop) RNA (shRNA). In one embodiment, these shRNAs are composed of a short (e.g., 19-25 nucleotide) antisense strand, followed by a 5-9 nucleotide loop, and the analogous sense strand. Alternatively, the sense strand can precede the nucleotide loop structure and the antisense strand can follow. These shRNAs can be contained in plasmids, retroviruses, and lentiviruses and expressed from, for example, the pol III U6 promoter, or another promoter (see. e.g., Stewart, et al. (2003) RNA Apr;9(4):493-501 incorporated by reference herein).

[00103] RNA interfering agents, e.g., siRNA molecules, can be administered to a patient having or at risk for having cancer, to inhibit expression of a biomarker gene which is overexpressed in cancer and thereby treat, prevent, or inhibit cancer in the subject.

[00104] The term “small molecule” can refer to molecules that are less than about 1000 molecular weight or less than about 500 molecular weight. In one embodiment, small molecules do not exclusively comprise peptide bonds. In another embodiment, small molecules are not oligomeric. Exemplary small molecule compounds which can be screened for activity include, but are not limited to, peptides, peptidomimetics, nucleic acids, carbohydrates, small organic molecules (e.g., polyketides) (Cane et al. (1998) Science 282:63), and natural product extract libraries. In another embodiment, the compounds are small, organic non-peptidic compounds. In a further embodiment, a small molecule is not biosynthetic. In a further embodiment, a small molecule is an inhibitor. In another embodiment, a small molecule is a degrader. For example, non-limiting examples of a small molecule comprise:

[00105] In embodiments, Ri can be Cl, or F.

[00106] Unless otherwise specified here within, the terms "antibody” and "antibodies” broadly encompass naturally occurring forms of antibodies (e.g. IgG, IgA, IgM, IgE) and recombinant antibodies, such as single-chain antibodies, chimeric and humanized antibodies and multi-specific antibodies, as well as fragments and derivatives of the foregoing, which fragments and derivatives have at least an antigenic binding site. Antibody derivatives can comprise a protein or chemical moiety conjugated to an antibody.

[00107] In addition, intrabodies are well-known antigen-binding molecules having the characteristic of antibodies, but that can be expressed within cells in order to bind and/or inhibit intracellular targets of interest (Chen et al. (1994) Human Gene Ther. 5:595-601). Methods are well-known in the art for adapting antibodies to target (e.g., inhibit) intracellular moieties, such as the use of single-chain antibodies (scFvs), modification of immunoglobulin VL domains for hyperstability, modification of antibodies to resist the reducing intracellular environment, generating fusion proteins that increase intracellular stability and/or modulate intracellular localization, and the like. Intracellular antibodies can also be introduced and expressed in one or more cells, tissues or organs of a multicellular organism, for example for prophylactic and/or therapeutic purposes (e.g., as a gene therapy) (see, at least PCT Pubis. WO 08/020079. WO 94/02610, WO 95/22618, and WO 03/014960; U.S. Pat. No. 7,004,940; Cattaneo and Biocca (1997) Intracellular Antibodies: Development and Applications (Landes and Springer-Verlag pubis.); Kontermann (2004) Methods 34: 163-170; Cohen et al. (1998) Oncogene 17:2445-2456; Auf der Maur et al. (2001) FEBS Lett. 508:407-412; Shaki- Loewenstein et al. (2005) J. Immunol. Meth. 303: 19-39).

[00108] The term “antibody” as used herein also includes an “antigen-binding portion” of an antibody (or simply “antibody portion”). The term “antigen-binding portion”, as used herein, can refer to one or more fragments of an antibody that retain the ability to specifically bind to an antigen (e.g., a biomarker polypeptide or fragment thereof). It has been shown that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody. Examples of binding fragments encompassed within the term “antigen-binding portion” of an antibody include (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CHI domains; (ii) a F(ab')2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (hi) a Fd fragment consisting of the VH and CHI domains; (iv) a Fv fragment consisting of the VL and VH domains of a single arm of an antibody, (v) a dAb fragment (Ward et al., (1989) Nature 341 :544-546), which consists of a VH domain; and (vi) an isolated complementarity determining region (CDR). Furthermore, although the two domains of the Fv fragment, VL and VH, are coded for by separate genes, they can be j oined, using recombinant methods, by a synthetic linker that allows for them to be made as a single protein chain in which the VL and VH regions pair to form monovalent polypeptides (known as single chain Fv (scFv); see e.g., Bird et al. (1988) Science 242:423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883; and Osbourn et al. 1998, Nature Biotechnology 1 : 778). Such single chain antibodies are also intended to be encompassed within the term “antigen-binding portion” of an antibody. Any VH and VL sequences of specific scFv can be linked to human immunoglobulin constant region cDNA or genomic sequences, in order to generate expression vectors encoding complete IgG polypeptides or other isot pes. VH and VL can also be used in the generation of Fab, Fv or other fragments of immunoglobulins using protein chemistry or recombinant DNA technology. Other forms of single chain antibodies, such as diabodies are also encompassed. Diabodies are bivalent, bispecific antibodies in which VH and VL domains are expressed on a single polypeptide chain, but using a linker that is too short to allow for pairing between the two domains on the same chain, thereby forcing the domains to pair with complementary domains of another chain and creating two antigen binding sites (see e.g., Holliger et al. (1993) Proc. Natl. Acad. Sci. U.S.A. 90:6444-6448; Poljak et al. (1994) Structure 2: 1121-1123).

[00109] Still further, an antibody or antigen-binding portion thereof can be part of larger immunoadhesion polypeptides, formed by covalent or noncovalent association of the antibody or antibody portion with one or more other proteins or peptides. Examples of such immunoadhesion polypeptides include use of the streptavidin core region to make a tetrameric scFv polypeptide (Kipriyanov et al. (1995) Human Antibodies and Hybridomas 6:93-101) and use of a cysteine residue, biomarker peptide and a C-termmal poly histidine tag to make bivalent and biotinylated scFv polypeptides (Kipriyanov et al. (1994) Mol. Immunol. 31: 1047-1058). Antibody portions, such as Fab and F(ab')2 fragments, can be prepared from whole antibodies using conventional techniques, such as papain or pepsin digestion, respectively, of whole antibodies. Moreover, antibodies, antibody portions and immunoadhesion polypeptides can be obtained using standard recombinant DNA techniques, as described herein.

[00110] Antibodies can be polyclonal or monoclonal; xenogeneic, allogeneic, or syngeneic; or modified forms thereof (e.g. humanized, chimeric, etc.). Antibodies can also be fully human. Antibodies of the invention bind specifically or substantially specifically to a biomarker polypeptide or fragment thereof. The terms “monoclonal antibodies” and “monoclonal antibody composition”, as used herein, refer to a population of antibody polypeptides that contain only one species of an antigen binding site that can immunoreact with a certain epitope of an antigen, whereas the term “polyclonal antibodies” and “polyclonal antibody composition” refer to a population of antibody polypeptides that contain multiple species of antigen binding sites that can interact with a certain antigen. A monoclonal antibody composition displays a single binding affinity for a certain antigen with which it immunoreacts. In embodiments, the antibody, or antigen binding fragment thereof, is murine, chimeric, humanized, mosaic, composite, or human.

[00111] Antibodies can be “humanized,” which is intended to include antibodies made by a non-human cell having variable and constant regions which have been altered to more closely resemble antibodies that can be made by a human cell. For example, by altering the non-human antibody amino acid sequence to incorporate amino acids found in human germline immunoglobulin sequences. The humanized antibodies of the invention can include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo), for example in the CDRs. The term “humanized antibody”, as used herein, also includes antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences.

[00112] The term “specific binding” can refer to antibody binding to a predetermined antigen. The antibody binds with an affinity (KD) of approximately less than 10-7 M, such as approximately less than 10-8 M, 10-9 M or 10-10 M or even lower when determined by surface plasmon resonance (SPR) technology in a BIACORE® assay instrument using an antigen of interest as the analyte and the antibody as the ligand, and binds to the predetermined antigen with an affinity’ that is at least about 1.1-, 1.2-, 1.3-. 1.4-, 1.5-, 1.6-, 1.7-, 1.8-, 1.9-, 2.0-. 2.5-, 3.0-, 3.5-, 4.0-, 4.5-, 5.0-, 6.0-, 7.0-, 8.0-, 9.0-, or 10.0-fold or greater than its affinity for binding to a non-specific antigen (e.g., BSA, casein) other than the predetermined antigen or a closely -related antigen. The phrases “an antibody recognizing an antigen” and “an antibody specific for an antigen” are used interchangeably herein with the term “an antibody which binds specifically to an antigen.” Selective binding is a relative term referring to the ability of an antibody to discriminate the binding of one antigen over another.

[00113] “Determining the level of a protein” can refer to the detection of a protein, or an mRNA encoding the protein, by methods known in the art, directly or indirectly. “Directly determining” can refer to performing a process (e.g., performing an assay or test on a sample or “analyzing a sample” as that term is defined herein) to obtain the physical entity or value. “Indirectly determining” can refer to receiving the physical entity or value from another party or source (e.g., a third-party⁷ laboratory⁷ that directly acquired the physical entity⁷ or value). For example, methods to measure protein level can include, but are not limited to, western blotting, immunoblotting, enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), immunoprecipitation, immunofluorescence, surface plasmon resonance, chemiluminescence, fluorescent polarization, phosphorescence, immunohistochemical analysis, matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry, liquid chromatography (LC)-mass spectrometry, microcytometry, microscopy, fluorescence activated cell sorting (FACS), and flow cytometry, as well as assays based on a property⁷ of a protein including, but not limited to, enzy matic activity or interaction with other protein partners. Methods to measure mRNA levels are known in the art.

[00114] '‘Reducing the level” of the SWI/SNF complex or component thereof can refer to decreasing the level of the complex or component, such as a SWI/SNF ATPase, in a cell or subject. The level of SWI/SNF complex or component thereof can be measured using any method known in the art.

[00115] "Level” can refer to a level of a protein, or mRNA encoding the protein, as compared to a reference. The reference can be any⁷ useful reference, as defined herein. By a “decreased level” or an “increased level” of a protein is meant a decrease or increase in protein level, as compared to a reference (e.g., a decrease or an increase by about 5%, about 1 0%, about 15%. about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 100%, about 1 50%, about 200%, about 300%, about 400%, about 500%, or more; a decrease or an increase of more than about 10%, about 15%, about 20%, about 50%, about 75%. about 100%, or about 200%, as compared to a reference; a decrease or an increase by less than about 0.01 -fold, about 0.02 -fold, about 0.1 -fold, about 0.3-fold, about 0.5-fold, about 0.8-fold, or less; or an increase by⁷ more than about 1 .2-fold, about 1 .4-fold, about 1 .5- fold, about 1 .8-fold, about 2.0-fold, about 3.0-fold, about 3.5-fold, about 4.5-fold, about 5.0- fold, about 10-fold, about 15-fold, about 20-fold, about 30-fold, about 40-fold, about 50-fold, about 100-fold, about 1000-fold, or more). A level of a protein can be expressed in mass/vol (e.g., g/dL, rng/mL, pg/mL, ng/ml_) or percentage relative to total protein or mRNA in a sample.

[00116] “Reducing the activity” of the SWI/SNF complex can refer to decreasing the level of an activity related to the SWI/SNF complex, a component thereof, or a related downstream effect. The activity level of the SWI/SNF complex can be measured using any method known in the art. In embodiments, an agent which reduces the activity of the SWI/SNF complex is a small molecule inhibitor. In embodiments, an agent which reduces the activity' of the SWI/SNF complex is a small molecule degrader.

[00117] The term "biomarker” can refer to a measurable entity of the invention that has been determined to be predictive of cancer therapy (e.g., at least one modulator of biomarkers listed in FIG. 1 and FIG. 3) effects. Biomarkers can include, without limitation, nucleic acids (e.g.. genomic nucleic acids and/or transcribed nucleic acids) and proteins, such as those involved shown in FIG. 1 and FIG. 3. Many biomarkers listed in FIG. 1 and FIG. 3 are also useful as therapeutic targets.

[00118] The term “altered amount'’ or “altered level” refers to increased or decreased copy number (e.g., germline and/or somatic) of a biomarker nucleic acid, e.g.. increased or decreased expression level in a cancer sample, as compared to the expression level or copy number of the biomarker nucleic acid in a control sample. The term “altered amount” of a biomarker also includes an increased or decreased protein level of a biomarker protein in a sample, e.g., a cancer sample, as compared to the corresponding protein level in a normal, control sample. Furthermore, an altered amount of a biomarker protein can be determined by detecting posttranslational modification such as methylation status of the marker, which can affect the expression or activity of the biomarker protein.

[00119] The amount of a biomarker in a subj ect is “significantly” higher or lower than the normal amount of the biomarker, if the amount of the biomarker is greater or less, respectively, than the normal level by an amount greater than the standard error of the assay employed to assess amount, and at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 300%, 350%, 400%, 500%, 600%, 700%, 800%, 900%, 1000% or than that amount. Alternately, the amount of the biomarker in the subject can be considered “significantly” higher or lower than the normal amount if the amount is at least about two, and at least about three, four, or five times, higher or lower, respectively, than the normal amount of the biomarker. Such “significance” can also be applied to any other measured parameter described herein, such as for expression, inhibition, cytotoxicity, cell growth, and the like.

[00120] The term “expression profile” can refer to a genomic expression profile. Profiles can be generated by any convenient means for determining a level of a nucleic acid sequence, non-limiting examples of which include quantitative hybridization of microRNA, labeled microRNA, amplified microRNA, cRNA, quantitative PCR, ELISA for quantitation, and the like. Expression profiles can allow for the analysis of differential gene expression between two samples. In embodiments, a subject or patient sample, e.g., cells or collections thereof, e.g., tissues, can be assayed. Samples can be collected by any convenient method, as known in the art.

[00121] The term “microarray” can refer to an ordered arrangement of hybridizable array elements, such as polynucleotide probes, on a substrate. [00122] The terms ‘‘level of expression"’ or “expression level” can be used interchangeably and can refer to the amount of a biomarker in a biological sample. A “biological sample” can refer to a sample that is of biologic origin or contains biologic elements. A biologic sample can contain whole cells (live or dead), parts of cells, cell debris, cell products (intracellular cell products or those that are secreted or excreted from a cell), compounds produced by a biologic entity or cell(s) thereof. The biological sample can be, contain, or be derived from a “bodily fluid” or “bodily gas”. The term “bodily fluid” can refer to any fluid produced by a biologic entity or subject and includes, amniotic fluid, aqueous humour, vitreous humour, bile, blood, blood serum, breast milk, cerebrospinal fluid, cerumen (earwax), chyle, chyme, endolymph, perilymph, exudates, feces, female ejaculate, gastric acid, gastric juice, lymph, mucus (including nasal drainage and phlegm), pericardial fluid, peritoneal fluid, pleural fluid, pus, rheum, saliva, sebum (skin oil), semen, sputum, synovial fluid, sweat, tears, urine, vaginal secretion, vomit, exhalant (respiratory), and mixtures of one or more thereof. Biologic gasses include, but are not limited to exhalant (respiratory), flatulence, decomposition gasses, and the like. Biological samples include cell cultures, bodily fluids, cell cultures from bodily fluids. Bodily fluids, gasses, or other biological samples can be obtained by any suitable collection method and/or technique, including but not limited to, those described in greater detail elsewhere herein, biopsy, puncturing (e.g., venous puncture), swabbing, scraping, plucking, pinching, cutting, catching (e.g., free catching urine or saliva after spitting by a subject), scooping, squeezing, expressing, extracting, sucking, passive sampling, and combinations thereof. Biologic samples can also those obtained from the environment, but contain cells or cell products secreted, released, or excreted from a subject. For example, methods as described herein can comprise detecting an analyte mRNA, protein, or genomic DNA in a biological sample in vitro as well as in vivo.

[00123] "‘Expression" can refer to the process by which information (for example, gene- encoded and/or epigenetic information) is converted into the structures present and operating in the cell. For example, “expression" can refer to transcription into a polynucleotide, translation into a polypeptide, or even polynucleotide and/or polypeptide modifications (for example, posttranslational modifications of a polypeptide). Fragments of the transcribed polynucleotide, the translated polypeptide, or polynucleotide and/or polypeptide modifications (for example, posttranslational modification of a polypeptide) can also be regarded as expressed whether they originate from a transcript generated by alternative splicing or a degraded transcript, or from a post-translational processing of the polypeptide, for example, by proteolysis. “Expressed genes” can include those that are transcribed into a polynucleotide as mRNA and then translated into a polypeptide, and also those that are transcribed into RNA but not translated into a polypeptide (for example, transfer and ribosomal RNAs).

[00124] '‘Increased expression,” ‘'increased expression level,” “increased levels," “elevated expression," "elevated expression levels,” or “elevated levels” can refer to an increased expression or increased levels of a biomarker in a subject or biological sample isolated from a subject relative to a control, such as a subject or subjects who are not suffering from the disease or disorder (for example, a cancer) or an internal control (for example, a housekeeping biomarker).

[00125] “Decreased expression," "decreased expression level," "decreased levels,” “reduced expression.” “reduced expression levels.” or “reduced levels” can refer to a decrease expression or decreased levels of a biomarker in a subject or biological sample isolated from a subject relative to a control, such as a subject or subjects who are not suffering from the disease or disorder (for example, a cancer) or an internal control (for example, a housekeeping biomarker).

[00126] The “normal” level of expression of a biomarker is the level of expression of the biomarker in cells of a subject, e.g., a human patient, not afflicted with a cancer. An “overexpression” or “significantly higher level of expression” of a biomarker refers to an expression level in a test sample that is greater than the standard error of the assay employed to assess expression, and is at least 10%, and 1.2, 1.3, 1.4, 1.5. 1.6, 1.7, 1.8. 1.9, 2.0, 2.1, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 times or more higher than the expression activity or level of the biomarker in a control sample (e.g., sample from a healthy subject not having the biomarker associated disease) and the average expression level of the biomarker in several control samples. A “significantly lower level of expression” of a biomarker refers to an expression level in a test sample that is at least 10%, and 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 12, 13, 14. 15, 16, 17, 18, 19, 20 times or more lower than the expression level of the biomarker in a control sample (e.g.. sample from a healthy subject not having the biomarker associated disease) and the average expression level of the biomarker in several control samples.

[00127] The term “altered level of expression” of a biomarker refers to an expression level or copy number of the biomarker in a test sample, e.g., a sample derived from a patient suffering from cancer, that is greater or less than the standard error of the assay employed to assess expression or copy number, and is at least twice, and but can be three, four, five or ten or more times the expression level or copy number of the biomarker in a control sample (e.g., sample from a healthy subjects not having the associated disease) and the average expression level or copy number of the biomarker in several control samples. The altered level of expression is greater or less than the standard error of the assay employed to assess expression or copy number, and is at least 20%, 30%, 40%, 50%, 60%, 70%. 80%, 90%, 100%, 150%, 200%, 300%, 350%, 400%, 500%. 600%, 700%, 800%, 900%, 1000% or more times the expression level or copy number of the biomarker in a control sample (e.g., sample from a healthy subjects not having the associated disease) and the average expression level or copy number of the biomarker in several control samples. In some embodiments, the level of the biomarker refers to the level of the biomarker itself, the level of a modified biomarker (e.g., phosphorylated biomarker), or to the level of a biomarker relative to another measured variable, such as a control (e.g., phosphorylated biomarker relative to an unphosphorylated biomarker). [00128] The term “altered activity” of a biomarker refers to an activity of the biomarker which is increased or decreased in a disease state, e.g., in a cancer sample, as compared to the activity of the biomarker in a normal, control sample. Altered activity’ of the biomarker can be the result of, for example, altered expression of the biomarker, altered protein level of the biomarker, altered structure of the biomarker, or, e.g., an altered interaction with other proteins involved in the same or different pathway as the biomarker or altered interaction with transcriptional activators or inhibitors.

[00129] The term “control” refers to any reference standard suitable to provide a comparison to the expression products in the test sample. In one embodiment, the control comprises obtaining a “control sample” from which expression product levels are detected and compared to the expression product levels from the test sample. Such a control sample can comprise any suitable sample, including but not limited to a sample from a control cancer patient (can be stored sample or previous sample measurement) with a known outcome; normal tissue or cells isolated from a subject, such as a normal patient or the cancer patient, cultured primary cells/tissues isolated from a subject such as a normal subject or the cancer patient, adjacent normal cells/tissues obtained from the same organ or body location of the cancer patient, a tissue or cell sample isolated from a normal subject, or a primary cells/tissues obtained from a depository. In another embodiment, the control can comprise a reference standard expression product level from any suitable source, including but not limited to housekeeping genes, an expression product level range from normal tissue (or other previously analyzed control sample), a previously determined expression product level range within a test sample from a group of patients, or a set of patients with a certain outcome (for example, survival for one, two, three, four years, etc.) or receiving a certain treatment (for example, standard of care cancer therapy). It will be understood by those of skill in the art that such control samples and reference standard expression product levels can be used in combination as controls in the methods of the invention. In one embodiment, the control can comprise normal or non-cancerous cell/tissue sample. In another embodiment, the control can comprise an expression level for a set of patients, such as a set of cancer patients, or for a set of cancer patients receiving a certain treatment, or for a set of patients with one outcome versus another outcome. In the former case, the specific expression product level of each patent can be assigned to a percentile level of expression, or expressed as higher or lower than the mean or average of the reference standard expression level. In another embodiment, the control can comprise normal cells, cells from patients treated with combination chemotherapy, and cells from patients having benign cancer. In another embodiment, the control can also comprise a measured value for example, average level of expression of a certain gene in a population compared to the level of expression of a housekeeping gene in the same population. Such a population can comprise normal subjects, cancer patients who have not undergone any treatment (i.e., treatment naive), cancer patients undergoing standard of care therapy, or patients having benign cancer. In another embodiment, the control comprises a ratio transformation of expression product levels, including but not limited to determining a ratio of expression product levels of two genes in the test sample and comparing it to any suitable ratio of the same two genes in a reference standard; determining expression product levels of the two or more genes in the test sample and determining a difference in expression product levels in any suitable control; and determining expression product levels of the two or more genes in the test sample, normalizing their expression to expression of housekeeping genes in the test sample, and comparing to any suitable control. In embodiments, the control comprises a control sample which is of the same lineage and/or type as the test sample. In another embodiment, the control can comprise expression product levels grouped as percentiles within or based on a set of patient samples, such as patients with cancer. In one embodiment a control expression product level is established wherein higher or lower levels of expression product relative to, for instance, a certain percentile, are used as the basis for predicting outcome. In another embodiment, a control expression product level is established using expression product levels from cancer control patients with a known outcome, and the expression product levels from the test sample are compared to the control expression product level as the basis for predicting outcome. [00130] The term “allele;’ which is used interchangeably herein with “allelic variant,’' refers to alternative forms of a gene or portions thereof. Alleles occupy the same locus or position on homologous chromosomes. When a subject has two identical alleles of a gene, the subject is said to be homozygous for the gene or allele. When a subject has two different alleles of a gene, the subject is said to be heterozy gous for the gene or allele. For example, biomarker alleles can differ from each other in a single nucleotide, or several nucleotides, and can include substitutions, deletions, and insertions of nucleotides. An allele of a gene can also be a form of a gene containing one or more mutations.

[00131] The term “allelic variant of a polymorphic region of gene” or “allelic variant”, used interchangeably herein, refers to an alternative form of a gene having one of several possible nucleotide sequences found in that region of the gene in the population. As used herein, allelic variant is meant to encompass functional allelic variants, non-functional allelic variants, SNPs, mutations and polymorphisms.

[00132] As used herein, the terms “gene” and “recombinant gene” refer to nucleic acid molecules comprising an open reading frame encoding a polypeptide corresponding to a marker of the invention. Such natural allelic variations can result in 1-5% variance in the nucleotide sequence of a given gene. Alternative alleles can be identified by sequencing the gene of interest in a number of different individuals. This can be readily carried out by using hybridization probes to identify the same genetic locus in a variety of individuals. Nucleotide variations and resulting ammo acid polymorphisms or variations that are the result of natural allelic variation and that do not alter the functional activity are intended to be within the scope of the invention.

[00133] The term “single nucleotide polymorphism” (SNP) refers to a polymorphic site occupied by a single nucleotide, which is the site of variation between allelic sequences. The site is preceded by and followed by highly conserved sequences of the allele (e.g., sequences that vary' in less than 1/100 or 1/1000 members of a population). A SNP arises due to substitution of one nucleotide for another at the polymorphic site. SNPs can also arise from a deletion of a nucleotide or an insertion of a nucleotide relative to a reference allele. The polymorphic site is occupied by a base other than the reference base. For example, where the reference allele contains the base “T” (thymidine) at the polymorphic site, the altered allele can contain a “C” (cytidine), “G” (guanine), or “A” (adenine) at the polymorphic site. SNP's can occur in protein-coding nucleic acid sequences, in which case they can give rise to a defective or otherwise variant protein, or generic disease. Such a SNP can alter the coding sequence of the gene and therefore specify another amino acid (a “missense” SNP) or a SNP can introduce a stop codon (a “nonsense” SNP). When a SNP does not alter the amino acid sequence of a protein, the SNP is called “silent.” SNP’s can also occur in noncoding regions of the nucleotide sequence. This can result in defective protein expression, e.g., as a result of alternative spicing, or it can have no effect on the function of the protein.

[00134] The “copy number” of a biomarker nucleic acid refers to the number of DNA sequences in a cell (e.g., germline and/or somatic) encoding a certain gene product. For a given gene, a mammal has two copies of each gene. The copy number can be increased, however, by gene amplification or duplication, or reduced by deletion. For example, germline copy number changes include changes at one or more genomic loci, wherein said one or more genomic loci are not accounted for by the number of copies in the normal complement of germline copies in a control (e.g., the normal copy number in germline DNA for the same species as that from which the specific germline DNA and corresponding copy number were determined). Somatic copy number changes include changes at one or more genomic loci, wherein said one or more genomic loci are not accounted for by the number of copies in germline DNA of a control (e.g., copy number in germline DNA for the same subject as that from which the somatic DNA and corresponding copy number were determined).

[00135] The term “predictive” includes the use of a biomarker nucleic acid and/or protein status, e.g., over- or under- activity⁷, emergence, expression, growth, remission, recurrence or resistance of tumors before, during or after therapy, for determining the likelihood of response of a cancer to modulators of T-cell mediated cytotoxicity alone or in combination with immunotherapy (e.g., treatment with a combination of an inhibitor of at least one biomarker described herein and an immunotherapy, such as an immune checkpoint inhibitor). Such predictive use of the biomarker can be confirmed by, e.g.. (1) increased or decreased copy number (e.g., by FISH, FISH plus SKY, single-molecule sequencing, e.g., as described in the art at least at J. Biotechnol., 86:289-301, or qPCR), overexpression or underexpression of a biomarker nucleic acid (e.g., by ISH, Northern Blot, or qPCR), increased or decreased biomarker protein (e.g., by IHC). or increased or decreased activity, e g., in more than about 5%, 6%, 7%. 8%, 9%, 10%. 11%. 12%, 13%, 14%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 100%, or more of assayed human cancers types or cancer samples; (2) its absolute or relatively modulated presence or absence in a biological sample, e.g., a sample containing tissue, whole blood, serum, plasma, buccal scrape, saliva, cerebrospinal fluid, urine, stool, or bone marrow, from a subject, e.g. a human, afflicted with cancer; (3) its absolute or relatively modulated presence or absence in clinical subset of patients with cancer (e.g.. those responding to a certain modulator of T-cell mediated cytotoxicity alone or in combination with immunotherapy or those developing resistance thereto).

[00136] The term '‘altered structure” of a biomarker refers to the presence of mutations or allelic variants within a biomarker nucleic acid or protein, e.g., mutations which affect expression or activity of the biomarker nucleic acid or protein, as compared to the normal or wild-type gene or protein. For example, mutations include, but are not limited to substitutions, deletions, or addition mutations. Mutations can be present in the coding or non-coding region of the biomarker nucleic acid.

[00137] The term “coding region” refers to regions of a nucleotide sequence comprising codons which are translated into amino acid residues, whereas the term “noncoding region” refers to regions of a nucleotide sequence that are not translated into amino acids (e.g., 5' and 3' untranslated regions).

[00138] The term “complementary” refers to the broad concept of sequence complementarity between regions of two nucleic acid strands or between two regions of the same nucleic acid strand. It is known that an adenine residue of a first nucleic acid region can form specific hydrogen bonds (“base pairing”) with a residue of a second nucleic acid region which is antiparallel to the first region if the residue is thymine or uracil. Similarly, it is known that a cytosine residue of a first nucleic acid strand can base pair with a residue of a second nucleic acid strand which is antiparallel to the first strand if the residue is guanine. A first region of a nucleic acid is complementary to a second region of the same or a different nucleic acid if, when the two regions are arranged in an antiparallel fashion, at least one nucleotide residue of the first region can base pair with a residue of the second region. The first region comprises a first portion and the second region comprises a second portion, whereby, when the first and second portions are arranged in an antiparallel fashion, at least about 50%, and at least about 75%, at least about 90%, or at least about 95% of the nucleotide residues of the first portion can base pair with nucleotide residues in the second portion. Nucleotide residues of the first portion can base pair with nucleotide residues in the second portion.

[00139] The terms “therapies” and/or “therapy” can refer to any protocol(s), method(s), compositions, formulations, and/or agent(s) that can be used in the prevention, treatment, management, or amelioration of a disease or disorder or a symptom associated therewith. In embodiments, the terms “therapies” and “therapy” can refer to biological therapy, supportive therapy, and/or other therapies useful in treatment, management, prevention, or amelioration of a disease or disorder or a symptom associated therewith known to one of skill in the art. [00140] The terms “therapeutic agent'’ and “therapeutic agents'’ can refer to any agent(s) which can be used in the prevention, treatment and/or management of a disease or disorder or a symptom associated therewith.

[00141] The term “therapeutic effect” can refer to a local or systemic effect in animals, such as mammals and humans, caused by a pharmacologically active substance. “Therapeutic effect” can refer to any substance intended for use in the diagnosis, cure, mitigation, treatment or prevention of disease or in the enhancement of desirable physical or mental development and conditions in subject.

[00142] The term "therapeutically effective amount" can refer to that amount of an embodiment of the composition or pharmaceutical composition being administered that will relieve to some extent one or more of the symptoms of the disease or condition being treated, and/or that amount that will prevent, to some extent, one or more of the symptoms of the condition or disease that the subject being treated has or is at risk of developing. For example, certain compounds encompassed by the methods of the invention can be administered in a sufficient amount to produce a reasonable benefit/risk ratio applicable to such treatment.

[00143] In embodiments, a therapeutically effective amount can comprise less than about 0. 1 mg/kg, about 0.1 mg/kg, about 0.5 mg/kg, about 1.0 mg/kg, about 2.5 mg/kg, about 5 mg/kg, about 7.5 mg/kg, about 10 mg/kg, about 15 mg/kg, about 20 mg/kg, about 25 mg/kg, about 30 mg/kg, about 35 mg/kg, about 40 mg/kg, about 45 mg/kg, about 50 mg/kg, about 55 mg/kg, about 60 mg/kg, about 70 mg/kg, about 80 mg/kg, about 90 mg/kg, about 100 mg/kg, about 120 mg/kg, about 135 mg/kg, about 150 mg/kg, about 175 mg/kg, about 200 mg/kg, about 225 mg/kg, about 250 mg/kg, about 275 mg/kg, about 300 mg/kg, about 325 mg/kg, about 350 mg/kg, about 375 mg/kg, about 400 mg/kg, about 425 mg/kg, about 450 mg/kg. about 475 mg/kg. about 500 mg/kg, about 525 mg/kg, about 550 mg/kg, about 575 mg/kg. about 600 mg/kg, about 625 mg/kg, about 650 mg/kg, about 675 mg/kg, about 700 mg/kg, about 725 mg/kg, about 750 mg/kg, about 775 mg/kg, about 800 mg/kg, about 825 mg/kg, about 850 mg/kg, about 875 mg/kg, about 900 mg/kg, about 1.0 g/kg, about 1.5 g/kg, about 2.0 g/kg, about 2.5 g/kg, about 5 g/kg, about 10 g/kg, about 25 g/kg, about 50 g/kg, or more than 50 g/kg of compound per body weight of a subject.

[00144] In embodiments, the therapeutically effective amount comprises less than about 0.1 mg, about 0.1 mg, about 0.5 mg, about 1.0 mg, about 2.5 mg, about 5 mg, about 7.5 mg, about 10 mg, about 15 mg, about 20 mg, about 25 mg, about 30 mg, about 35 mg, about 40 mg, about 45 mg. about 50 mg, about 55 mg, about 60 mg. about 70 mg, about 80 mg, about 90 mg, about 100 mg, about 120 mg, about 135 mg, about 150 mg, about 175 mg, about 200 mg, about 225 mg, about 250 mg, about 275 mg, about 300 mg, about 325 mg, about 350 mg, about 375 mg, about 400 mg, about 425 mg, about 450 mg, about 475 mg, about 500 mg. about 525 mg, about 550 mg, about 575 mg, about 600 mg, about 625 mg, about 650 mg, about 675 mg, about 700 mg, about 725 mg, about 750 mg, about 775 mg, about 800 mg, about 825 mg, about 850 mg, about 875 mg, about 900 mg, about 1.0 g, about 1.5 g, about 2.0 g, about 2.5 g, about 5 g, about 10 g, about 25 g, about 50 g. or more than 50 g.

[00145] A therapeutically effective amount of the SWI/SNF complex modulator will depend on the age and weight of the subject and the concentration and/or formulation of the inhibitor. [00146] The term “subject” or “patient” can refer to any organism to which aspects of the invention can be administered, e.g., for experimental, diagnostic, prophylactic, and/or therapeutic purposes. For example, subjects to which compounds of the disclosure can be administered include animals, such as mammals. Non-limiting examples of mammals include primates, such as humans. For veterinary applications, a wide variety of subjects will be suitable, e.g.. livestock such as cattle, sheep, goats, cows, swine, and the like; poultry such as chickens, ducks, geese, turkeys, and the like; and domesticated animals for example pets such as dogs and cats. For diagnostic or research applications, a wide variety of mammals will be suitable subjects, including rodents (e.g., mice, rats, hamsters), rabbits, primates, and swine such as inbred pigs and the like. The term “living subject” can refer to a subject noted herein or another organism that is alive. The term “living subject” can refer to the entire subject or organism and not just a part excised (e.g., a liver or other organ) from the living subject. In embodiments, the SWI/SNF complex inhibitor and/or degrader can comprise an antibody directed to the SWI/SNF complex, a nucleic acid molecule targeting the SWI/SNF complex, a compound or prodrug thereof that binds to the SWI/SNF complex, or a pharmaceutically acceptable salt or ester of said compound or prodrug.

[00147] In another embodiment of the methods of the invention, the subject has not undergone treatment, such as chemotherapy, radiation therapy, targeted therapy, and/or immunotherapies. In still another embodiment, the subject has undergone treatment, such as chemotherapy, radiation therapy, targeted therapy, and/or immunotherapies.

[00148] In certain embodiments, the subj ect has had surgery to remove cancerous or pre- cancerous tissue. In other embodiments, the cancerous tissue has not been removed, e.g., the cancerous tissue can be located in an inoperable region of the body, such as in a tissue that is essential for life, or in a region where a surgical procedure can cause considerable risk of harm to the patient. [00149] The methods of the invention can be used to determine the responsiveness to cancer therapy (e.g., at least one modulator of biomarkers listed in FIG. 1 or FIG. 3) of many different cancers in subjects such as those described herein. Tn one embodiment, the cancer comprises lung cancer. For example, the lung cancer comprises non-small cell lung cancer or EGFR-mutant lung cancer.

[00150] In embodiments, the SWI/SNF complex modulator can comprise a degrader directed to the SWI/SNF complex, a nucleic acid molecule targeting the SWI/SNF complex, a compound or prodrug thereof that binds to the SWI/SNF complex, or a pharmaceutically acceptable salt or ester of said compound or prodrug.

[00151] A compound can refer to any chemical entity, pharmaceutical, drug, and the like that can be used to treat or prevent a disease, illness, sickness, or disorder of bodily function (for example, a cancer). The term “compound” as used herein can include but is not limited to peptides, nucleic acids, carbohydrates, natural product extract libraries, organic molecules, such as small organic molecules, inorganic molecules, including but not limited to chemicals, metals, and organometallic molecules. For example, non-limiting examples of a compound comprise:

[00152] In embodiments, Ri can be Cl, or F.

[00153] Embodiments of the invention can also comprise pharmaceutically acceptable salts. “Pharmaceutically acceptable salts” can refer to a salt prepared by combining a compound of the invention with an acid whose anion, or a base whose cation, is considered suitable for human consumption. Non-limiting examples of pharmaceutically acceptable salts comprise mineral acid salts, such as hydrochlorides, hydrobromides, phosphates and sulphates, or salts of organic acids, such as acetates, propionates, malonates and benzoates. [00154] "Pharmaceutically acceptable derivatives" of a compound can include salts, esters, enol ethers, enol esters, acetals, ketals, orthoesters, hemiacetals, hemiketals, acids, bases, solvates, hydrates or prodrugs thereof. Such derivatives can be readily prepared by those of skill in this art using know n methods for such derivatization. The compounds produced can be administered to animals or humans without substantial toxic effects as pharmaceutically active compounds or as prodrugs.

[00155] In embodiments, the compound can be an antagonist. The term “antagonist” can refer to a compound or composition that can decrease, block, inhibit, abrogate, or interfere with a biological response by binding to or blocking a cellular constituent.

[00156] In embodiments, the compound can be an agonist. The term “agonist” can refer to a compound or composition that interacts with a cellular constituent and elicits an observable response. For example, an agonist can stimulate an activity at a receptor or receptors normally stimulated by naturally occurring substances, thus triggering a response.

[00157] Aspects of the invention can comprise administering to a subj ect pharmaceutical compositions comprising a SWI/SNF complex modulator. The phrase "pharmaceutical composition" or a “pharmaceutical formulation” can refer to a composition or pharmaceutical composition suitable for administration to a subject, such as a mammal, especially a human and that can refer to the combination of an active agent(s), or ingredient with a pharmaceutically acceptable earner or excipient, making the composition suitable for diagnostic, therapeutic, or preventive use in vitro, in vivo, or ex vivo. A “pharmaceutical composition” can be sterile and can be free of contaminants that can elicit an undesirable response within the subject (e.g., the compound(s) in the pharmaceutical composition is pharmaceutical grade). Pharmaceutical compositions can be designed for administration to subjects or patients in need thereof via a number of different routes of administration including oral, intranasal, topical, intravenous, buccal, rectal, parenteral, intraperitoneal, intradermal, intratracheal, intramuscular, subcutaneous, by stent-eluting devices, catheters-eluting devices, intravascular balloons, inhalational and the like.

[00158] A "pharmaceutically acceptable excipient," "pharmaceutically acceptable diluent," "pharmaceutically acceptable carrier," or "pharmaceutically acceptable adjuvant" can refer to an excipient, diluent, carrier, and/or adjuvant that are useful in preparing a pharmaceutical composition that are safe, non-toxic and neither biologically nor otherwise undesirable, and include an excipient, diluent, carrier, and adjuvant that are acceptable for veterinary use and/or human pharmaceutical use. "A pharmaceutically acceptable excipient. diluent, carrier and/or adjuvant" as used herein can include one and more such excipients, diluents, carriers, and adjuvants.

[00159] Pharmaceutical composition can also be included, or packaged, with other non-toxic compounds, such as pharmaceutically acceptable carriers, excipients, diluents, binders and fillers including, but not limited to, glucose, lactose, gum acacia, gelatin, mannitol, xanthan gum, locust bean gum. galactose, oligosaccharides and/or polysaccharides, starch paste, magnesium trisilicate, talc, com starch, starch fragments, keratin, colloidal silica, potato starch, urea, dextrans, dextrins, and the like. For example, the pharmaceutically acceptable carriers, excipients, binders, and fillers for use in the practice of the invention are those which render the compounds of the invention amenable to intranasal delivery, oral delivery, parenteral delivery, intravitreal delivery, intraocular delivery, ocular delivery, subretinal delivery, intrathecal delivery, intravenous delivery, subcutaneous delivery, transcutaneous delivery, intracutaneous delivery, intracranial delivery, topical delivery' and the like. Moreover, the packaging material can be biologically inert or lack bioactivity, such as plastic polymers or silicone, and can be processed internally by the subject without affecting the effectiveness of the composition/formulation packaged and/or delivered therewith.

[00160] In embodiments, the pharmaceutical compositions can comprise pharmaceutically acceptable salts. Pharmaceutically acceptable salts can include, but are not limited to, amine salts, such as but not limited to N,N'-dibenzylethylenediamine, chloroprocaine, choline, ammonia, diethanolamine and other hydroxyalkylamines, ethylenediamine, N- methylglucamine, procaine, N-benzy 1 phenethyl amine, l -para-chlorobenz l-2-pyrrolidin- l '- ylmethylbenzimidazole, diethylamineand other alkylamines, piperazine and tris(hydroxymethyl) aminomethane; alkali metal salts, such as but not limited to lithium, potassium and sodium; alkali earth metal salts, such as but not limited to barium, calcium and magnesium; transition metal salts, such as but not limited to zinc; and other metal salts, such as but not limited to sodium hydrogen phosphate and disodium phosphate; and also including, but not limited to, salts of mineral acids, such as but not limited to hydrochlorides and sulfates; and salts of organic acids, such as but not limited to acetates, lactates, malates, tartrates, citrates, ascorbates, succinates, butyrates, valerates and fumarates.

[00161] Different forms of the pharmaceutical composition can be calibrated in order to adapt both to different subjects and to the different needs of a single subject. However, the pharmaceutical composition need not counter every cause in every subject. Rather, by countering the necessary causes, the pharmaceutical composition will restore the body to its normal function. Then the body will correct the remaining deficiencies. [00162] For oral preparations, the pharmaceutical composition can be used alone or in combination with appropriate additives to make tablets, powders, granules or capsules, for example, with conventional additives, such as lactose, mannitol, com starch or potato starch; with binders, such as crystalline cellulose, cellulose derivatives, acacia, com starch or gelatins; with disintegrators, such as com starch, potato starch or sodium carboxymethylcellulose; with lubricants, such as talc or magnesium stearate; and optionally, with diluents, buffering agents, moistening agents, preservatives and flavoring agents.

[00163] Embodiments of the pharmaceutical composition can be formulated into preparations for injection by dissolving, suspending, or emulsifying them in an aqueous or nonaqueous solvent, such as vegetable or other similar oils, synthetic aliphatic acid glycerides, esters of higher aliphatic acids or propylene glycol; and optionally, with conventional additives such as solubilizers, isotonic agents, suspending agents, emulsifying agents, stabilizers and preservatives.

[00164] Embodiments of the composition or pharmaceutical composition can be utilized in aerosol formulation to be administered via inhalation. Embodiments of the composition or pharmaceutical composition can be formulated into pressurized acceptable propellants such as dichlorodifluoromethane, propane, nitrogen and the like.

[00165] Unit dosage forms for oral administration, such as syrups, elixirs, and suspensions, can be provided wherein each dosage unit, for example, teaspoonful, tablespoonful, tablet or suppository, contains a predetermined amount of the composition containing one or more compositions. Similarly, unit dosage forms for injection or intravenous administration can comprise the pharmaceutical composition as a solution in sterile water, normal saline or another pharmaceutically acceptable carrier.

[00166] The term "administering" can refer to introducing a substance into a subject. Any route of administration can be utilized including, for example, intranasal, topical, oral, parenteral, intravitreal, intraocular, ocular, subretinal, intrathecal, intravenous, subcutaneous, transcutaneous, intracutaneous, intracranial and the like administration. For example, “parenteral administration" can refer to administration via injection or infusion. Parenteral administration includes, but is not limited to, subcutaneous administration, intravenous administration, and intramuscular administration. For example, the inhibitor and/or degrader can be administered intranasally, by inhalation, intrapulmonarily, or by injection (e.g., intravenous or subcutaneous).

[00167] In embodiments, "administering" can also refer to providing a therapeutically effective amount of a formulation or pharmaceutical composition to a subject. The formulation or pharmaceutical compound can be administered alone, but can be administered with other compounds, excipients, fillers, binders, carriers or other vehicles selected based upon the chosen route of administration and standard pharmaceutical practice.

[00168] Administration can be by way of carriers or vehicles, such as injectable solutions, including sterile aqueous or non-aqueous solutions, or saline solutions; creams; lotions; capsules; tablets; granules; pellets; powders; suspensions, emulsions, or microemulsions; patches; micelles; liposomes; vesicles; implants, including microimplants; eye drops; other proteins and peptides; synthetic polymers; microspheres; nanoparticles; and the like.

[00169] In embodiments, the compound can be administered alone, or can be administered as a pharmaceutical composition together with other compounds, excipients, carriers, diluents, fdlers, binders, or other vehicles selected based upon the chosen route of administration and standard pharmaceutical practice. Administration can be by way of carriers or vehicles, such as injectable solutions, including sterile aqueous or non-aqueous solutions, or saline solutions; creams; lotions; capsules; tablets; granules; pellets; powders; suspensions, emulsions, or microemulsions; patches; micelles; liposomes; vesicles; implants, including microimplants; eye drops; other proteins and peptides; synthetic polymers; microspheres; nanoparticles; and the like.

[00170] Embodiments can be administered to a subject in one or more doses. The dose level can vary as a function of the specific composition or pharmaceutical composition administered, the severity of the symptoms and the susceptibility of the subject to side effects. Dosages for a given compound are readily determinable by a variety of means. For example, dosages can be determined by standard clinical techniques. In addition, in vitro or in vivo assays can be employed to help identify optimal dosage ranges. The precise dose to be employed can also depend on the route of administration and can be decided according to the judgment of the practitioner and each patient's circumstances.

[00171] In an embodiment, multiple doses of the pharmaceutical composition can be administered. The frequency of administration and the duration of administration of the pharmaceutical composition can vary depending on any of a variety of factors, e.g., patient response, severity of the symptoms, and the like. For example, in an embodiment, the pharmaceutical composition can be administered once per month, twice per month, three times per month, every other week (qow), once per week (qw), twice per week (biw), three times per week (tiw). four times per week, five times per week, six times per week, every’ other day (qod), daily (ad), twice a day (qid). three times a day (tid), or four times a day. In an embodiment, the pharmaceutical composition can be administered 1 to 4 times a day over a period of time, such as 1 to 10-day time period, or longer than a 10-day period of time.

[00172] In embodiments, the pharmaceutical composition can be administered in combination with one or more additional active agents. For example, a first agent (e.g., a prophylactic or therapeutic agent) can be administered prior to (e.g., 5 minutes, 15 minutes, 30 minutes, 45 minutes. 1 hour. 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks before), concomitantly with, or subsequent to (e.g., 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks after) the administration of a second agent (e.g.. a prophylactic or therapeutic agent) to a subject with a disease or disorder or a symptom thereof.

[00173] Embodiments as described herein further comprise administering one or more additional active agents to a subject together with the SWI/SNF modulator. Non-limiting examples of such additional active agents can comprise a vaccine, an anti-inflammatory agent, cancer therapeutic agents or regimens, (e.g.. a targeted therapy, a chemotherapy, a radiation therapy, surgery, immunotherapy, and/or hormonal therapy), a pain reliever, a steroid, or any combination thereof. In embodiments, a cancer therapeutic agent or regimen can comprise a ty rosine kinase inhibitor. For example, the tyrosine kinase inhibitor can comprise osimertinib, gefitinib, or trametinib. Accordingly, embodiments of the invention comprise administering one or more additional active agents, such as a tyrosine kinase inhibitor, to a subject together with a SWI/SNF modulator.

[00174] In embodiments, the SWI/SNF complex modulator and additional active agent can be administered sequentially, such as one before the other, or concurrently or simultaneously, such as at about the same time. In embodiments, a SWI/SNF complex modulator can be administered together with a tyrosine kinase inhibitor. In embodiments, a SWI/SNF complex modulator can be administered before a tyrosine kinase inhibitor.

[00175] The term "simultaneous administration’⁷ can refer to a first agent and a second agent, when together in the therapeutic combination therapy, are administered less than about 15 minutes, e.g., less than about 10, 5, or 1 minute. For example, the first agent can be a SWI/SNF complex modulator and the second agent can be a ty rosine kinase inhibitor. When the first agent and the second agent are administered simultaneously, the first and second treatments can be in the same composition (e.g., a composition comprising both the first and second therapeutic agents) or separately (e.g., the first therapeutic agent is contained in one composition and the second treatment is contained in another composition). The term “sequential administration⁷’ can refer to a first agent and a second agent administered to a subject greater than about 15 minutes apart, such as greater than about 20, 30, 40, 50, 60 minutes, or greater than 60 minutes apart. Any agent can be administered first. For example, the first agent and the second agent can be included in separate compositions, which can be included in the same or different packages or kits.

[00176] The terms “co-administration” or the like, as used herein, can refer to the administration of a first active agent, such as a SWI/SNF complex modulator, and at least one additional active agent, such as a tyrosine kinase inhibitor, to a single subject, and is intended to include treatment regimens in which the compounds and/or agents are administered by the same or different route of administration, in the same or a different dosage form, and at the same or different time.

[00177] The term “in combination” can refer to the use of more than one therapies (<?.g., one or more prophylactic and/or therapeutic agents). The use of the term “in combination” does not restrict the order in which therapies are administered to a subject with a disease or disorder, or the route of administration.

[00178] “Amplification” can refer to the process of producing multiple copies of a sequence. “Multiple copies” can refer to at least two copies. A “copy” does not necessarily mean perfect sequence complementarity or identity to the template sequence. For example, copies can include nucleotide analogs such as deoxyinosine, intentional sequence alterations (such as sequence alterations introduced through a primer comprising a sequence that is hybridizable, but not complementary, to the template), and/or sequence errors that occur during amplification.

[00179] In embodiments, primary cells can undergo amplification in order to be tested for the impact of ATPase inhibition. In embodiments, the nucleic acid amplification can include polymerase chain reaction (PCR), reverse-transcription PCR, quantitative PCR, real-time PCR, isothermal amplification, linear amplification, or isothermal linear amplification, quantitative fluorescent PCR (QF-PCR), multiplex fluorescent PCR (MF-PCR), single cell PCR, restriction fragment length polymorphism PCR(PCR-RFLP), PCR-RFLP/RT-PCR-RFLP, hot start PCR, nested PCR, in situ colony PCR, in situ rolling circle amplification (RCA), bridge PCR (bPCR), picotiter PCR, digital PCR, droplet digital PCR, or emulsion PCR (emPCR). Other suitable amplification methods include ligase chain reaction (LCR (oligonucleotide ligase amplification (OLA)), transcription amplification, cycling probe technology (CPT), molecular inversion probe (MIP)PCR, self-sustained sequence replication, selective amplification of target polynucleotide sequences, consensus sequence primed polymerase chain reaction (CP-PCR), arbitrarily primed polymerase chain reaction (AP-PCR), transcription mediated amplification (TMA), degenerate oligonucleotide-primed PCR (DOP-PCR), multiple-displacement amplification (MDA), strand displacement amplification (SDA), and nucleic acid based sequence amplification (NABS A).

[00180] The technique of "polymerase chain reaction” or "PCR " a procedure wherein minute amounts of a specific piece of nucleic acid, RNA and/or DNA, are amplified as described, for example, in U.S. Pat. No. 4, 683, 195. In embodiments, sequence information from the ends of the region of interest or beyond is available, such that oligonucleotide primers can be designed; these primers will be identical or similar in sequence to opposite strands of the template to be amplified. The 5' terminal nucleotides of the two primers can coincide with the ends of the amplified material. PCR can be used to amplify specific RNA sequences, specific DNA sequences from total genomic DNA, and cDNA transcribed from total cellular RNA, bacteriophage, or plasmid sequences, etc. See Mullis et al., Cold Spring Harbor Symp. Quant. Biol. 51 : 263 (1987) and Erlich, ed., PCR Technology. (Stockton Press. NY, 1989). As used herein, PCR is one, but not the only, example of a nucleic acid polymerase reaction method for amplify ing a nucleic acid test sample, comprising the use of a known nucleic acid (DNA or RNA) as a primer and utilizes a nucleic acid polymerase to amplify⁷ or generate a specific piece of nucleic acid or to amplify or generate a specific piece of nucleic acid which is complementary to a nucleic acid.

[00181] The term “multiplex-PCR” can refer to a single PCR reaction carried out on nucleic acid obtained from a single source (e.g., an individual) using more than one primer set for the purpose of amplifying two or more DNA sequences in a single reaction.

[00182] "Quantitative real-time polymerase chain reaction" or "qRT-PCR” can refer to a form of PCR wherein the amount of PCR product is measured at each step in a PCR reaction. This technique has been described in various publications including, for example, Cronin et al., Am. J. Pathol. 164 (1): 35-42 (2004) and Ma et al., Cancer Cell 5 : 607-616 (2004).

[00183] In some embodiments, nucleic acid amplification can include digital PCR. It will be appreciated that digital PCR can include any method, process, and/or protocol, using instruments and/or kits associated with performing such, that can discretely amplify and quantitate a nucleic acid(s) w ithin individual partitions of a sample. In some embodiments, the individual partitions for a digital PCR can be generated by a microfluidic process, such as by using a microfluidic device, and/or by a droplet generating process. Generation of individual partitions by a microfluidic process, such as by using a microfluidic device, and/or a droplet generating process to provide a plurality' of partitions in the form of droplets and performing nucleic acid amplification thereon has been described in the art as "droplet digital PCR." The droplets generated for droplet digital PCR can be provided in, for example, a water-in-oil emulsion. In some embodiments, the methods, processes, and/or protocols, and instruments and/or kits for performing nucleic acid amplification on partitions in the form of droplets generated using a microfluidic device/process and/or a droplet generating process, are commercially available, for example, but not limited to, those provided by Bio-Rad, 10X Genomics, Qiagen, and/or ThermoFisher. In an exemplary embodiment, nucleic acid amplification includes droplet digital PCR (ddPCR™) using Bio-Rad's QX100™ or QX200™ Droplet Digital PCR systems, and analysis of nucleic acid amplification products produced by the same, but is not limited thereto.

[00184] "Sequencing", "sequence determination" and the like can refer to biochemical methods that can be used to determine the order of nucleotide bases in a nucleic acid. Targeted sequencing can include the ability to detect complex variation, avoiding clonal errors, and analysis that is less computationally burdensome (e.g., de novo sequencing). There are several embodiments of targeted sequencing.

[00185] The term “targeted sequencing” can refer to efficient sequencing of a small subset of the genome. In clinical settings, sequencing a subset of the genome not only reduce costs, but also focuses on the relevant regions. The main challenge for clinical targeted resequencing methods is obtaining complete and uniform coverage of target regions. Methods for target enrichment rely on lengthy and inefficient hybrid capture or multiplexed PCR techniques, resulting in lower coverage and more off-target sequencing reads.

[00186] The terms “whole genome sequencing.” “full genome sequencing”, “complete genome sequencing”, and “entire genome sequencing” can refer to a laboratory process that determines the complete DNA sequence of an organism's genome at a single time. This entails sequencing of an organism's chromosomal DNA as well as DNA contained in the mitochondria and, for plants, in the chloroplast.

[00187] Aspects of the invention are also drawn to a variety of diagnostic, prognostic, and therapeutic methods utilizing the molecular biomarkers described herein, such as those listed in FIG. 1 and FIG. 3. In any method described herein, such as a diagnostic method, prognostic method, therapeutic method, or combination thereof, the steps of the method can be performed by a single actor or, alternatively, by more than one actor. For example, diagnosis of an EGFR- mutant cancer can be performed directly by the actor providing therapeutic treatment. Alternatively, a person providing a therapeutic agent can request that a diagnostic assay be performed. The diagnostician and/or the therapeutic interventionist can interpret the diagnostic assay results to determine a therapeutic strategy. Similarly, such alternative processes can apply to other assays, such as prognostic assays.

[00188] In embodiments, one or more molecular biomarkers, such as those listed in FIG. 1 or FIG. 3, can provide a “molecular signature” that can be used in a variety of diagnostic, prognostic, and therapeutic methods. For example, the molecular signature can be used to diagnose a cancer. For example, the molecular signature can be used to prognose a cancer. For example, the molecular signature can be used to identify a subject afflicted with a drug-resistant cancer or afflicted with a cancer in need of sensitization. For example, the molecular signature can be used to track the efficacy of a treatment.

[00189] One aspect of the invention is drawn towards screening assays, including non-cell based assays. In one embodiment, the assays provide a method for utilizing at least one of the biomarkers listed in FIG. 1 or FIG. 3, or a molecular signature comprising the same for identifying whether a cancer can respond to cancer therapy and/or whether an agent can inhibit the growth of or kill a cancer cell that is unlikely to respond to cancer therapy. In embodiments, the cancer therapy can be at least one modulator of biomarkers listed in FIG. 1 or FIG. 3.

[00190] In one embodiment, the invention is directed towards assays for screening test agents which bind to, or modulate the biological activity of, at least one biomarker listed in FIG. 1 or FIG. 3. In one embodiment, a method for identifying such an agent entails determining the ability of the agent to modulate, e.g. inhibit, the at least one biomarker listed in FIG. 1 or FIG. 3

[00191] In one embodiment, an assay is a cell-free or cell-based assay, comprising contacting at least one biomarker listed in FIG. 1 or FIG. 3, with a test agent, and determining the ability of the test agent to modulate (e.g. inhibit or enhance) the molecular function (e.g.. enzymatic activity) of the biomarker, such as by measuring direct binding of substrates or by measuring indirect parameters as described herein. In embodiments, the cell-based assay further comprises determining cancer cell proliferation, cancer cell killing, epithelial-to-mesenchymal transition, epithelial cell differentiation, and/or NRF2 signaling. In embodiments, the step of contacting occurs in vivo, ex vivo, or in vitro.

[00192] The invention further pertains to agents identified by the screening assays described herein. Accordingly, it is within the scope of this invention to further use an agent identified as described herein in an appropriate animal model. For example, an agent identified as described herein can be used in an animal model to determine the efficacy, toxicity, or side effects of treatment with such an agent. Alternatively, an antibody identified as described herein can be used in an animal model to determine the mechanism of action of such an agent. [00193] The invention also pertains to the field of predictive medicine in which diagnostic assays, prognostic assays, and monitoring clinical trials are used for prognostic (predictive) purposes to thereby treat an individual prophy tactically. Accordingly, one aspect of the invention tis drawn towards diagnostic assays for determining the presence, absence, amount, and/or activity level of a biomarker described herein, such as those listed in FIG. 1 or FIG. 3, or a molecular signature comprising the same, in the context of a biological sample (e.g., blood, serum, cells, or tissue) to thereby determine whether a subject afflicted with a cancer or at risk for developing a cancer can benefit from increasing sensitivity of the cancer to a tyrosine kinase inhibitor. Such assays can be used for prognostic or predictive purpose to thereby prophylactically treat an individual prior to the onset or after recurrence of a disorder characterized by or associated with biomarker polypeptide, nucleic acid expression or activity. The skilled artisan will appreciate that any method can use one or more (e.g., combinations) of biomarkers described herein, such as those listed in FIG. 1 or FIG. 3. In embodiments, the biological sample can comprise a cancerous or pre-cancerous subject sample.

[00194] Another aspect of the invention pertains to monitoring the influence of agents (e.g., drugs, compounds, and small nucleic acid-based molecules) on the expression or activity' of a biomarker listed in FIG. 1 or FIG. 3, or a molecular signature comprising the same. These and other agents are described in further detail in the following sections.

[00195] An exemplary^ method for identifying a subject afflicted with a cancer or at risk for developing a cancer that can benefit from increasing sensitivity' of the cancer to a tyrosine kinase inhibitor involves obtaining the cancerous or pre-cancerous sample from the subject and contacting the sample with an agent, such as a protein-binding agent like an antibody or antigen-binding fragment thereof, or a nucleic acid-binding agent like an oligonucleotide, can detect the amount or activity' of the biomarker in the sample. In embodiments, the method further comprises determining in a control sample the amount and/or activity of at least one control biomarker; and comparing the amount and/or activity of the at least one biomarker from the cancerous or pre-cancerous subject sample with the amount and/or activity of least one control biomarker from the control sample. In one embodiment, the amount and/or activity of at least one control biomarker is determined from a cancerous, pre-cancerous, or non-cancerous sample from the subject or a member of the same species to which the subject belongs.

[00196] In another embodiment, the presence of or a significant change in the amount and/or activity of the at least one biomarker listed in FIG. 1 or FIG. 3, or a molecular signature comprising the same from the cancerous or pre-cancerous subject sample relative to the amount and/or activity of the at least one control biomarker from the control sample is indicative of the subject having or at risk of developing a cancer that can benefit from increasing sensitivity of the cancer cells to a tyrosine kinase inhibitor.

[00197] In another embodiment, the diagnosis of a subject is followed by recommending, prescribing or administering an agent that modulates the at least one biomarker listed in FIG. 1 or FIG. 3 to a subject. In embodiments, the agent can comprise a SWI/SNF complex modulator.

[00198] In one embodiment, the diagnosis of a subject can be followed by recommending, prescribing or administering at least one cancer therapeutic agent or regimen to the subject. In embodiments, the cancer therapeutic agent or regimen can comprise a targeted therapy, chemotherapy, radiation therapy, surgery, immunotherapy, and/or hormonal therapy. In one embodiment, the cancer therapeutic agent or regimen can comprise a ty rosine kinase inhibitor. For example, the tyrosine kinase inhibitor can comprise osimertinib or gefitinib.

[00199] Another aspect of the invention provides a method for predicting the clinical outcome of a subject afflicted with a cancer or at risk for developing cancer. An exemplary method for predicting the clinical outcome of a subject afflicted with a cancer or at risk for developing a cancer involves determining in a cancerous or pre-cancerous subject sample the amount and/or activity of at least one biomarker listed in FIG. 1 or FIG. 3, or a molecular signature comprising the same. In embodiments, the method further comprises determining the amount and/or activity⁷ of at least one control biomarker having a good clinical outcome and comparing the amount and/or activity of the at least one sample biomarker and the at least one control biomarker. In embodiments, the presence of or a significant change in the amount and/or activity of the at least biomarker listed in FIG. 1 or FIG. 3, or a molecular signature comprising the same from the subject sample relative to the at least one control biomarker indicates that the subject afflicted with the cancer or at risk for developing the cancer has a poor clinical outcome.

[00200] The methods described herein can furthermore be utilized to monitor the progression of a cancer in a subject. In embodiments, the method can comprise detecting in a subject sample at a first point in time the amount and/or activity of at least one sample biomarker listed in FIG. 1 or FIG. 3, or a molecular signature comprising the same. In embodiments, the method further comprises repeating the detecting step at one or more subsequent points in time and comparing the amount and/or activity of the at least one sample biomarker from the subsequent points in time to monitor the progression of the cancer in the subject. In embodiments, the sample can comprise cells, serum, peritumoral tissue, and/or intratumoral tissue obtained from the subject. In embodiments, between the first point in time and the subsequent point in time, the subject has undergone treatment, completed treatment, and/or is in remission for the cancer. In embodiments, the first and/or at least one subsequent sample is selected from the group consisting of ex vivo and in vivo samples. In another embodiment, the first and/or at least one subsequent sample is obtained from an animal model of cancer. In a further embodiment, the first and/or at least one subsequent sample can comprise a portion of a single sample or pooled samples obtained from the subject.

[00201] Another aspect of the invention provides a method of assessing the efficacy of an agent for treating a cancer in a subject. An exemplary’ method of assessing the efficacy of an agent for treating a cancer in a subject comprises detecting in a subject sample at a first point in time the amount and/or activity of at least one biomarker listed in FIG. 1 or FIG. 3, or a molecular signature comprising the same and repeating the detecting step at one or more subsequent points in time after administration of the agent. In embodiments, the method further comprises comparing the amount and/or activity of the at least one biomarker from the subsequent points in time, wherein the presence of or a significant change in the amount and/or activity' of the at least one biomarker indicates that the agent treats the cancer in the subject.

[00202] Another aspect of the invention pertains to methods of modulating the expression or activity of one or more biomarkers described herein (e.g., those listed in FIG. 1 or FIG. 3, or a molecular signature comprising the same, and the Examples, or fragments thereof,) for therapeutic purposes. The biomarkers of the invention have been demonstrated to correlate with cancers. Accordingly, the activity’ and/or expression of the biomarker, as well as the interaction between one or more biomarkers or a fragment thereof and its natural binding partner(s) or a fragment(s) thereof, can be modulated in order to treat cancers.

[00203] Modulatory methods of the invention involve contacting a cell with one or more modulators of a biomarker of the invention, including one or more biomarkers of the invention, including one or more biomarkers listed in FIG. 1, FIG. 3, and the Examples, or a fragment thereof or agent that modulates one or more of the activities of biomarker activity associated with the cell. An agent that modulates biomarker activity can be an agent as described herein, such as a nucleic acid or a polypeptide, a naturally-occurring binding partner of the biomarker, an antibody against the biomarker, a combination of antibodies against the biomarker and antibodies against other immune related targets, one or more biomarkers agonist or antagonist, a peptidomimetic of one or more biomarkers agonist or antagonist, one or more biomarkers peptidomimetic, other small molecule, or small RNA directed against or a mimic of one or more biomarkers nucleic acid gene expression product.

[00204] An agent that modulates the expression of one or more biomarkers of the invention, including one or more biomarkers of the invention, including one or more biomarkers listed in FIG. 1, FIG. 3, and the Examples, or a fragment thereof is, e.g.. an antisense nucleic acid molecule, RNAi molecule. shRNA, mature miRNA, pre-miRNA. pri-miRNA, miRNA*. anti- miRNA, or a miRNA binding site, or a variant thereof, or other small RNA molecule, triplex oligonucleotide, ribozyme, or recombinant vector for expression of one or more biomarkers polypeptide. For example, an oligonucleotide complementary to the area around one or more biomarkers polypeptide translation initiation site can be synthesized. One or more antisense oligonucleotides can be added to cell media at 200 pg/ml, or administered to a patient to prevent the synthesis of one or more biomarkers polypeptide. The antisense oligonucleotide is taken up by cells and hybridizes to one or more biomarkers mRNA to prevent translation. Alternatively, an oligonucleotide which binds double-stranded DNA to form a triplex construct to prevent DNA unwinding and transcription can be used. As a result, synthesis of biomarker polypeptide is blocked. When biomarker expression is modulated, such modulation occurs by a means other than by knocking out the biomarker gene.

[00205] Agents which modulate expression, by virtue of the fact that they control the amount of biomarker in a cell, also modulate the total amount of biomarker activity in a cell.

[00206] In one embodiment, the agent stimulates one or more activities of one or more biomarkers of the invention, including one or more biomarkers listed in FIG. 1, FIG. 3, and the Examples or a fragment thereof. Examples of such stimulatory' agents include active biomarker polypeptide or a fragment thereof and a nucleic acid molecule encoding the biomarker or a fragment thereof that has been introduced into the cell (e.g., cDNA, mRNA, shRNAs, siRNAs, small RNAs, mature miRNA, pre-miRNA, pri-miRNA, miRNA*, anti- miRNA, or a miRNA binding site, or a variant thereof, or other functionally equivalent molecule known to a skilled artisan). In another embodiment, the agent inhibits one or more biomarker activities. In one embodiment, the agent inhibits or enhances the interaction of the biomarker with its natural binding partner(s). Examples of such inhibitory agents include antisense nucleic acid molecules, anti-biomarker antibodies, biomarker inhibitors, and compounds identified in the screening assays described herein.

[00207] These modulatory methods can be performed in vitro (e.g., by contacting the cell with the agent) or, alternatively, by contacting an agent with cells in vivo (e.g.. by administering the agent to a subject). As such, the invention provides methods of treating an individual afflicted with a condition or disorder that can benefit from up- or down-modulation of one or more biomarkers of the invention listed in FIG. 1, FIG. 3, and the Examples or a fragment thereof, e.g., a disorder characterized by unwanted, insufficient, or aberrant expression or activity of the biomarker or fragments thereof. In one embodiment, the method involves administering an agent (e.g., an agent identified by a screening assay described herein), or combination of agents that modulates (e.g., upregulates or downregulates) biomarker expression or activity'. In another embodiment, the method involves administering one or more biomarkers polypeptide or nucleic acid molecule as therapy to compensate for reduced, aberrant, or unwanted biomarker expression or activity⁷.

[00208] Stimulation of biomarker activity is desirable in situations in which the biomarker is abnormally downregulated and/or in which increased biomarker activity can have a beneficial effect. Likewise, inhibition of biomarker activity is desirable in situations in which biomarker is abnormally upregulated and/or in which decreased biomarker activity can have a beneficial effect.

[00209] In addition, these modulatory agents can also be administered in combination therapy with, e.g., chemotherapeutic agents, hormones, anti-angiogens, radiolabels, compounds, or with surgery, cry otherapy, and/or radiotherapy. The preceding treatment methods can be administered in conjunction with other forms of conventional therapy (e.g., standard-of-care treatments for cancer well-known to the skilled artisan), consecutively with, pre- or post-conventional therapy. For example, these modulatory agents can be administered with a therapeutically effective dose of chemotherapeutic agent. In another embodiment, these modulatory⁷ agents are administered in conjunction with chemotherapy to enhance the activity' and efficacy of the chemotherapeutic agent. The Physicians’ Desk Reference (PDR) discloses dosages of chemotherapeutic agents that have been used in the treatment of various cancers. The dosing regimen and dosages of these aforementioned chemotherapeutic drugs that are therapeutically effective will depend on the melanoma, being treated, the extent of the disease and other factors familiar to the physician of skill in the art and can be determined by the physician.

[00210] Aspects of the invention are also directed towards kits, such as kits comprising compositions as described herein. For example, the kit can comprise therapeutic combination compositions described herein.

[00211] In one embodiment, the kit includes (a) an agent, such as a SWI/SNF complex modulator described herein, and optionally (b) informational material. The informational material can be descriptive, instructional, marketing or other material that is drawn to the methods described herein and/or the use of the agents for therapeutic benefit.

[00212] In an embodiment, the kit includes two or more agents. For example, the kit includes a container comprising a SWI/SNF complex modulator, and a second container comprising a second active agent. In embodiments, the second active ingredient can comprise a tyrosine kinase inhibitor. For example, the tyrosine kinase inhibitor can comprise osimertinib or gefitinib.

[00213] In embodiments, the kit further comprises a third container comprising a third active agent.

[00214] The informational material of the kits is not limited in its form. In one embodiment, the informational material can include information about production of the compound, molecular weight of the compound, concentration, date of expiration, batch or production site information, and so forth. In one embodiment, the informational material comprises methods of administering the therapeutic combination composition, e.g., in a suitable dose, dosage form, or mode of administration (e.g., a dose, dosage form, or mode of administration described herein), to treat a subject who has cancer). The information can be provided in a variety of formats, include printed text, computer readable material, video recording, or audio recording, or information that provides a link or address to substantive material.

[00215] The composition in the kit can include other ingredients, such as a solvent or buffer, a stabilizer, or a preservative. The SWI/SNF complex modulator can be provided in any form, e.g., liquid, dried or lyophilized form, or for example, substantially pure and/or sterile. When the agents are provided in a liquid solution, the liquid solution is an aqueous solution. When the agents are provided as a dried form, reconstitution can be by the addition of a suitable solvent. The solvent, e.g., sterile water or buffer, can optionally be provided in the kit.

[00216] The kit can include one or more containers for the composition or compositions containing the agents. In some embodiments, the kit contains separate containers, dividers or compartments for the composition and informational material. For example, the composition can be contained in a bottle, vial, or syringe, and the informational material can be contained in a plastic sleeve or packet. In other embodiments, the separate elements of the kit are contained within a single, undivided container. For example, the composition is contained in a bottle, vial or syringe that has attached thereto the informational material in the form of a label. In some embodiments, the kit includes a plurality (e.g., a pack) of individual containers, each containing one or more unit dosage forms (e.g., a dosage form described herein) of the agents. The containers can include a combination unit dosage, e.g., in a given ratio. For example, the kit includes a plurality of syringes, ampules, foil packets, blister packs, or medical devices, e.g., each containing a single combination unit dose. The containers of the kits can be airtight, waterproof (e.g., impermeable to changes in moisture or evaporation), and/or light-tight. The kit optionally includes a device suitable for administration of the composition, e.g., a syringe or other suitable delivery' device. The device can be provided pre-loaded with one or both of the agents or can be empty, but suitable for loading.

[00217] Other Embodiments

[00218] While the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

[00219] The invention will be further described in the following examples, which do not limit the scope of the invention described in the claims.

EXAMPLES

[00220] Examples are provided herein to facilitate a more complete understanding of the invention. The following examples illustrate the exemplary' modes of making and practicing the invention. However, the scope of the invention is not limited to specific embodiments disclosed in these Examples, which are for purposes of illustration only, since alternative methods can be utilized to obtain similar results.

EXAMPLE 1

[00221] Mammalian SWI/SNF chromatin remodeling complexes promote tyrosine kinase inhibitor resistance in EGFR-mutant lung cancer

[00222] Summary

[00223] Acquired resistance to tyrosine kinase inhibitors (TKI). such as osimertinib used to treat EGF7?-mutant lung adenocarcinomas, limits long-term efficacy and is frequently caused by non-mutational mechanisms. Here, we define the chromatin accessibility and gene regulatory signatures (e.g., a molecular signature) of osimertinib sensitive and resistant EGFR- mutant cell and patient-derived models and uncover a role for mammalian SWI/SNF chromatin remodeling complexes in TKI resistance. By profiling mSWI/SNF genome-wide localization in these cell line pairs, we identify both common and cancer cell line-specific gene targets underlying the resistant state. Importantly, genetic and pharmacologic disruption of the SMARCA4/SMARCA2 mSWI/SNF ATPases re-sensitizes a subset of resistant cell lines and an in vivo tumor model to osimertinib via inhibition of mSWI/SNF-mediated regulation of cellular programs governing cell proliferation, epithelial-to-mesenchymal transition, epithelial cell differentiation, and NRF2 signaling. These data highlight the role of mSWI/SNF complexes in supporting TKI resistance and indicate utility of mSWI/SNF inhibitors in TKI- resistant lung cancers.

[00224] Introduction

[00225] Over the past two decades, targeted therapies such as tyrosine kinase inhibitors (TKIs) directed against mutant or hyperactive tyrosine kinases, have transformed clinical management across a range of cancer types, bringing precision medicine to the forefront of modem oncology. More than 70 different targeted therapies have been approved by the FDA (Food and Drug Administration) with utility across a broad range of cancers, from lung cancers, chronic myelogenous leukemia (CML), melanoma and others ¹’⁵. Indeed, decreases in lung cancer mortality⁷ observed in recent years are, in part, attributed to the use of targeted therapies used to treat specific subsets of oncogene-driven lung adenocarcinomas ⁶ such as epidermal growth factor receptor (EGFR) -mutant lung adenocarcinomas ⁷. Mutations in exons encoding the tyrosine kinase domain of EGFR account for -15% of lung adenocarcinomas in the US, -40-50% in East Asians and 14-51% in Latin America ⁸'¹⁰. Tumors harboring most EGFR mutations respond to TKIs which have been approved for the first-line treatment of the disease and have significantly improved outcomes for patients ⁿ.

[00226] Despite their undeniable efficacy, targeted therapies face a maj or drawback: the emergence of acquired resistance which significantly limits their long-term efficacy and curative potential ¹². Although individual targeted therapies act on different targets, the mechanisms by which tumor cells become resistant are often shared. Mutations in the gene encoding the drug target (e.g. kinase) or point mutations and copy number variations (CNVs) in genes encoding pathway members that bypass the target oncogene are amongst the most common mechanisms of resistance¹³. However, in a significant fraction of resistant tumors, the mechanism of resistance cannot readily be identified, making the clinical management of these tumors a challenge. These issues are apparent in EGFA-mutant lung adenocarcinomas that develop acquired resistance to TKIs, including the third-generation TKI osimertinib ¹⁴, often used as the first-line therapy for EGAZ?-mutant lung cancer ¹⁵. In contrast to tumors treated with earlier-generation TKIs, the proportion of tumors that develop on-target EGFR mutations at acquired resistance is lower with osimertinib ^16,17 Emerging evidence instead reveals that tumors with off-target resistance mechanisms are not only more common, but they also have worse outcomes on TKI treatment compared to those with on-target mutations ¹⁸. For these reasons, understanding the cellular processes underpinning the emergence of off-target mechanisms of resistance is critical to identify actionable alterations and offer alternative therapeutic approaches for an increasingly large patient population.

[00227] Epigenetic processes can mediate resistance to targeted therapies, especially in tumors lacking clear mutational mechanisms of resistance across different cancer types which in turn, then exhibit vulnerabilities to inhibitors of such epigenetic processes ¹⁹. For example, the histone lysine methyltransferase EZH2 has been shown to play a role in the neuroendocrine differentiation of both prostate and lung cancer cells, leading to resistance to anti-androgens and TKIs, respectively ^20,21. Underscoring the therapeutic relevance of this pathway, EZH2 inhibition overcomes resistance to anti-androgens in castration resistant prostate cancer ²² Additionally, in EGAA-mutant lung cancer, the histone demethylase KDM5A was found to be upregulated in cells that persist upon TKI treatment (drug-tolerant persisters) and KDM5A inhibition suppressed the growth of these cells ^23,24 Overall, however, the exact mechanisms and requirements for epigenetic reprogramming to occur and how epigenetic processes contribute to resistance are poorly understood.

[00228] Mammalian SWI/SNF complexes (or BAF complexes) modulate chromatin architecture by altering DNA-nucleosome contacts and enabling chromatin accessibility ^{2 28}. These complexes are 11 -15-subunit entities pieced together from the products of 29 genes into three major forms: cBAF, PBAF, and ncBAF, each demarcated by unique subunits ^25,29 Two ATPases, SMARCA4 and SMARCA2 hydrolyze ATP to power mSWI/SNF complexes. SMARCA4 is mutated in ~8% ofNSCLC, the majority' of which are truncating/ early frameshift mutations resulting in a loss of protein. Importantly these mutations occur in a mutually exclusive manner with EGFR mutations or AL K rearrangements raising the possibility that mSWI/SNF complexes can play a different role in these molecular subsets of lung cancer^30,31. [00229] Importantly, an emerging body of evidence implicates SMARCA4 in tumor maintenance and oncogenicity' ³²‘³⁸, findings which have prompted the development of highly specific, potent small molecule inhibitors targeting the activity of mSWI/SNF complexes, which are currently being evaluated in Phase I clinical trials ³⁹’ ⁴¹(NCTO4879O17, NCT04891757). For example, overexpression of SMARCA4 is associated with more aggressive prostate tumors that underwent neuroendocrine differentiation on therapy ³³. Targeting mSWI/SNF ATPases has been shown to be an effective anti-tumor strategy for castration-resistant prostate tumors in mice that rely on SMARCA2/4 for activation of oncogenic gene expression programs via interactions with lineage-specific transcription factors (TFs) ⁴² Like prostate cancer, EGFR mutant lung adenocarcinomas can also undergo neuroendocrine differentiation following exposure to targeted agents. Altogether, these findings raise the intriguing possibility that SMARCA4 can play a tumor supportive role in EGFR-driven lung cancer.

[00230] Here, we discover that mSWI/SNF complexes, and specifically the ATPase activity' supported by the SMARCA4/A2 subunits, play pivotal roles in mediating the osimertinib-resistance gene regulatory profile in AGAA-mutant lung adenocarcinoma. Genetic and pharmacologic inhibition of mSWI/SNF complexes in cells and in vivo results in improved osimertinib efficacy via, in part, reversal of the resistance profile, thus presenting mSWI/SNF complexes as new therapeutic vulnerabilities in TKI-resistant lung cancers (e.g., osimertinib- resistant lung cancers).

[00231] Results

[00232] Unique chromatin accessibility changes underpin osimertinib-resistant gene expression programs in EGFR-mutant lung cancer cells [00233] To investigate mechanisms of resistance to osimertinib, we generated five pairs of parental and osimertinib-resistant /T/7’7 -mutant lung cancer cell lines using increasing concentrations of osimertinib for PC9, Hl 975 and HCC827 cell lines, or continuous exposure to osimertinib in PC9* and HCC4006 cell lines. (FIG. 1, panel A). To broaden our scope beyond osimertinib, we included the HCC827* cell line treated with increasing doses of gefitinib (HCC827* and HCC827GR6 cell line pair), as generated previously ⁴³. Parental cell lines exhibited EC50s to osimertinib of lOnM or less while their resistant counterparts showed >90-fold higher EC50s (FIG. 1, panels B-C, Table 1). Exome sequencing studies performed on osimertinib-resistant cell lines did not reveal additional mutations in EGFR. Instead, we detected a previously-described loss of CIC in the H1975-OR cells (via an R422* mutation) (FIG. 6, panel A) and an epithelial to mesenchymal transition (EMT) profile in the HCC827- OR and HCC4006-OR cells (FIG. 6, panels B-C) ^44,45. RAFI amplification was detected in the PC9-OR cells (FIG. 6, panel D), but neither knock-dow n of RAFI nor trametinib (MEKi) treatment attenuated cell line proliferation in combination with osimertinib (FIG. 6, panels E- I) ⁴⁶. Similarly, PC9-OR* cells acquired a clinically relevant BRAF G469A TKI resistance mutation ⁴⁷ which was not detected in PC9 parental cells, but PC9-OR* cells remained resistant to the combination of osimertinib and trametinib, indicating a resistance mechanism independent of MAPK pathway reactivation (FIG. 6, panel J). Of note, HCC827GR6 cells are characterized by a MET amplification⁴³. Taken together, these data indicate that coding gene mutations do not play a key role in mediating TKI-resistance in these cell lines which rather can be due to non-mutational mechanisms.

[00234]

[00235] We next performed RNA-seq on the cell line pairs to define the gene regulatory profiles of parental and resistant cells. We identified differentially up- and down-regulated genes, many of which w ere shared between at least 2 cell lines (FIG. 1, panel D; FIG. 6, panels K-L). Clusters 3 and 8 contained genes downregulated and upregulated, respectively, in each of the five osimertimb- but not gefitinib-resistant states, including genes such as MMP2. and FGF1 as downregulated, and ZEB2, ATF3, ETS-1 and FYN as upregulated. Of note, we identified many genes that were differentially up- and down- regulated uniquely in one resistant cell line, underscoring the heterogeneity and complexity of the resistant state in different lines (FIG. 1, panel D; FIG. 6, panel L). Furthermore, pathways such as epithelial to mesenchymal transition and inflammatory response were upregulated whereas MYC targets, interferon alpha/gamma response signaling pathways were downregulated in more than one cell line (FIG. 1, panel E)

[00236] We next performed ATAC-seq across four of the cell line pairs described herein, including PC9*, HCC4006, HCC827* and PC9 as they represent a diverse set of resistance- associated transcriptional programs (FIG. 1, panel F). We identified differentially-accessible chromatin regions which characterize resistant cells amongst the cell line pairs (FIG. 1, panel F). Integrating these data w ith RNA-seq data, we found that >50% of gene expression changes occurred in a concordant manner with DNA accessibility changes at or near gene promoters or enhancers (FIG. 1, panels G-H; FIG. 6, panel M) Examination of these ’primary’ gene targets revealed key gene sets/pathways that are hallmarks of the resistant states, including those pertaining to the upregulation of epithelial to mesenchymal transition (EMT) and RTK signaling pathways, and downregulation of epithelial cell differentiation and cell-cell adhesion (FIG. 6, panel N). Taken together, these findings establish that chromatin accessibility changes between resistant and parental states in A'G7’7 -mutant NSCLC cancer cell lines underlie the non-mutational gene regulatory' programs that characterize osimertinib- (and gefitinib-) resistance.

[00237] Mammalian SWI/SNF complexes are upstream regulators of osimertinib resistance and target to a subset of accessible regions [00238] Given our findings that changes in chromatin accessibility underlie a subset of key gene regulatory features of the TKI-resistant state, we next sought to predict potential chromatin-associated regulators that can govern these changes. Intriguingly, across cell line pairs, Ingenuity Pathway Analysis (IP A) performed on differential gene expression profiles (RNA-seq) in the osimertinib-resistant (OR) (or gefitinib-resistant) states revealed SMARC A4, the ATPase subunit of mSWI/SNF complexes, as the top hit (FIG. 2, panel A). Other top hits included TFs such as TP63, STAT3, and SOX2, several of which are tethered to chromatin by mSWI/SNF complexes ⁴⁸‘⁴⁹. as well as ARID 1 A, the defining subunit of the canonical BAF (cBAF) mSWI/SNF subcomplex ²³ (FIG. 2, panel A). Beta-catenin (CTNNB) was also within the top genes, and has been previously implicated in TKI resistance ⁵⁰. SMARCA4 expression levels did not account for it emerging as the top hit. as we did not detect differences in mRNA or protein levels between the parental and resistant states (FIG. 7, panels A-B).

[00239] To understand how SMARCA4 regulates gene expression, we next profiled the occupancy of mSWI/SNF complexes genome-wide across the PC9*/OR*, HCC4006/OR, and HCC827*/GR6 cell lines using CUT&RUN (FIG. 2, panel B; FIG. 7, panels C-D). We integrated these data with ATAC-seq profiles and identified a subset of sites that were gained or lost specifically in the resistant state (FIG. 2, panel C; FIG. 7, panels E-F). Importantly, gained and lost sites were largely promoter-distal in nature, consistent with the positioning of cBAF complexes at distal enhancer sites ²⁵, and were enriched over API -family motifs (FIG. 2, panels B-C; FIG. 7, panels G-H) We then ranked differentially-expressed genes that exhibited differential mSWI/SNF binding as well as concordant changes in accessibility in the resistant state (FIG. 2, panel D; FIG. 7, panels I-J) Genes corresponding to EMT (e.g., TWIST! . and ZEP2). cell migration (MMP13. BMP4, COL4A1) and RTK signaling were upregulated, while genes encoding members of epithelial cell differentiation and signaling (i.e. FGFR1/2, JAG1. and NOTCH 1) were down regulated in the resistant state. Expression of genes involved in MAPK signaling (MAP2K6, MAPK1, MAP4K1, I)IISP6. SPRY4 etc) were altered in a cell-line specific manner (FIG. 2, panel D; FIG. 7, panel J). Concordant changes in mSWI/SNF occupancy, accessibility’, and gene expression between the parental and resistant states are exemplified at the ETV1 and TWIST1 loci (FIG. 2, panel E). These findings point toward a key role for mSWI/SNF complex activity' at resistance associated gene loci across TKI-resistant A’G77 -mutant cell lines.

[00240] Pharmacologic inhibition of mSWI/SNF ATPase activity reverses the osimertinib-resistant state in a subset of /:G/7 -mutant lung cancer cell lines [00241] Recently, several small molecule inhibitors and degraders targeting the ATPase components of mSWI/SNF (BAF) complexes. SMARCA2/A4 . have been developed and have even entered the clinic in Phase I clinical trials in oncology (i.e. FHD-286 in NCT04879017. NCT04891757) ³⁹'⁴¹. We therefore next sought to investigate the impact of mSWI/SNF pharmacologic inhibition on the resistance associated chromatin and gene regulatory signature using a specific, allosteric SMARCA4/2 ATPase inhibitor, Compound 14 (Cmpl4) ³⁹ To complement SMARCA4 knockdown experiments in selected cell line pairs, we used the PC9*, HCC4006*, and HCC827* cell line systems for these studies. Cell viability assays performed with Cmpl4 alone across parental-resistant cell line pairs revealed modest effects (FIG. 8, panel A). To assess whether BAF inhibition can sensitize resistant cell lines to osimertinib treatment, we measured drug synergy using combenefit assays (FIG. 3, panel A; see Methods). Of note, previous work from our group implicated ERK reactivation as a major determinant of EGFR TKI treatment failure, which can be circumvented by co-treatment with a MEK inhibitor ^{51 52} As such, since pERK was reactivated upon Cmpl4 and osimertinib treatment in PC9-OR* cells at later timepoints (FIG. 8, panel B). PC9* and PC9-OR* cells were treated with both osimertinib and trametinib (OT) in the presence or absence of Cmpl4 for combenefit assays (FIG. 3, panel A; FIG. 8, panel C). Conversely, osimertinib alone was used for HCC827GR6 cells because the addition of Cmpl4 was sufficient to dampen the pERKl/2 hyperactivation observed with osimertinib treatment alone (FIG. 8, panel D).

[00242] Importantly, combenefit assays revealed substantial synergy between OT and Cmpl4 (as compared to without Cmpl4), specific to PC9-OR* cells (FIG. 3, panel A). We also observ ed striking synergy⁷ in HCC827GR6 cells, but not HCC4006-OR cells (FIG. 8, panel E). The addition of Cmpl4 to OT augmented cellular apoptosis in PC9-OR* as compared to OT alone but was unsuccessful at eliciting further degrees of apoptosis in HCC827GR6 (FIG. 3, panel B; FIG. 8, panels F-G). This difference in mechanism is attributable to the MET amplification in HCC827GR6 cells that can bypass apoptotic signaling, as previously reported ^4?. These combination treatment experiments indicate that mSWI/SNF ATPase inhibitors can re-sensitize a subset of resistant cell lines to TKIs.

[00243] To define the transcriptional and chromatin accessibility changes underlying the observed drug synergy in PC9-OR* cells, we next performed RNA-seq and ATAC-seq experiments (FIG. 3, panel C). Of note, protein levels for mSWI/SNF complex subunits, SMARCA4 and SMARCC1, were unaltered across conditions (FIG. 8, panel H). Clustering analyses performed on differentially regulated genes between DMSO, OT. and OT+Cmpl4 (Cmpl4 alone did not have an effect on cell viability so was not included in this analysis) revealed four key sets of genes (FIG. 3, panel D; FIG. 8, panels I-J). Cluster 1 (Cl) included genes that switched from downregulated in no treatment or OT conditions to strongly upregulated upon inclusion of Cmpl4 (‘Up switch’). Cluster 2 (C2) contained genes that were activated upon OT treatment but strongly downregulated upon 0T+Cmpl4 (‘Inverse Response’), Cluster 3 (C3) included genes for which expression was mildly reduced by OT treatment (relative to control) but strongly reduced by 0T+Cmpl4 treatment (‘Enhanced down’), and Cluster 4 (C4) included genes that were on in both control and OT treatment conditions but strongly downregulated upon combination with Cmpl4 (‘Down switch’) (Figure 3, panel D). Notably, integration of these data with ATAC-seq generated for each treatment condition, revealed that the chromatin accessibility of loci (largely TSS-distal sites) corresponding to genes in these four clusters correlated with gene expression changes (FIG. 3, panel D; FIG. 8, panel K).

[00244] We next characterized the transcriptional responses across the four clusters (C 1 - C4) of Cmpl4 synergy genes, highlighting both 'primary' targets (those with concordant chromatin accessibility’ (AT AC) changes), and ‘secondary’ targets (those lacking concordant changes in accessibility) (FIG. 3, panel E). Examples of genes that were altered or reversed (in the downregulated direction) by Cmpl4 treatment included those involved in cell cycle and apoptosis, cell migration and adhesion, metabolic processes, and nuclear receptor pathwayfactors. including NRF2 signaling and metabolism of toxins, exemplified by genes such as EPHA4, RAC 2. CDC25C, and GPX2 (FIG. 3, panels D-F). Upregulated genes (Cl) included genes involved in processes such as protein kinase activity, immune signaling, and nuclear receptors such as ATF3, and CYP1B1 (FIG. 3, panels E-F). Intriguingly, of the 8344 sites exhibiting reductions in accessibility upon OT+Cmpl4 (but not OT alone), we found that over 40% contained a BAF complex peak in the PC9-OR* TKI-resistant setting (FIG. 3, panel G, top). Further, a subset of these sites (green circle) mapped to genes that were selectively upregulated in the resistant state (purple circle) but downregulated only upon combined OT+Cmpl4 treatment (magenta circle) (FIG. 3, panel G, bottom). This is exemplified at the CES1 locus at which we observed heightened BAF complex occupancy and accessibility in the OR* (resistant) setting relative to the parental cell context that is substantially reduced upon OT+Cmpl4 treatment (FIG. 3, panel H). Notably, CES1 is a critical NRF2-regulated enzyme which mediates xenobiotic metabolism ⁵³ and has previously been linked to chemotherapy resistance in hepatocellular carcinoma ⁵⁴ Our results indicate that BAF inhibition can resensitize a subset of osimertinib resistant cells in part via antagonizing NRF2 signaling. [00245] Finally, we aimed to identify whether genes that were uniquely up- or down- regulated in the resistant state (i.e. PC9-OR* versus PC9*) can be reversed selectively in the 0T+Cmpl 4 combination treatment setting relative to OT only. Excitingly, we identified n=60 genes for which expression was down regulated in the resistant state, unaltered by OT alone, but reversed in expression (upregulated) upon OT+Cmpl4 treatment, such as TGI-A. GDF15, and NDRG1 (FIG. 3, panel I). In parallel, we identified n=76 genes with the opposite patern for which resistance-associated activation was reversed selectively in the 0T+Cmpl4 combination treatment, such as COL4A1, MAP2K6, and EPHX1 (FIG. 3, panel I; FIG. 8, panels L-M). In parallel, we defined a collection of genes sensitive to OT treatment in PC9* cells (up- or down-regulated genes), which failed to respond in a similar manner in PC9-OR* cells. Among these genes, we found that the combination of Cmpl4+OT in PC9-OR* cells was able to reverse their expression and ‘re-sensitize’ cells as observed in the parental state via cell proliferation processes and cytokine production pathways (FIG. 3, panel J; FIG. 8, panel N). These transcriptional programs were broadly consistent with those observed upon SMARCA4 knockdown and osimertinib treatment.

[00246] Overall, these results demonstrate that the Cmp 14 mSWI/SNF ATPase inhibitor can synergize with OT to re-sensitize resistant PC9-OR* cells to TKI treatment in part via rewiring chromatin accessibility to reverse a portion of the transcriptional programs underlying the drug resistant state.

[00247] Attenuation of reactive oxygen species by SMARCA4 correlates with resensitization to osimertinib of resistant lung cancer cell lines

[00248] We next sought to identify potential markers or hallmark signatures of EGFR- mutant cell lines re-sensitized to osimertinib by SMARCA4/2 inhibition or SMARCA4 knockdown. We thus analyzed the differences in the resistant state DEGs between the cell lines that were re-sensitized to osimertinib and those that were not following SMARCA4 KD or pharmacologic inhibition. This revealed the differences between the PC9-OR (sensitizing) cell line and the H1975-OR and HCC827-OR (non-sensitizing) cell lines as well as between the PC9-OR* (sensitizing) cell line and the HCC4006-OR (non-sensitizing) cell lines (FIG. 4, panels A-B). GSEA pathway analysis identified that the sensitized cell lines, PC9-OR and PC9-OR*, both enriched for pathways such as PIK3 signaling and Reactive Oxygen Species pathways relative to their non-sensitized cell lines (FIG. 4, panel B). Each sensitized cell line also exhibited specific positive or negative enrichment for Interferon a/y signaling, indicating cell line specific pathway function (FIG. 4, panel B). In parallel, to identify gene targets which can potentiate the re-sensitization response to osimertinib or OT treatment in PC9-OR and PC9- OR* cells, we performed a differential gene analysis which enriches for DEGs specifically upregulated and downregulated in PC9-OR* and PC9-OR cells as compared to HCC4006-OR, Hl 975-OR or HCC827-OR DEGs (FIG. 4, panel C). While these cell line specific differential gene sets were enriched for their respective pathways (FIG. 9, panel A), we overlapped upregulated and downregulated differential genes to identify common regulators which can underpin or contribute to the re-sensitization of PC9-OR* and PC9-OR cells to osimertinib/OT upon SMARCA4 inhibition or KD (FIG. 9, panel B). Many terms are associated with MAPK signaling and detoxification (FIG. 9, panel C) and include upregulated genes MAPK CRKL, CES1, as well as downregulated genes PLK3 and ARNT2 (FIG. 4, panel D; FIG. 9, panel D). [00249] We next determined the underlying factors governing BAF-mediated gene expression changes correlating with accessibility changes upon SMARCA4 inhibition or knockout in PC9-OR* cells, PC9-OR cells and YU-005C cells (FIG. 4, panels E-F; FIG. 9, panels E-F). At sites with reduced accessibility nearby Cmpl4 synergy' genes in PC9-OR* cells, we found motifs corresponding to AP- 1 factors and NRF2 as putative candidates involved in the synergy response (FIG. 4, panel E) (gained sites can reflect indirect effects (FIG. 9, panel E)). Similarly, motif analysis of sites losing accessibility upon SMARCA4 KD and osimertinib treatment in PC9-OR and YU-005C cells also revealed NRF2 as a candidate factor (gained accessible sites represent indirect effects) (FIG. 4, panel F; FIG. 9, panel F).

[00250] The NRF2 signaling pathway is responsible for scavenging ROS through the activation of antioxidant response elements under oxidative stress conditions ⁵⁵. Therefore, to validate the association between SMARCA4/2-mediated osimertinib resensitization and reactive oxygen species and detoxification genes and pathways, we investigated how osimertinib affects the levels of ROS in PC9-OR and YU-005C TKI-resistant cells that rely on SMARCA4 activity for resistance. We measured ROS levels using the fluorescent probe CellROX™ (Thermo Fisher), a fluorogenic probe that exhibits fluorescence upon oxidation by reactive oxygen species. Under osimertinib treatment, the levels of ROS dramatically increased in PC9 cells, which is consistent with a global decrease in NRF2 signaling (FIG. 4, panels G- H; FIG. 9, panel G). In contrast, the amount of baseline ROS was significantly higher in PC9- OR cells and osimertinib did not profoundly affect ROS levels, consistent with active NRF2 signaling (FIG. 4, panels G-H; FIG. 9, panel G). SMARCA4 knock-down in osimertinib- treated PC9-OR cells further increased the levels of ROS w hile decreasing the activity' of the NRF2 pathway, indicating that SMARCA4 plays a key role in ROS neutralization (FIG. 4, panels H-l). Consistent with these observations, the levels of ROS in YU-005C subcutaneous tumors were highest in osimertinib-treated tumors upon SMARCA4 knock-out (FIG. 4, panel

J; FIG. 9, panels H-I)

[00251] We next examined the functional role of NRF2 in the resistant phenotype. In line with our previous observations, NRF2 knock-down reduced the sensitivity of PC9-OR cells to osimertinib by approximately half (FIG. 4, panel K), confirming that NRF2 is an important factor in maintaining resistance. Additionally, we tested if the presence of ROS had a direct effect on how the cells respond to osimertinib. For this, we performed dose-response assays in the presence of a non-toxic concentration of the ROS scavenger N-acetyl-L-cysteine (NAC) ⁵⁶. The incorporation of NAC significantly shifted the EC50 of osimertinib in PC9, PC9-OR and YU-005C cells to a similar extent (FIG. 9, panel J). However, the magnitude of the effect was small and very similar within the three cell lines independently of their prior response to osimertinib, indicating that the accumulation of ROS is a consequence of the toxicity created by the drug in the cells and it is not directly involved in the resistance mechanism. Finally, we examined the levels of NRF2 and SMARCA4 in a tissue microarray of EGFR-m utan t tumors treated with TKIs. The levels of SMARCA4 in these tumors were positively correlated with NRF2 nuclear expression, further supporting that these two proteins can function together to regulate oxidative stress (FIG. 4, panel L). Altogether, these results confirm a pivotal role for SMARCA4 chromatin remodeling activity in controlling the levels of osimertinib-induced oxidative stress in osimertinib resistant cells, viaNRF2 activation.

[00252] Pharmacological inhibition of mSWI/SNF ATPase activity attenuates tumor growth in an osimertinib-resistant PDX mouse model

[00253] We next explored the utility of pharmacological inhibition of mSWI/SNF ATPase activity in the SMARCA4-dependent YU-005 patient-derived model. YU005C cells were resistant to the treatment of Compound 14 (Cmpl4) or FHD-286 (a clinical-stage SMARCA4/SMARCA2 inhibitor alone (FIG. 10, panels A-B). However, both Cmpl4 and FHD-286 sensitized YU005C cells to osimertinib (FIG. 5, panels A-C) consistent with our findings in isogenic cell lines (FIG. 3 and FIG. 8). We, therefore, investigated the potential of combining SMARCA4/2 inhibition with osimertinib in vivo using FHD-286. This four-arm study used n=7 mice per treatment group (FIG. 5, panel D). Tumor bearing mice were treated with vehicle, osimertinib, FHD-286 or the combination of osimertinib and FHD-286 for 18 days. While each drug alone modestly slowed tumor growth, the osimertinib+FHD-286 combination treatment significantly suppressed tumor growth as compared to baseline (FIG.

5, panel E; FIG. 10, panel C). [00254] Discussion

[00255] N on-mutational mechanisms are responsible for TKI resistance in a large fraction of human tumors, yet detailed mechanistic understanding is lacking. In this study, we uncover that mSWI/SNF complexes are retargeted genome-wide in TKI-resistant lung cancer cell lines, thus promoting chromatin accessibility changes which underlie the transcriptional programs that define the resistant state. mSWI/SNF inhibition allowed for a reversal of the resistant state and the resensitization to osimertinib, which promoted cell death in a subset of both cell line and mouse model systems. Together, these data indicate that a subset of osimertinib-resistant tumors can have evolved during treatment to require SMARCA4 for survival in the presence of osimertinib. These findings demonstrate that SMARCA4 can have a pro-growth role in cancer cells, in addition to its well-known role as a tumor suppressor in lung cancer ³⁰’⁵⁷'⁵⁹. Several recent studies have indicated the tumor-promoting roles for SMARCA4 in cancers such as liver ³⁶, prostate ³³-³⁸-⁶⁰, leukemia ³², pancreas ³⁷, sarcomas ^61,62, neuroblastoma ³⁵ and lung ³⁴ In some cases, the role of SMARCA4 is dependent on the differentiation state of the tumors ³⁷ It is well established that mSWI/SNF complexes are involved in several differentiation processes during normal development and cancer ^26,28’⁶³, including during the process of EMT ⁶⁴'⁶⁶. Our results in the EMT-like HCC827-OR cells and HCC4006-OR cells indicate that disruption of mSWI/SNF activity does not affect proliferation or the resistance phenotype in these cells (FIG. 9). However, it is possible that BAF complexes participate in EMT, perhaps underpinning the role for SMARCA4 in regulating the transcriptome of these cells (FIG. 2, panel A). Moreover, SMARCA4 knock-down upon osimertinib treatment, does not have a significant effect on the proliferation of H1975-OR cells, which indicates that mSWI/SNF activity is not the only factor supporting the resistant state. Instead, these cells exhibit CIC loss (FIG. 6, panel B). Similarly, inhibition of BAF in HCC827GR6 cells (gefitinib resistant cells), which harbor MET amplification ⁴³, does not lead to cell death, despite synergizing with osimertinib treatment to attenuate the growth of these gefitinib resistant cells. In these cell lines, it is plausible that SMARCA4 contributes to the drug-tolerant persister states which allow the cells to survive in the presence of osimertinib ⁶⁷ or gefitinib until a genetic alteration or another mechanism leads to overt resistance. Future studies in models that capture the tolerant persister phase will be required to further explore these mechanisms.

[00256] Our studies here indicate that mSWI/SNF complexes target to and act over many genomic sites in the TKI-resistant state. Previous studies from our group and others have indicated that such retargeting can be a result of many mechanisms, alone or in combination, including interactions with the histone landscape ⁶⁸ or binding to transcription factors which target complexes to specific motifs genome wide ⁶⁰’^61,69. Further, it is possible that kinases alter post-translational modifications on the chromatin landscape or on BAF complex subunits themselves which in turn allow or inhibit interactions with other factors such as transcription factors. This is, in part, in agreement with our findings for the AP-1 family of TFs being amongst the most enriched motifs at sites for which BAF occupancy is changed in the resistant state. As the AP-1 family of TFs govern many biological processes deemed hallmarks of cancer, it is not surprising. The interplay between these TFs and other TF effectors of downstream signaling cascades affected by TKIs or BAF inhibition (immune, PI3K, cytokine, MAPK etc), or of bromodomains on SMARCA4 and other mSWI/SNF subunits can be the fine-tuning events that can modulate therapeutic sensitivity.

[00257] In this study we found that chromatin accessibility of genes with NRF2 binding motifs are regulated by mSWI/SNF complexes. Our data indicate that mSWI/SNF increases accessibility at these loci, which facilitates expression of antioxidant response genes and allow s cells to withstand osimertinib-induced oxidative stress and survive (FIG. 5, panel F). In line with these findings, we recently reported that Keapl loss is protective for EGFE-mutant mouse tumors treated with osimertinib ⁷⁰. Finally, other groups have also observed activation of antioxidant programs to counter osimertinib resistance including in drug-tolerant persister cells ⁵⁶-⁷¹. These studies reveal that resistant tumors develop mechanisms to withstand the damaging effects of oxidative stress. Here, we show that the oxidative stress caused by osimertinib is partially attenuated by SMARCA4-mediated chromatin remodeling and transcriptional regulation (FIG. 4). It has been reported that SMARC A4 can physically interact with NRF2 to regulate oxidative stress ⁷² which is consistent with the concordance in the levels of the two proteins that we observed in EGER-mutant tumors (FIG. 4, panel L). Given that SMARCA4 knock-down does not modify NRF2 levels, NRF2 and SMARCA4 can both translocate to the nucleus and cooperate on chromatin to activate expression of antioxidant genes. In contrast to our observations, in KRAS mutant LUAD cell lines and a squamous cell carcinoma cell line, loss of mSWI/SNF chromatin remodeling was found to cause increased NRF2 activity ⁷³. While this further substantiates a link betw een the two pathways, it also highlights how the role of SMARCA4 can be different in different biological contexts (e.g. EGFR vs. KRAS mutant lung cancer). Together these results underscore how' epigenetics can play a role in the regulation of oxidative stress in osimertinib resistant tumors.

[00258] Our finding that EG? E-mutant tumors can rely on SMARC A4 for their survival is consistent with the observation that mutations in SMARCA4 (mostly loss-of-function in lung cancer) and EGFR are mutually exclusive (LUAD/TCGA, PanCancer Atlas, P <0.030 ⁷⁴ NSCLC/GENIE Cohort v9. 1-public P<0.001 ⁷⁵; cbioportal.org) (FIG. 10, panel C) ^76,77 Although occasional cases of SMARCA4 mutations have been reported in EGF7?-mutant tumors ⁷⁸, these events are rare. In support of this, to date, broad studies of osimertinib-resistant tumors have not uncovered SMARCA4 mutations ^l6-'°-⁷⁹-⁸« although it is possible that these occasionally occur if tumors lose dependence on SMARCA4 for survival as they evolve. This mutual exclusivity of mutations in these genes in lung cancer indicates that SMARCA4 function is important for EG E-mutant tumors. Interestingly, a large-scale analysis of SMARCA4 mutations in thousands of solid tumors showed that they are not only mutually exclusive with EGFR alterations in lung adenocarcinomas, but also with other common oncogenic driver alterations in this disease⁸¹, indicating potentially more wide-spanning impact of the findings here. Evaluation of SMARCA4 dependence along with mutational analyses at baseline and resistance will be important across oncogene (mutant kinase)-driven tumors.

[00259] EXPERIMENTAL MODEL AND SUBJECT DETAILS

[00260] Animal Models

[00261] In vivo experiments were performed in NOD.Cg-Prkdc^scld I12rg^tmlw-’¹/SzJ (NSG) mice (Jackson Labs, IMSR_J AX: 005557) Tumors to generate patient-derived xenografts (PDXs) were digested according to the manufacturer instructions (Miltenyi Biotec, Cat# 130- 095-92) prior to subcutaneous injection. Cells were counted, re-suspended in PBS and mixed 1 : 1 with matrigel (Coming, Cat#356237) and injected in the right flanks of the mice. Tumor volume was measured using calipers and calculated with the formula [(Length x Width x Width)/2], For the CRISPR/Cas9 knock-out of SMARCA4 in vivo experiment, cells were transduced with lentiviral particles as described herein, selected with 1 pg/mL puromycin in culture for one week, and 5x 10⁶ cells were injected per mouse. After monitoring tumor growth for 10 days, the diet was changed to doxycycline to activate Cas9 (Envigo, Cat#TD.00426). Treatment was initiated after 7 days on a doxycycline diet. The mice were treated daily with vehicle (5% DMSO + 40% PEG300 + 5%Tween 80 + 50% MilliQ-H2O) or osimertinib (25 mg/kg, Selleckchem. Cat#S7297) by oral gavage. Tumor volume was measured twice a week and the mice were euthanized after 2 weeks of treatment. Six mice per group were used in two independent replicates (n=12) from which: 4 tumors were used to obtain paraffin-embedded tissue, 4 were used for flow cytometry CellROX™ (Thermo Fisher, Cat#C 10422) experiments and 4 were flash frozen to archive at -80 °C.

[00262] In vivo experiments were performed in female NOD.Cg-Prkdc^scld 112rg^tmlw-’^I/SzJ (NSG) mice (Jackson Labs, #005557) YU-005C cells were counted, re-suspended in PBS and mixed 1 :3 with matrigel (Coming, #356237) and 5* 10⁶ cells were injected in the right flank of each mouse. Tumor volume was measured using a caliper and calculated with the formula [(Length x Width x Width)/2], Treatment was initiated 16-days after injection when the tumors reached an average size of ~50 mm³. The mice were treated daily with vehicle (5% DMSO + 40% PEG300 + 5%Tween 80 + 50% MilliQ-H2O), osimertinib (25 mg/kg, Selleckchem, # S7297), FHD-286 (1.5 mg/kg, Jun Qi laboratory), or combination (osimertinib and FHD-286) by oral gavage (n=7 mice per group). Tumor volume was measured twice a w eek and the mice were euthanized 18-days posttreatment initiation.

[00263] Cell Lines

[00264] Isogenic osimertinib-sensitive and resistant cell lines were generated independently and analyses of the lines were integrated as described in the manuscript. PC9. H1975, HCC827 and YU-005C cells were maintained in RPMI 1640 Medium (Thermo Fisher, Cat#A1049101) supplemented with 10% Fetal Bovine Serum (Thermo Fisher, Cat#16140-071) and 1% Penicillin-Streptomycin (Thermo Fisher, Cat#15140122). These cell lines were authenticated using the GenePrint 10 System (Promega. Cat#B9510). Cells were passaged using 0.25% Trypsin with EDTA (Thermo Fisher, Cat#25200056) when necessary and routinely tested for mycoplasma (Lonza, Cat#LT07-118). To generate osimertinib resistant PC9, H1975 and HCC827 cells , sensitive parental cells were sequentially treated with increasing concentrations of osimertinib. The starting concentration was 25 nM and the drugcontaining complete media was changed every 2-3 days. When the surviving cell population exhibited signs of proliferation and reached confluence in 10 cm plates, the cells were transferred to anew plate and the drug concentration was subsequently increased by 25 nM, 50 nM and 100 nM. The process was repeated until EC50 of the cells increased by ~100-fold compared to the sensitive cells. The final concentrations were 1 pM for PC9-OR, 1 pM for HCC827-OR and 2 pM for H1975-OR cells. OR cells were maintained in the respective final concentration of the drug to ensure the survival of the resistant population. PC9*, PC9-OR*, HCC4006, HCC4006-OR, HCC827*, and HCC827GR6 were maintained in RMPI-1640 (Gibco) supplemented with 10% fetal bovine serum (GeminiBio) and 1% penicillinstreptomycin (Gibco). Cell line identity was confirmed for PC-9*, HCC4006, and HCC827* by DNA fingerprinting. PC9-OR* and HCC4006-OR cell lines were generated by continuously culturing the respective parental cell lines in 100 nM osimertinib for at least 2 months before being characterized . Unless stated otherwise in the figure legends, these OR lines were maintained in 1 0 nM osimertinib but the drug was removed for 1 week before use in drug experiments to account for acute effects of treatment. HCC827GR6 w as previously established ⁴³. Cells were routinely tested and confirmed to be mycoplasma negative (ATCC Cat#30- 1012K).

[00265] METHOD DETAILS

[00266] Cell Growth, Viability and Drug Synergy Assays

[00267] For PC9, H1975 and HCC827 cell line pairs and YU-005C, dose-response curves and cell viability assays were performed in 96-well plates in three technical replicates. For dose-response curves, cells were plated at 20-40% confluency and treated for 72 hours with the drugs suspended in DMSO. The media was replaced after the treatment and viability was measured by a fluorescence-based viability⁷ assay (Promega, Cat#G8081). Conditions without drug and 0.1% Triton X-100 were used as 100% and 0% viability controls, respectively. IC50 values and dose-response models were calculated using the following formula in GraphPad Prism software: normalized viability= 100/(1+ [inhibitor]/IC50). For proliferation assays, the viability was measured using the same system at the indicated times. Colony formation assays were performed in 6-well plates in which 1000-3000 cells/well were plated and treated with the specified conditions for 10-14 days. Cells were washed with PBS, fixed with 10% NBF and stained with Crystal Violet solution (5 mg/mL cry stal violet powder, 20% methanol in water). Copy number assays were performed following the manufacturer instructions. The following TaqMan assays from Thermo Fisher were used: EGFR (Cat#Hs02088787_cn & Cat#Hs02190396_cn) and RAFI (Cat#Hs02614899_cn & Cat#Hs04252880_cn). 3-4 biological replicates were performed for in vitro functional experiments as indicated in each specific figure. Quantitative RT-PCRs were performed using the Power SYBR Green Master mix (Thermo Fisher, Cat#4367659) and custom designed primers (https://primer3.ut.ee/. RRID:SCR_003139) in a Viia 7 Real-Time PCR System (Thermo Fisher, RRID:SCR_019582).

[00268] For PC9*, HCC4006 and HCC827* cell line pairs, 500 cells per well were seeded into 384-well plates and 2000 cells per well were seeded into 96-well plates. Cells were drugged the following morning in triplicate. Cell viability was assessed after 72 hours with CellTiter-Glo® Luminescent Cell Viability Assay (Promega) according to manufacturer's instructions. Plates were read using a POLARstar Omega microplate reader (BMG Labtech). Drug synergy was assessed with Combenefit software as previously described ⁸² Cells were drugged in a 6-by-6 drug concentration matrix in triplicate and viability was assessed by CellTiter-Glo® after 72 hours. Bliss synergy scores were calculated for each drug combination and were mapped relative to cell proliferation using Combenefit v2.021.

[00269] Caspase Assays [00270] Apoptosis was measured in real-time using CellEvent™ Caspase- 3/7 Green Ready Probes™ Reagent (Invitrogen) as previously described ⁸³. Briefly, 2000 cells per well were seeded in 96- ell plates. The following morning, media was replaced with CellEvent™ dye and drug containing media. Plates were housed in a BioSpa 8 automated incubator (Agilent) and were scanned at regular intervals using a Cytation5 cell imaging multimode reader (Agilent) or were housed and scanned with an Incucyte S3 (Sartorius). Refer to figure legends for further details on specific experiments. Fluorescent signal was normalized to cellular confluence at each timepoint.

[00271] DNA Sequencing

[00272] Genomic DNA for whole-exome sequencing (WES) or Sanger sequencing of the sensitive and the osimertinib resistant PC9, H1975 and HCC827 cell line pairs was extracted from cells in culture using the DNeasy Blood & Tissue kit (Qiagen, Cat#69504). cDNA used for Sanger sequencing was obtained from RNA using SuperScript III reverse transcriptase (Thermo Fisher, Cat# 18080093) following the manufacturer instructions. Sanger sequencing was performed in the Yale Keck Biotechnology Resource Laboratory following their guidelines (htps://medicine.vale.edu/keck/). ABI sequencing trace files were visualized using the software ApE (htps://jorgensen.biology.utah.edu/wayned/ape/). WES library preparation and Illumina sequencing was performed at the Y ale Center for Genome Analysis (YCGA, htps://medicine.yale.edu/keck/ycga/). One ug of genomic DNA was sheared to a mean fragment length of about 140 base pairs using focused acoustic energy (Covaris E210). Exome sequencing was performed by exome capture using the IDT xGen capture probe panel with an additional "spike-in" of -2,500 regions, totaling -620 kb, of RefGene coding regions that were not included or were poorly covered by the IDT panel. Captured fragments were sequenced using 101 bp paired-end sequencing reads in an Illumina NovaSeq 6000 with an S4 flowcell according to Illumina protocols. Sequencing reads were aligned to human genome build 38 (GRCh38/hg38) using the BWA-MEM, aggregated into a BAM file, and further processed to produce somatic variants with GATK v3.4 and MuTect, following the GATK Best Practices workflow ⁸⁴ Identified variants were further filtered based on their presence in repositories of common variations (1000 Genomes, NHLBI exome variant server, and 2,577 noncancer exomes sequenced at Yale). CNV analysis was based on the read depth ratio differences between tumor and normal using custom scripts.

[00273] Genomic DNA for PC9*, PC9-OR*, HCC4006. and HCC4006-OR was extracted using the DNeasy Blood & Tissue kit (Qiagen, Cat#69504) and was submited for WES at the Broad Institute of MIT and Harvard using the “Express Somatic Human WES v6” workflow. Captured fragments were sequenced on a NovaSeq 6000 with an S4 flow cell according to standard Illumina protocols using 151 bp paired-end sequencing reads and achieved a median target coverage of -200X. Sequencing reads were aligned to human genome build 37 (GRCh37/hgl9), aggregated into a BAM file, and further processed to identify somatic variants with GATK v4.0.4.0, MuTect2, and Oncotator vl.9.8.0. A panel of normal tissue samples were used to filter out germline variants, identify sequencing artifacts, and to serve as a reference for CNV analysis. For follow up studies, RNA was extracted from PC9*, PC9* treated with 100 nM osi for 4 days, and PC9-OR* cells using the RNeasy Plus Mini Kit (Qiagen, Cat#74136) and was reverse transcribed to cDNA using the QuantiTect Reverse Transcription kit (Qiagen, Cat#205313). PCR amplicons encompassing the BRAF G469A site were generated using the Platinum SuperFi II PCR mix (Invitrogen. Cat #12368250). and were submitted for Sanger sequencing at Genewiz according to standard protocols.

[00274] siRNA Design and Expression Vectors

[00275] The siRNAs were designed using i-Score Designer and siRNA Scales ^85,86 and obtained from Sigma-Aldrich. For siRNA-mediated inhibition, cells were plated on unsupplemented RPMI media and transfected with Lipofectamine and 30 nM of siRNA following the manufacturer instructions (Thermo Fisher, Cat# 13778150). The SMARCA4 shRNAs were custom designed (http://katahdin.mssm.edu/siRNA/RNAi. cgi?type=shRNA, ⁸⁷ and cloned into the pINDUCERlO lentiviral construct following the standard protocol ⁸⁸. For NRF2 shRNA inhibition, we used the pLKO. l-puro vector (Addgene_8453; Sigma Millipore) and the empty vector as control. The SMARCA4 sgRNA sequences were previously described ⁸⁹ and cloned into the TLCV2 (Addgene_87360) lentiviral construct following the standard protocol ⁹⁰ The sequences are in Table 2. Lentiviral particles were produced in 293T cells using the pMD2.G (Addgene_12259) and psPAX2 (Addgene_12260) vector system and a DNA transfection reagent optimized for 293T cells following the vendor instructions (Minis, Cat#MIR2704). Doxycycline inducible expression was achieved by adding 1 pg/mL (shRNAs/PC9-OR, sgRNAs/YU-005C) or 2 pg/mL (shRNAs/YU-005C) DMSO-dissolved doxycycline into the culture media and replacing the media every 2-3 days. Knock-down of SMARCA4 with shRNAs was performed for a week prior to treatment in the experiments unless stated otherwise in the figure legends.

[00276] Western Blotting

[00277] Whole cell and nuclear lysates were generated using RIPA lysis buffer (50 mmol/L Tris, pH 8.0, 150 mmol/L NaCl, 5 mmol/L MgC12, 1% Triton X-100, 0.5% sodium deoxy cholate, 0.1% SDS) and no salt EB0 buffer (50mM Tris, 0.1%NP-40, ImM EDTA, ImM MgCh) followed by high salt EB300 buffer (50mM Tris, 1% NP-40, ImM EDTA, ImM MgC12, 300mM NaCl) supplemented with protease and phosphatase inhibitor cocktail (Thermo Fisher, Cat#78440), respectively. Equal amounts of total protein were separated by SDS-PAGE and blots were probed as indicated (antibodies are found in KRT). Signals were detected using SuperSignal West Pico PLUS (Thermo Fisher, Cat#34579) or Femto chemiluminescent substrates (Thermo Fisher, Cat# 34096) or imaged on LI-COR Odyssey CLx.

[00278] IHC and IF

[00279] Subcutaneous tumors were collected, fixed in 4% paraformaldehyde overnight at room temperature (RT), and rehydrated in 70% ethanol. Paraffm-embedding and sectioning w as performed by the Yale Pathology Tissue Services (YPTS). Four-micrometer sections w ere used for hematoxylin and eosin (H&E) staining and immunohistochemistry (IHC) or immunofluorescence (IF) staining using standard protocols. The YTMA-356 preparation has been previously described ⁹¹ and purchased from YPTS. Antigen retrieval was performed by steam heating of slides at 95°C for 30 minutes using citrate-based pH 6.0 (Vector, Cat#H-3300- 250) or Tris-based pH 9.0 (Vector, Cat#H-3301-250) solutions. Biotinylated molecule detection (Vector Cat#PK-4001, Cat#PK-4010) and peroxidase reactions (Vector, Cat#SK- 4800) were performed using the manufacturer protocols. For IF staining, permeabilization was achieved by adding 0.25% Triton X-100 (Sigma Aldrich, Cat#X-100) in PBS for 45 minutes at RT and blocking was performed with 3% BS A + 0.05% Tween 20 in PBS for 1 hour. Nuclear staining was performed by adding Hoechst dye (2 pg/mL, Thermo Fisher, Cat#62249) using the manufacturer's instructions prior to mounting the slides.

[00280] The following antibodies were used for IHC: SMARCA4 (Cat#abl08318; AB 10889900), NRF2 (Cat#abl37550; AB_2687540) and yH2A.X (Cat#2577; AB_2118010, CST). For IF staining of cultured cells, cells were plated in 8-well chambers previously coated with collagen and standard protocols were applied for the stainings. The following antibodies were used for IF staining: CDH1-AF594 (Cat#7687; AB_2797633), VIM-AF488 (Cat#9854; ABJ0829352) and yH2A.X-AF647 (Cat#9720; AB_10692910) from Cell Signaling Technology'. ImageJ was used to quantify the bands of the immunoblots and QuPath ⁹² to quantify IHC and IF staining. Antibodies were used at the dilutions indicated by the manufacturer. Primary and secondary antibody incubations were performed overnight at 4°C and for 1 hour at RT, respectively.

[00281] Drug Treatments for Genomics Assays (ATAC-seq and RNA-seq)

[00282] PC9* and PC9-OR* cells were treated with DMSO, luM Compound 14 (Cmpl4), lOOnM Osimertinib (Osi) and lOOnM Osimertinib + 30nM Trametinib (OT) for 24hrs before harvesting for ATAC-seq and RNA-seq. The PC9-OR* cells were washed to remove continuous Osimertinib growth media for 7 days prior to experimental setup and then re-challenged with osimertinib or OT as appropriate (see FIG. 3 and FIG. 8).

[00283] CellROX™ Oxidative Stress Detection

[00284] CellROX™ Green (Thermo Fisher, Cat#C 10444) and Deep Red (Thermo Fisher, Cat#C10422) were used for IF imaging and flow cytometry quantification respectively. For IF imaging, cells were plated on 8-well collagen-coated plates and treated as specified for 72 hours. The cells were washed with PBS, incubated with 5 pM CellROX™ Green in culture media at concentration for 30 minutes at 37 °C, washed with PBS again and incubated with Hoechst dye (2 ug/mL) for 15 minutes in the dark at RT. The slides were mounted with ProLong™ Gold Antifade Mountant (Thermo Fisher, P10144) and imaged in an inverted fluorescence microscope (Zeiss). The quantification of the IF signal was performed using QuPath ⁹² For in vitro flow cytometry experiments, cells were treated as described in this section in 6-well plates. Cells were trypsinized, collected, resuspended in FACS buffer (2% FBS in PBS) and kept on ice. For in vivo flow cytometry experiments, subcutaneous tumors were extracted, weighed, and digested as previously described. After 3 washes with PBS, tumor cells were suspended in complete RPMI culture media containing 5 pM CellROX™ DeepRed and incubated for 30 minutes at 37 °C. Cells were washed with PBS. resuspended in FACS buffer and kept on ice. Experiments were performed in a BD LSRII flow cytometer and analyzed using FlowJo version 10. Quantification of the MFI for YU-005C in vivo tumors was performed after gating GFP+ (Cas9/sgRNA+) and APC+ (CellROX™ DeepRed+) cells.

[00285] CUT&RUN

[00286] CUT&RUN was performed following EpiCypher’s protocol for their CUT ANA ChIC/CUT&RUN kit with slight modifications to day 1. Prior to wash steps, cells were lysed using a nuclear extraction buffer 20mM HEPES-KOH, pH 7.9, lOmM KC1, 0.1% Triton X- 100 and 20% glycerol, supplemented with fresh 0.5mM spermidine, and cOmplete Mini, EDTA-free protease inhibitor (Sigma). Next, cells were pelleted at 500g for 3 mins. Cell pellets were then washed using protocol’s permeabilization buffer and subsequently incubated with activated Concanavalin A beads at room temperature for 15 mins. Next, beads mixed in Antibody Buffer were incubated with appropriate primary antibodies overnight (antibodies in key resource table). Library amplification was carried out using Epicypher’s CUT&RUN library kit using 12 cycles of amplification. Quality of CUT&RUN libraries were assessed by TapeStation (Agilent). Libraries were sequenced on NextSeq 500 (Illumina) using 37 bp paired-end sequencing.

[00287] ATAC-Seq

[00288] The Omni-ATAC protocol ⁹³ was used with slight modifications as follows. 100,000 cells per condition were used and washed with PBS before lysis. Cell pellets were lysed in 50pl of cold ATAC-seq resuspension buffer containing lOmM Tris-HCl pH 7.4, lOmM NaCl and 3mM MgC12, supplemented with fresh NP-40 (final 0.1% v/v), Tween-20 (final 0.1% v/v) and digitonin (final 0.01% v/v) for 3-5min on ice. Next cells were washed with 1ml of resuspension buffer supplemented with Tween-20 (final 0. 1% v/v) and pelleted at 500g for lOmin at 4 °C. Cell pellets were resuspended in 50pl of transposition reaction mix containing 25pl of 2x TD buffer. 2.5pl of transposase. 16.5 pl of 1 x PBS, 0.5pl of 1% digitonin (final 0.01% v/v), 0.5 pl of 10% Tween-20 (final 0.1% v/v) and 5 pl of nuclease-free water. The transposition reaction was incubated at 37°C for 30 minutes with constant shaking on a thermomixer. The Qiagen MinElute Reaction Cleanup Kit was used for DNA purification. A standard ATAC-seq amplification protocol with 5-7 cycles of amplification was used to amplify the tagmented library⁹⁴. ATAC-seq libraries were sequenced on NovaSeq 6000 (Illumina) using 37-bp paired-end sequencing or performed by YCGA following their standard protocols (https://medicine.yale.edu/keck/vcga/). ATAC-seq experiments were performed in biological duplicates for cell lines and three replicates for YU-005C cells.

[00289] RNA-Seq [00290] One million cells were harvested and washed with cold PBS to remove trypsin and stored in RLT buffer (Qiagen) until further processing. RNA was purified using the Qiagen RNeasy kit (Qiagen). PC9*/OR*, HCC4006/QR and HCC827*/GR6 RNA was further processed using the Illumina NEBNext Ultra II Directional RNA Library' Prep Kit and quality control was assessed by TapeStation (Agilent) and librairies were quantified by Qubit Fluorometer. These RNA-seq libraries were sequenced on the Illumina NextS eq 500 with 75- bp single-end sequencing. Quality control and library preparation for RNA from PC9/OR, H1975/OR and HCC827/OR cell lines was performed at the Yale Center for Genome Analysis (Y CGA) following their standard protocols. Replicates for specific RNA-seq experiments are as indicated in figure legends.

[00291] QUANTIFICATION AND STATISTICAL ANALYSIS

[00292] Enrichment and Statistical Analysis

[00293] The Upstream Regulator & Cellular Functions enrichment analysis were generated using Ingenuity' Pathway Analysis

(https://www.qiagenbioinformatics.com/products/ingenuitv-pathway-analysis). Student t-tests for paired and unpaired samples, depending on the experimental setting, were performed for in vitro experiments in which we performed 3-4 biological replicates. Shapiro-Wilk and Kolmogorov-Smirnov normality' tests were performed for datasets with an n>10. Datasets following a normal distribution were analyzed with a parametric Student t-test and whilst datasets that did not meet normality were analyzed with the non-parametric Mann- Whitney test. Statistical P values for RNA-seq and ATAC-seq data were obtained from the DESeq2 analyses. The Coefficient of Drug Interaction (CDI) was calculated as follows: CDI = AB/(AxB); where AB is % combinatorial effect of osimertinib and SMARCA4 knock-down, A is the % effect of osimertinib alone and B is the % effect of SMARCA4-knock down alone. Other specific tests, different sample sizes as well as depicted values and error bars are indicated in figure legends. Tests were performed in GraphPad Prism 9.1.1.

[00294] Data processing for RNA, ATAC and CUT & RUN samples

[00295] RNA-seq reads were mapped to the hg 19 human genome assembly using STAR v2.3.1 ⁹⁵ with default parameters. Alignment files in BAM format were generated using samtools vO.1.19 ⁹⁶. ATAC-seq paired-end reads were processed as follows: reads were trimmed to 30bp using Trimmomatic v0.35 ⁹⁷, mapped to the hg!9 human genome assembly using Bowtie2 v2.1.0 (-X2000), and filtered for duplicates using Picard MarkDuplicates . Cut&Run reads were processed using the Cut&RunTools pipeline with default parameters ⁹⁸. [00296] RNA-seq gene counts for cell-line data were generated with using STAR (- quantMode GeneCounts, last column of GeneReadsOut.tab) against the hgl9 refFlat annotation. The raw counts were prefiltered to exclude genes with less than one read per sample on average. These prefiltered raw count matrices were converted to RPKMS using a hgl9 refFlat annotation with the median isoform length for gene length and the total gene counts per sample (in millions) for the per-million scaling factor.

[00297] Raw counts for Cut&Run, and ATAC samples across a subset of sites were generated using bedtools intersect with default parameters on coverage bed files. Paired-end reads for Cut&Run and ATAC samples were converted to the appropriate paired-end bed file using samtools view (-h) and a custom perl script to filter the SAM entries. Raw counts were converted to RPKMs using site widths and total million mapped reads from samtools idxstats as the scaling factor.

[00298] Peaks were called using MACS2 v2. 1.0 (-q 0.001) ⁹⁹ with the narrow peak caller for marks in this study and broad peak caller for ATAC. Duplicate reads were excluded using samtools rmdup and used for downstream analyses. ATAC and Cut&Run tracks were generated using deepTools bamCoverage (-normalizeUsing CPM -bs 50 -smoothLength 600 -ignoreDuplicates — samFlaglnclude 64) and RNA tracks were generated using deepTools bamCoverage (— normalizeUsingRPKM).

[00299] CUT & RUN, ATAC-seq and RNA-seq Data Analysis

[00300] SMARCA4, SMARCC 1 , H3K27ac, IgG and ATAC peaks were merged across conditions for each antibody using the default bedtools merge call. Upon peak calling and visualization of IgG negative control samples, IgG signal was negligible and no further processing to account of IgG was needed. Venn diagrams of peak overlaps were generated using the ChIPpeakAnno package in R with the findOverlapsOfPeaks and makeVennDiagram functions with default parameters. Unless otherwise noted, gained and lost ATAC or SMARCA4 Cut&Run sites, and upregulated or downregulated genes across the indicated conditions were determined using edgeR (glmQLFit, log2FC=l, FDR=0.05). Perceived batch effects were modeled as covariates in the analysis if necessary. Volcano plots for changes in expression and accessibility across the indicated conditions were visualized as scatter plots in -loglO(adj.p) vs logFC using matplotlib. Interaction terms were modelled to assess significant differences in accessibility and expression between the parental and resistance state between the indicated cell lines for the quadrant analyses plots. Quadrant analyses were visualized as scatter plots of the logFCs between the parental and resistant state for the indicated cell lines. [00301] Changes in SMARCA4 occupancy across the indicated conditions were determined using logFCs of the log RPKM values (logFC=l). These changes were visualized as a scatterplot of the log RPKM signal in each condition and colored by gains or losses in BAF occupancy using matplotlib. FASTA sequences across these sets of sites were generated using site centers with flanking windows of 200bp (total window size of 400bp). Enriched motifs across these sets of sites were determined using HOMER fmdMotifsGenome.pl ¹⁰⁰ against genome-background (-size 400). HOMER motif known results were visualized as barplots using matplotlib. Unless otherw ise noted, barplots and Venn diagrams were visualized using matplotlib, and heatmaps were created using seaborn clustermap.

[00302] The Metascape web server was used to assess the enrichment of different pathways and gene sets using an input gene list. These gene lists were determined by up and downregulated genes across the indicated conditions, and the overlap of these deregulated genes with coordinated or anti-coordinated changes in accessibility and BAF localization (using genes assigned to changing peaks by nearest distance to TSS) as indicated in the figures. The significance of enriched pathways and gene sets were visualized as horizontal bar charts using the description column of the metascape_results.xlsx files. To elaborate, sites of interest were annotated to their nearest protein-coding gene against the hg!9 refFlat gene annotation and distance to transcription start site (TSS) were calculated with a custom perl script. Gene intersections with gene subsets were visualized as volcano plots of logFC vs -log(adj.p). Gene intersections were input into downstream gene enrichment analysis such as Metascape with default parameters. Gene enrichment results from Metascape were visualized as barplots of - log(p-val) by each enriched term, process, or pathw ay as horizontal bar charts using matplotlib. RPKMs of different genes w ere visualized as bar plots across the indicated conditions using matplotlib or ggplot in R. Different sets of sites were assigned to their nearest protein-coding gene using distances to TSS. These distances were visualized as stacked bar charts using ggplot in R, highlighting the proportion of promoter, promoter proximal, and distal enhancer regions. PCA plots were generated using the quantile-normalized log RPKM signal of the expression and accessibility profiles as input and visualized as scatterplots using seaborn scatterplot. Heatmaps and metaplots were generated for each antibody over subsets of sites using ngs.plot.r with lOkb wdndow-s centered on each site (-G hgl9 -FL 150 -L 5000 -R bed). Heatmaps and metaplots were visualized on the RPM-scale using matplotlib.

[00303] Unless otherwise noted, gene heatmaps were visualized as z-scored RPKMs across the samples using a blue-white-red heatmap in the clustermap function of seaborn. For gene heatmaps with rectangular black boxes within it, the RPKMs were z-scored across the samples within each box separately and horizontally concatenated together to accentuate the different between the indicated conditions and to help visualize the most biologically meaningful differences between the samples. Whenever indicated, k-means clustering was used cluster the genes with similar expression profiles using the scikit-leam k-means clustering utility, and clusters were reordered and colored to present the data more meaningfully.

[00304] The number of common and specific deregulated genes between the parental and resistant state across the six cell lines were visualized as a bar chart using matplotlib (FIG. 6, panel L). The number and proportion of deregulated genes that have coordinated changes in accessibility were visualized as pie charts using matplotlib.

[00305] LogFCs estimated by EdgeR across the indicated conditions (such as parental vs resistant in each cell line) were used to rank genes for input into GSEA in pre-ranked mode (-norm meandiv -nperm 20000 -scoring_scheme weighted -set_ max 500 -setymin 15) against a variety of MSigDB databases, including Hallmark ¹⁰¹, BioCarta, GO Biological Processes, and Wikipathways ¹⁰² databases. GSEA results were visualized as bubble charts of normalized enrichment scores (NES) and FDR values using ggplot in R or as blue-white-red clustered heatmaps of NES values using seaborn clustermap. Differences in logFCs between the parental and resistant state between cell lines were also used as input into GSEA in pre-rank mode using the same parameters and the logFC difference as the ranking metric.

[00306] Changes in the expression profiles characterizing resistance for each cell line were also visualized as an ordered scatter plot (e.g. hockey stick plot) of the logFC between the parental and resistant state and the rank of the logFC for each cell line and colored by coordinated changes in BAF localization and accessibility.

[00307] Gene overlaps between the indicated conditions were visualized as venn diagrams using matplotlib.

[00308] In FIG. 3, panel D, the expression profiles of a subset of deregulated genes as defined in the text were z-scored across the indicated samples (RPKM z-score values) and k- means clustered using scikit-leam (k=4). The largest coordinated change in accessibility for the ATAC peaks assigned to these genes (by nearest distance to TSS analysis) were plotted as a blue-purple-yellow heatmap using seaborn heatmap. Genes with no matched ATAC peak were assigned an ATAC logFC of 0.

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EXAMPLE 2

[00415] Highlights

[00416] Osimertinib resistance is associated with chromatin accessibility changes.

[00417] mSWI/SNF complex targeting and activity govern the resistance signature.

[00418] mSWI/SNF sustains a proliferative and ROS-attenuating profile in resistant cells.

[00419] Genetic and pharmacological inhibition of SMARCA4/2 reverses osimertinib resistance.

[00420] This study finds that mammalian SWI/SNF chromatin remodeling complexes promote osimertinib resistance in EGFE-mutant lung cancer model systems via changes in chromatin accessibility which underlie resistance-associated gene expression and cancer cell proliferation. Genetic and pharmacological inhibition of mSWI/SNF complexes leads to Osimertinib resensitization in a subset of osimertinib-resistant cells and in vivo tumor models.

EQUIVALENTS

[00421] Those skilled in the art will recognize, or be able to ascertain, using no more than routine experimentation, numerous equivalents to the specific substances and procedures described herein. Such equivalents are considered to be within the scope of this invention, and are covered by the following claims.

Claims

What is claimed:

1. A method of sensitizing tyrosine kinase inhibitor (TKI)-resistant cancer cells or tumors in a subject, the method comprising: administering to the subject a therapeutically effective amount of a SWI/SNF complex modulator, thereby sensitizing the cells to the tyrosine kinase inhibitor.

2. The method of claim 1, wherein the TKI is osimertinib, gefitinib, or trametinib.

3. The method of claim 1, wherein the cancer is lung cancer or any cancer with an EGFR mutation.

4. The method of claim 3, wherein the lung cancer comprises non-small cell lung cancer.

5. The method of claim 3, wherein the lung cancer is EGFR-mutant lung cancer.

6. The method of claim 1, wherein the SWI/SNF complex modulator comprises an inhibitor or a degrader.

7. The method of claim 1, wherein the SWI/SNF complex modulator comprises a chromatin modifying agent.

8. The method of claim 1, wherein the SWI/SNF complex modulator comprises a modulator of a cBAF subunit.

9. The method of claim 8, wherein the cBAF subunit comprises ARID1A, ARID1B, DPF2, DPF3, BCL11A, BCL11B or any combination thereof.

10. The method of claim 1, wherein the SWI/SNF complex modulator comprises an ATPase modulator.

11. The method of claim 10, wherein the SWI/SNF complex modulator comprises a SWI/SNF ATPase modulator.

12. The method of claim 11, wherein the SWI/SNF ATPase comprises SMARCA2, SMARCA4, or a combination thereof.

13. The method of claim 1, wherein the SWI/SNF complex modulator comprises a nucleic acid molecule, a small molecule, a peptide, or a polypeptide.

14. The method of claim 13, wherein the small molecule comprises a structure according to:

Formula I, or a derivative or analog thereof.

15. The method of claim 14, wherein R1 is Cl, or F.

16. The method of claim 13, wherein the small molecule comprises:

- Il l - or a derivative or analog thereof.

17. The method of claim 13. wherein the polypeptide comprises an antibody or antigen binding fragment thereof.

18. The method of claim 17. wherein the antibody is murine, chimeric, humanized, mosaic, composite, or human.

19. The method of claim 1, wherein the SWI/SNF complex modulator comprises a degrader directed to the SWI/SNF complex, a nucleic acid molecule targeting the SWI/SNF complex, a compound or prodrug thereof that binds to the SWI/SNF complex, or a pharmaceutically acceptable salt or ester of said compound or prodrug.

20. The method of claim 1, wherein the SWI/SNF complex comprises canonical BAF (cBAF), polybromo-associated BAF (PBAF) or non-canonical BAF (ncBAF).

21. The method of claim 1, wherein sensitization is indicated by reducing cancer cell proliferation, inducing cancer cell killing, inhibiting epithelial-to-mesenchymal transition, inhibiting epithelial cell differentiation, and/or modulating NRF2 signaling which exceeds that observed in cancer cells not treated with the SWI/SNF complex modulator.

22. A method of treating a subject afflicted with a tyrosine kinase inhibitor (TKI)resistant cancer, the method comprising: administering to the subject a SWI/SNF complex modulator to sensitize cells of the TKI-resistant cancer to a tyrosine kinase inhibitor; and administering to the subject a tyrosine kinase inhibitor.

23. The method of claim 22, wherein sensitization is indicated by the amount and/or activity of at least one sample biomarker listed in Figure 1 or Figure 3.

24. A method of identifying a subject afflicted with a cancer or at risk for developing a cancer that would benefit from increasing sensitivity of the cancer to a tyrosine kinase inhibitor, the method comprising: determining in a cancerous or pre-cancerous subject sample the amount and/or activity of at least one biomarker listed in Figure 1 or Figure 3; wherein the amount and/or activity of at least one biomarker listed in Figure 1 or Figure 3 is indicative of a subject afflicted with cancer or at risk for developing cancer that would benefit from increasing sensitivity of the cancer to a ty rosine kinase inhibitor.

25. The method of claim 24, further comprising obtaining the cancerous or pre-cancerous sample from the subject.

26. The method of claim 24, further comprising determining in a control sample the amount and/or activity of at least one control biomarker; and comparing the amount and/or activity' of the at least one biomarker from the cancerous or pre-cancerous subject sample with the amount and/or activity of least one control biomarker from the control sample.

27. The method of claim 26, wherein the presence of or a significant change in the amount and/or activity of the at least one biomarker from the cancerous or precancerous subject sample relative to the amount and/or activity of the at least one control biomarker from the control sample is indicative of the subject having or at risk of developing a cancer that would benefit from increasing sensitivity of the cancer cells to a tyrosine kinase inhibitor.

28. The method of claim 24 or claim 26, further comprising recommending, prescribing, or administering an agent that modulates the at least one biomarker listed in Figure 1 or Figure 3.

29. The method of claim 28. wherein the agent comprises a SWI/SNF complex modulator.

30. The method of 24 or claim 26, further comprising administering at least one additional cancer therapeutic agent or regimen.

31. The method of claim 30, wherein the at least one additional cancer therapeutic agent or regimen comprises a targeted therapy, chemotherapy, radiation therapy, surgery', immunotherapy, and/or hormonal therapy.

32. The method of claim 30, wherein the at least one additional cancer therapeutic agent or regimen comprises a ty rosine kinase inhibitor.

33. The method of claim 32, wherein the tyrosine kinase inhibitor comprises osimertinib, gefitinib, or trametinib.

34. The method of claim 26, wherein the amount and/or activity' of at least one control biomarker is determined from a cancerous, pre-cancerous, or non-cancerous sample from either the subject or a member of the same species to which the subject belongs.

35. The method of claim 24 or 26, wherein the cancer is a tyrosine kinase inhibitor (TKI)- resistant cancer or is at risk of developing resistance to a tyrosine kinase inhibitor.

36. A method for predicting the clinical outcome of a subject afflicted with a cancer or at risk for developing cancer, the method comprising: determining in a cancerous or pre-cancerous subject sample the amount and/or activity of at least one biomarker listed in Figure 1 or Figure 3; determining the amount and/or activity' of at least one control biomarker having a good clinical outcome; and comparing the amount and/or activity of the at least one sample biomarker and the at least one control biomarker; wherein the presence of or a significant change in the amount and/or activity of the at least biomarker from the subject sample relative to the at least one control biomarker indicates that the subject afflicted with the cancer or at risk for developing the cancer has a poor clinical outcome.

37. A method for monitoring the progression of a cancer in a subject, the method comprising: detecting in a subject sample at a first point in time the amount and/or activity' of at least one sample biomarker listed in Figure 1 or Figure 3; repeating the detecting step at one or more subsequent points in time; and comparing the amount and/or activity⁷ of the at least one sample biomarker from the subsequent points in time to monitor the progression of the cancer in the subject.

38. A method of assessing the efficacy of an agent for treating a cancer in a subject, the method comprising: detecting in a subject sample at a first point in time the amount and/or activity⁷ of at least one biomarker listed in Figure 1 or Figure 3; repeating the detecting step at one or more subsequent points in time after administration of the agent; and comparing the amount and/or activity of the at least one biomarker from the subsequent points in time, wherein the presence of or a significant change in the amount and/or activity of the at least one biomarker indicates that the agent treats the cancer in the subject.

39. The method of either claim 37 or claim 38, wherein between the first point in time and the subsequent point in time, the subject has undergone treatment, completed treatment, and/or is in remission for the cancer.

40. The method of either claim 37 or claim 38, wherein the first and/or at least one subsequent sample is selected from the group consisting of ex vivo and in vivo samples.

41. The method of either claim 37 or claim 38, wherein the first and/or at least one subsequent sample is obtained from an animal model of cancer.

42. The method of either claim 37 or claim 38, wherein the first and/or at least one subsequent sample is a portion of a single sample or pooled samples obtained from the subject.

43. The method of either claim 37 or claim 38, wherein the sample comprises cells, serum, peritumoral tissue, and/or intratumoral tissue obtained from the subject.

44. A cell-based assay for screening for agents that sensitize a cancer cell to a tyrosine kinase inhibitor, comprising contacting the cancer cell with a test agent, and determining the ability⁷ of the test agent to change the amount and/or activity of at least one biomarker listed in Figure 1 or Figure 3.

45. The cell-based assay of claim 44, wherein the step of contacting occurs in vivo, ex vivo, or in vitro.

46. The cell-based assay of claim 44, further comprising determining cancer cell proliferation, cancer cell killing, epithelial-to-mesenchymal transition, epithelial cell differentiation, and/or NRF2 signaling.