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Purpose: In patients with metastatic prostate cancer (mPC), ATM and BRCA2 mutations dictate differences in PARPi inhibitor response and other therapies. We interrogated the molecular features of ATM- and BRCA2-mutated mPC to explain the divergent clinical outcomes and inform future treatment decisions. Experimental design: We examined a novel set of 1,187 mPCs after excluding microsatellite-instable (MSI) tumors. We stratified these based on ATM (n = 88) or BRCA2 (n = 98) mutations. As control groups, mPCs with mutations in 12 other homologous recombination repair (HRR) genes were considered non-BRCA2/ATM HRR-deficient (HRDother, n = 193), whereas lack of any HRR mutations were considered HRR-proficient (HRP; n = 808). Gene expression analyses were performed using Limma. Real-world overall survival was determined from insurance claims data. Results: In noncastrate mPCs, only BRCA2-mutated mPCs exhibited worse clinical outcomes to AR-targeted therapies. In castrate mPCs, both ATM and BRCA2 mutations exhibited worse clinical outcomes to AR-targeted therapies. ATM-mutated mPCs had reduced TP53 mutations and harbored coamplification of 11q13 genes, including CCND1 and genes in the FGF family. BRCA2-mutated tumors showed elevated genomic loss-of-heterozygosity scores and were often tumor mutational burden high. BRCA2-mutated mPCs had upregulation of cell-cycle genes and were enriched in cell-cycle signaling programs. This was distinct from ATM-mutated tumors. Conclusions: Tumoral ATM and BRCA2 mutations are associated with differential clinical outcomes when patients are stratified by treatments, including hormonal or taxane therapies. ATM- and BRCA2-mutated tumors exhibited differences in co-occurring molecular features. These unique molecular features may inform therapeutic decisions and development of novel therapies.

Chimeric antigen receptor (CAR) T-cell therapy can lead to dramatic clinical responses in B-cell malignancies. However, early clinical trials with CAR T-cell therapy in non–B-cell malignancies have been disappointing to date, suggesting that tumor-intrinsic features contribute to resistance. To investigate tumor-intrinsic modes of resistance, we performed genome scale CRISPR-Cas9 screens in mesothelin (MSLN)-expressing pancreatic cancer cells. Co-culture with MSLN-targeting CAR T cells identified both antigen-dependent and antigen-independent modes of resistance. In particular, loss of the majority of the genes involved in the pathway responsible for GPI-anchor biosynthesis and attachment abrogated the ability of CAR T cells to target pancreatic cancer cells, suggesting that disruption of this pathway may permit MSLN CAR T-cell evasion in the clinic. Antigen-independent mediators of CAR T-cell response included members of the death receptor pathway as well as genes that regulate tumor transcriptional responses, including TFAP4 and INTS12. TFAP4-mediated CAR T resistance depended on the NFκB transcription factor p65, indicating that tumor resistance to CAR T-cell therapy likely involves alterations in tumor-intrinsic states. Overall, this study uncovers multiple antigen-dependent and -independent mechanisms of CAR T-cell evasion by pancreatic cancer, paving the way for overcoming resistance in this disease that is notoriously refractory to immunotherapy.Significance:The identification and validation of key determinants of CAR T-cell response in pancreatic cancer provide insights into the landscape of tumor cell intrinsic resistance mechanisms and into approaches to improve therapeutic efficacy.

Tumor mutation burden (muts/Mb) of ATM and BRCA2 mutant mPCs.

Comparisons of WTS profiles between ATMm and BRCA2m to the HRP group.

AR activities and NEPC signatures in ATM- and BRCA2- mutated tumors.

Overall survival of ATM, BRCA2 mutant, and HRDother mPCs.

Composition and rates of all detectable genomic features detectable through the Caris diagnostic platform.

Purpose:In patients with metastatic prostate cancer (mPC), ATM and BRCA2 mutations dictate differences in PARPi inhibitor response and other therapies. We interrogated the molecular features of ATM- and BRCA2-mutated mPC to explain the divergent clinical outcomes and inform future treatment decisions.Experimental design:We examined a novel set of 1,187 mPCs after excluding microsatellite-instable (MSI) tumors. We stratified these based on ATM (n = 88) or BRCA2 (n = 98) mutations. As control groups, mPCs with mutations in 12 other homologous recombination repair (HRR) genes were considered non-BRCA2/ATM HRR-deficient (HRDother, n = 193), whereas lack of any HRR mutations were considered HRR-proficient (HRP; n = 808). Gene expression analyses were performed using Limma. Real-world overall survival was determined from insurance claims data.Results:In noncastrate mPCs, only BRCA2-mutated mPCs exhibited worse clinical outcomes to AR-targeted therapies. In castrate mPCs, both ATM and BRCA2 mutations exhibited worse clinical outcomes to AR-targeted therapies. ATM-mutated mPCs had reduced TP53 mutations and harbored coamplification of 11q13 genes, including CCND1 and genes in the FGF family. BRCA2-mutated tumors showed elevated genomic loss-of-heterozygosity scores and were often tumor mutational burden high. BRCA2-mutated mPCs had upregulation of cell-cycle genes and were enriched in cell-cycle signaling programs. This was distinct from ATM-mutated tumors.Conclusions:Tumoral ATM and BRCA2 mutations are associated with differential clinical outcomes when patients are stratified by treatments, including hormonal or taxane therapies. ATM- and BRCA2-mutated tumors exhibited differences in co-occurring molecular features. These unique molecular features may inform therapeutic decisions and development of novel therapies.

Supplemental figures 1-6

expression of Hallmark IFNγ/IFNα gene sets in HupT3 cells following TFAP4 knockout and overexpression

Raw data for follow-up xenograft studies. Note different tabs for each xenograft type.

Malignant rhabdoid tumors (MRT) are highly aggressive pediatric cancers that respond poorly to current therapies. In this study, we screened several MRT cell lines with large-scale RNAi, CRISPR-Cas9, and small-molecule libraries to identify potential drug targets specific for these cancers. We discovered MDM2 and MDM4, the canonical negative regulators of p53, as significant vulnerabilities. Using two compounds currently in clinical development, idasanutlin (MDM2-specific) and ATSP-7041 (MDM2/4-dual), we show that MRT cells were more sensitive than other p53 wild-type cancer cell lines to inhibition of MDM2 alone as well as dual inhibition of MDM2/4. These compounds caused significant upregulation of the p53 pathway in MRT cells, and sensitivity was ablated by CRISPR-Cas9–mediated inactivation of TP53. We show that loss of SMARCB1, a subunit of the SWI/SNF (BAF) complex mutated in nearly all MRTs, sensitized cells to MDM2 and MDM2/4 inhibition by enhancing p53-mediated apoptosis. Both MDM2 and MDM2/4 inhibition slowed MRT xenograft growth in vivo, with a 5-day idasanutlin pulse causing marked regression of all xenografts, including durable complete responses in 50% of mice. Together, these studies identify a genetic connection between mutations in the SWI/SNF chromatin-remodeling complex and the tumor suppressor gene TP53 and provide preclinical evidence to support the targeting of MDM2 and MDM4 in this often-fatal pediatric cancer.Significance:This study identifies two targets, MDM2 and MDM4, as vulnerabilities in a deadly pediatric cancer and provides preclinical evidence that compounds inhibiting these proteins have therapeutic potential.

Purpose:Rhabdoid tumors are devastating pediatric cancers in need of improved therapies. We sought to identify small molecules that exhibit in vitro and in vivo efficacy against preclinical models of rhabdoid tumor.Experimental Design:We screened eight rhabdoid tumor cell lines with 481 small molecules and compared their sensitivity with that of 879 other cancer cell lines. Genome-scale CRISPR–Cas9 inactivation screens in rhabdoid tumors were analyzed to confirm target vulnerabilities. Gene expression and CRISPR–Cas9 data were queried across cell lines and primary rhabdoid tumors to discover biomarkers of small-molecule sensitivity. Molecular correlates were validated by manipulating gene expression. Subcutaneous rhabdoid tumor xenografts were treated with the most effective drug to confirm in vitro results.Results:Small-molecule screening identified the protein-translation inhibitor homoharringtonine (HHT), an FDA-approved treatment for chronic myelogenous leukemia (CML), as the sole drug to which all rhabdoid tumor cell lines were selectively sensitive. Validation studies confirmed the sensitivity of rhabdoid tumor to HHT was comparable with that of CML cell lines. Low expression of the antiapoptotic gene BCL2L1, which encodes Bcl-XL, was the strongest predictor of HHT sensitivity, and HHT treatment consistently depleted Mcl-1, the synthetic-lethal antiapoptotic partner of Bcl-XL. Rhabdoid tumor cell lines and primary-tumor samples expressed low BCL2L1, and overexpression of BCL2L1 induced resistance to HHT in rhabdoid tumor cells. Furthermore, HHT treatment inhibited rhabdoid tumor cell line and patient-derived xenograft growth in vivo.Conclusions:Rhabdoid tumor cell lines and xenografts are highly sensitive to HHT, at least partially due to their low expression of BCL2L1. HHT may have therapeutic potential against rhabdoid tumors.

Expanded methodological detail on large-scale data analysis (RNAi, CRISPR-Cas9, p53 status annotations, RNA-seq, and primary tumor expression).

S1: Sources, media, and other information on all cell lines. S2: p53 status annotations for all cell lines. S3: sgRNA sequences for TP53 inactivation. S4: Primers for TP53 allele screening and RT-qPCR. S5: Sources, catalog numbers, and dilutions of all antibodies. S6: RNA-seq data for p53 target genes. S7: GSEA from RNA-seq data.

Combined supplementary figures with accompanying legends.

S1: RNAi data and CRISPR-Cas9 data divided by p53 status. S2: Expression of MDM2 and MDM4 in MRT and control cell lines. S3: CRISPR-Cas9 mediated inactivation of TP53 in MRT cell lines. S4: Immunoblots for p53 pathway activation upon MDM2 and MDM2/4 inhibition in MRT and control cell lines. S5: Cell counts, flow cytometry, and senescence assays in MRT cells following MDM2 and MDM2/4 inhibition. S6: Characterization of MRT cells expressing SMARCB1 or p16. S7: MRT xenograft growth characteristics and pharmacodynamic responses.

Chimeric antigen receptor (CAR) T-cell therapy can lead to dramatic clinical responses in B-cell malignancies. However, early clinical trials with CAR T-cell therapy in non–B-cell malignancies have been disappointing to date, suggesting that tumor-intrinsic features contribute to resistance. To investigate tumor-intrinsic modes of resistance, we performed genome scale CRISPR-Cas9 screens in mesothelin (MSLN)-expressing pancreatic cancer cells. Co-culture with MSLN-targeting CAR T cells identified both antigen-dependent and antigen-independent modes of resistance. In particular, loss of the majority of the genes involved in the pathway responsible for GPI-anchor biosynthesis and attachment abrogated the ability of CAR T cells to target pancreatic cancer cells, suggesting that disruption of this pathway may permit MSLN CAR T-cell evasion in the clinic. Antigen-independent mediators of CAR T-cell response included members of the death receptor pathway as well as genes that regulate tumor transcriptional responses, including TFAP4 and INTS12. TFAP4-mediated CAR T resistance depended on the NFκB transcription factor p65, indicating that tumor resistance to CAR T-cell therapy likely involves alterations in tumor-intrinsic states. Overall, this study uncovers multiple antigen-dependent and -independent mechanisms of CAR T-cell evasion by pancreatic cancer, paving the way for overcoming resistance in this disease that is notoriously refractory to immunotherapy.Significance:The identification and validation of key determinants of CAR T-cell response in pancreatic cancer provide insights into the landscape of tumor cell intrinsic resistance mechanisms and into approaches to improve therapeutic efficacy.

Chimeric antigen receptor (CAR) T cell therapy can lead to dramatic clinical responses in B cell malignancies. However, early clinical trials with CAR T cell therapy in non-B cell malignancies have been disappointing to date, suggesting that tumor intrinsic features contribute to resistance. To investigate tumor intrinsic modes of resistance, we performed genome scale CRISPR-Cas9 screens in mesothelin (MSLN)-expressing pancreatic cancer cells. Co-culture with MSLN-targeting CAR T cells identified both antigen-dependent and antigen-independent modes of resistance. In particular, loss of the majority of the genes involved in the pathway responsible for GPI-anchor biosynthesis and attachment abrogated the ability of CAR T cells to target pancreatic cancer cells, suggesting that disruption of this pathway may permit MSLN CAR T cell evasion in the clinic. Antigen independent mediators of CAR T cell response included members of the death receptor pathway as well as genes that regulate tumor transcriptional responses, including TFAP4 and INTS12. TFAP4-mediated CAR T resistance depended on the NFκB transcription factor p65, indicating that tumor resistance to CAR T cell therapy likely involves alterations in tumor intrinsic states. Overall, this study uncovers multiple antigen-dependent and -independent mechanisms of CAR T cell evasion by pancreatic cancer, paving the way for overcoming resistance in this disease that is notoriously refractory to immunotherapy.

The determination of molecular features that mediate clinically aggressive phenotypes in prostate cancer remains a major biological and clinical challenge1,2. Recent advances in interpretability of machine learning models as applied to biomedical problems may enable discovery and prediction in clinical cancer genomics3–5. Here we developed P-NET—a biologically informed deep learning model—to stratify patients with prostate cancer by treatment-resistance state and evaluate molecular drivers of treatment resistance for therapeutic targeting through complete model interpretability. We demonstrate that P-NET can predict cancer state using molecular data with a performance that is superior to other modelling approaches. Moreover, the biological interpretability within P-NET revealed established and novel molecularly altered candidates, such as MDM4 and FGFR1, which were implicated in predicting advanced disease and validated in vitro. Broadly, biologically informed fully interpretable neural networks enable preclinical discovery and clinical prediction in prostate cancer and may have general applicability across cancer types. A biologically informed, interpretable deep learning model has been developed to evaluate molecular drivers of resistance to cancer treatment, predict clinical outcomes and guide hypotheses on disease progression.

Metastatic castration-resistant prostate cancers (mCRPCs) are treated with therapies that antagonize the androgen receptor (AR). Nearly all patients develop resistance to AR-targeted therapies (ARTs). Our previous work identified CREB5 as an upregulated target gene in human mCRPC that promoted resistance to all clinically approved ART. The mechanisms by which CREB5 promotes progression of mCRPC or other cancers remains elusive. Integrating ChIP-seq and rapid immunoprecipitation and mass spectroscopy of endogenous proteins, we report that cells overexpressing CREB5 demonstrate extensive reprogramming of nuclear protein–protein interactions in response to the ART agent enzalutamide. Specifically, CREB5 physically interacts with AR, the pioneering actor FOXA1, and other known co-factors of AR and FOXA1 at transcription regulatory elements recently found to be active in mCRPC patients. We identified a subset of CREB5/FOXA1 co-interacting nuclear factors that have critical functions for AR transcription (GRHL2, HOXB13) while others (TBX3, NFIC) regulated cell viability and ART resistance and were amplified or overexpressed in mCRPC. Upon examining the nuclear protein interactions and the impact of CREB5 expression on the mCRPC patient transcriptome, we found that CREB5 was associated with Wnt signaling and epithelial to mesenchymal transitions, implicating these pathways in CREB5/FOXA1-mediated ART resistance. Overall, these observations define the molecular interactions among CREB5, FOXA1, and pathways that promote ART resistance.

Metastatic castration resistant prostate cancers (mCRPC) are treated with therapies that antagonize the androgen receptor (AR). Nearly all patients develop resistance to AR-targeted therapies (ART). Our previous work identified CREB5 as an upregulated target gene in human mCRPC that promoted resistance to all clinically-approved ART. The mechanisms by which CREB5 promotes progression of mCRPC or other cancers remains elusive. Integrating ChIP-seq and rapid immunoprecipitation and mass spectroscopy of endogenous proteins (RIME), we report that cells overexpressing CREB5 demonstrate extensive reprogramming of nuclear protein-protein interactions in response to the ART agent enzalutamide. Specifically, CREB5 physically interacts with AR, the pioneering actor FOXA1, and other known co-factors of AR and FOXA1 at transcription regulatory elements recently found to be active in mCRPC patients. We identified a subset of CREB5/FOXA1 co-interacting nuclear factors that have critical functions for AR transcription (GRHL2, HOXB13) while others (TBX3, NFIC) regulated cell viability and ART resistance and were amplified or overexpressed in mCRPC. Upon examining the nuclear protein interactions and the impact of CREB5 expression on the mCRPC patient transcriptome, we found CREB5 was associated with TGFβ and Wnt signaling and epithelial to mesenchymal transitions, implicating these pathways in ART resistance. Overall, these observations define the molecular interactions among CREB5, FOXA1, and pathways that promote ART resistance.

Bladder carcinoma has a 6% 5-year survival-rate for metastatic disease, with poorly understood links between genetic and environmental drivers of disease development, progression, and treatment response. Rhode Island has among the highest annual age-adjusted incidence rate of bladder cancer at 26.0/100,000, compared to 20.0 in the US, with a paucity of known driver genes for targeted therapies or predictive biomarkers. Bladder carcinomas have the highest frequency of alterations in CDKN1A/p21(WAF1) (10%) across all cancer types analyzed in The Cancer Genome Atlas (TCGA) PanCancer Atlas Studies, displaying a predominance of truncating mutations (86%). We found that lung carcinomas lack CDKN1A truncating mutations, despite the shared role of tobacco as a risk factor for bladder cancer. Bladder carcinomas also have the highest frequency of RB1 alterations in TCGA (25%). We find that chromophobe renal cell carcinomas with CDKN1A and RB1 mutations are 100% truncating. Analysis of 1,868 bladder tumors demonstrated that truncating CDKN1A mutations co-occur with truncating RB1 mutations, suggesting an environmental exposure signature. Moreover, we found that HRNR and FLG mutations are enriched in tumors with CDKN1A alteration, suggesting potential novel roles in promoting bladder tumorigenesis. Association of HRNR with AKT activation offers possible therapeutic avenues, and FLG may provide insight into carcinogen exposure within the bladder. We suggest that because APOBEC mutations largely shape the bladder cancer mutational landscape, these events likely contribute to dysfunctional DNA repair genes, leading to frameshifts and the predominance of truncations in CDKN1A, RB1, ARID1A, or other drivers. We propose that patients with co-occurrence of CDKN1A and RB1 truncations may display enhanced responsiveness to targeted therapies combining cisplatin with ATR, ATM, CHK1, and CHK2 inhibitors, expanding therapeutic options for patients in need of improved precision treatments.

Malignant rhabdoid tumors (MRT) are highly aggressive pediatric cancers that respond poorly to current therapies. In this study, we screened several MRT cell lines with large-scale RNAi, CRISPR-Cas9, and small-molecule libraries to identify potential drug targets specific for these cancers. We discovered MDM2 and MDM4, the canonical negative regulators of p53, as significant vulnerabilities. Using two compounds currently in clinical development, idasanutlin (MDM2-specific) and ATSP-7041 (MDM2/4-dual), we show that MRT cells were more sensitive than other p53 wild-type cancer cell lines to inhibition of MDM2 alone as well as dual inhibition of MDM2/4. These compounds caused significant upregulation of the p53 pathway in MRT cells, and sensitivity was ablated by CRISPR-Cas9–mediated inactivation of TP53. We show that loss of SMARCB1, a subunit of the SWI/SNF (BAF) complex mutated in nearly all MRTs, sensitized cells to MDM2 and MDM2/4 inhibition by enhancing p53-mediated apoptosis. Both MDM2 and MDM2/4 inhibition slowed MRT xenograft growth in vivo, with a 5-day idasanutlin pulse causing marked regression of all xenografts, including durable complete responses in 50% of mice. Together, these studies identify a genetic connection between mutations in the SWI/SNF chromatin-remodeling complex and the tumor suppressor gene TP53 and provide preclinical evidence to support the targeting of MDM2 and MDM4 in this often-fatal pediatric cancer. Significance: This study identifies two targets, MDM2 and MDM4, as vulnerabilities in a deadly pediatric cancer and provides preclinical evidence that compounds inhibiting these proteins have therapeutic potential.

While the transcription factor MYC is amplified in all breast cancer subtypes, nearly 60% of patients with triple-negative tumors have elevated MYC copy number and expression. Patients with triple-negative breast cancer (TNBC) typically have increased metastasis, decreased response to therapies, and poor outcomes, highlighting MYC’s causal association with disease aggressiveness and low survival rates. Unfortunately, MYC is not considered directly pharmacologically tractable. Therefore, we sought to identify collateral “genetic dependencies,” downstream of oncogenic MYC. Using comparative genome-scale CRISPR/Cas9 screening in isogenic human mammary epithelial cells (HMECs), we have identified two mitochondrial membrane transporter genes, TIMM17A and MTCH2, specifically required for MYC-dependent proliferation and survival. Since MYC is suspected to drive metabolic reprogramming in cancers, we assessed how MYC affected mitochondrial protein content by quantitative mass spectrometry. This revealed an increase in N-acetylaspartate (NAA) in HMECs with MYC amplification compared to other genetic backgrounds. Interestingly, increased NAA levels are dependent on TIMM17A and MTCH2 presence. Loss of either of these genes results in cell death coupled with decreased NAA. NAA supplementation in the media of MYC-HMECs following loss of TIMM17A or MTCH2 can rescue the cell death. Importantly, NAA supplementation in cells with guides targeting GFP (negative control) or general essential gene RPL11 did not show increased cell growth/viability - showing that the NAA supplementation is specifically compensating for loss of mitochondrial transport function in MYC-HMECs. Based on these data, it appears that MYC-amplified TNBCs are uniquely dependent on TIMM17A and MTCH2 mitochondrial transporter function because they drive metabolic reprogramming resulting in addiction to N-acetylaspartate production. To conclude these studies, we are evaluating the dependence on mitochondrial transport and NAA synthesis in MYC-amplified TNBCs by assessing the function of TIMM17A and MTCH2 as MYC-specific genetic dependencies in patient derived xenografts of TNBC and determining if TNBCs are addicted to increased NAA synthesis by overexpressing aspartoacylase to breakdown NAA. Successful completion of this work will provide novel drug targets required for survival of aggressive MYC-amplified breast cancers. Citation Format: Sydney M. Moyer, Nina Ilic, Sydney Gang, Taylor E. Arnoff, William C. Hahn. MYC-driven breast cancer tumorigenesis is dependent on normal mitochondrial function [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2361.

Melanoma has a five-year survival rate of 27% for distant metastatic disease, and there remains a paucity of targeted therapies for metastatic disease and biomarkers that predict metastasis to specific sites. We analyzed 1081 primary melanoma samples and 358 metastatic melanoma samples and found that metastatic disease is enriched for amplifications in both MDM2 and MDM4 compared to primary disease, and these amplifications are associated with a lower probability of overall survival. Two additional negative regulators of TP53, namely USP7 and PPM1D, are enriched for alterations in metastatic melanoma compared to primary melanoma. MDM4 amplifications are associated with a higher rate of metastasis to the brain, liver, and lungs, while MDM2 amplifications are associated with a higher rate of metastasis to the brain, liver, and adrenal glands. These findings suggest that patients with metastatic melanoma show an enhanced dysregulation of the TP53 pathway compared to primary disease; though still under ongoing preclinical evaluation to assess therapeutic implications, we propose that patients with metastatic melanoma and TP53 wild-type status may be more likely to benefit from MDM2, MDM4, USP7, and PPM1D inhibitors, both alone and in combination, compared to those with primary disease. Additionally, we found that patients with MDM2 alterations were more likely to have a deep deletion in CDKN2A, alterations that are also associated with a higher rate of metastasis to the brain. We found that patients with a CDKN2A deep deletion had a statistically significant higher rate of alterations in TTN, MUC16, LRP1B, NF1, and SERPINB4, alterations that have all been previously associated with a favorable response to immune checkpoint inhibitors in melanoma. We therefore propose that CDKN2A deletion may serve as a biomarker to predict response to immunotherapy in melanoma. Moreover, given prior documented cases of patients diagnosed with both melanoma and glioblastoma multiforme (GBM), we found that GBM displays the highest rate of deep deletions in CDKN2A (54.39%) across all cancer types screened. We analyzed 619 GBM samples and found that 9.16% display an MDM2 amplification and 9.52% display an MDM4 amplification. Given the genomic similarities between melanoma and glioblastoma, we suggest that patients with melanoma or GBM and amplifications in MDM2/4 and CDKN2A deletions may need the development of combinations of targeted inhibitors of MDM2/4, CDK’s and immunotherapy. We are currently pursuing these translational directions. Citation Format: Taylor E. Arnoff, Wafik S. El-Deiry. MDM2/MDM4 amplification and CDKN2A deletion in melanoma brain metastases and GBM may have implications for targeted therapeutics and immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr LB516.

Lineage plasticity, the ability of a cell to alter its identity, is an increasingly common mechanism of adaptive resistance to targeted therapy in cancer. An archetypal example is the development of neuroendocrine prostate cancer (NEPC) after treatment of prostate adenocarcinoma (PRAD) with inhibitors of androgen signaling. NEPC is an aggressive variant of prostate cancer that aberrantly expresses genes characteristic of neuroendocrine (NE) tissues and no longer depends on androgens. Here, we investigate the epigenomic basis of this resistance mechanism by profiling histone modifications in NEPC and PRAD patient-derived xenografts (PDXs) using chromatin immunoprecipitation and sequencing (ChIP-seq). We identify a vast network of cis-regulatory elements (N~15,000) that are recurrently activated in NEPC. The FOXA1 transcription factor (TF), which pioneers androgen receptor (AR) chromatin binding in the prostate epithelium, is reprogrammed to NE-specific regulatory elements in NEPC. Despite loss of dependence upon AR, NEPC maintains FOXA1 expression and requires FOXA1 for proliferation and expression of NE lineage-defining genes. Ectopic expression of the NE lineage TFs ASCL1 and NKX2-1 in PRAD cells reprograms FOXA1 to bind to NE regulatory elements and induces enhancer activity as evidenced by histone modifications at these sites. Our data establish the importance of FOXA1 in NEPC and provide a principled approach to identifying cancer dependencies through epigenomic profiling., The molecular processes that lead to neuroendocrine prostate cancer after treating prostate adenocarcinoma (PRAD) are not well understood. Here the authors show that regulation by FOXA1 and changes in the epigenomic profile drive the transition from PRAD to a neuroendocrine phenotype.

Determination of molecular features that mediate clinically aggressive phenotypes in prostate cancer (PrCa) remains a major biological and clinical challenge. Here, we developed a biologically informed deep learning model (P-NET) to stratify PrCa patients by treatment resistance state and evaluate molecular drivers of treatment resistance for therapeutic targeting through complete model interpretability. Using a molecular cohort of 1,238 prostate cancers, we demonstrated that P-NET can predict cancer state using molecular data that is superior to other modeling approaches. Moreover, the biological interpretability within P-NET revealed established and novel molecularly altered candidates, such as MDM4 and FGFR1, that were implicated in predicting advanced disease and validated in vitro. Broadly, biologically informed fully interpretable neural networks enable preclinical discovery and clinical prediction in prostate cancer and may have general applicability across cancer types.

SUMMARY Metastasis is a complex and poorly understood process. In pancreatic cancer, loss of the transforming growth factor (TGF)-β/BMP effector SMAD4 is correlated with changes in altered histopathological transitions, metastatic disease, and poor prognosis. In this study, we use isogenic cancer cell lines to identify SMAD4 regulated genes that contribute to the development of metastatic colonization. We perform an in vivo screen identifying FOSL1 as both a SMAD4 target and sufficient to drive colonization to the lung. The targeting of these genes early in treatment may provide a therapeutic benefit., Graphical abstract, In brief Loss of SMAD4 is associated with poor outcome in pancreatic cancer. Using an in vivo, isogenic metastatic colonization assay, Dai et al. identified SMAD4-regulated genes that affect metastasis but not primary tumor growth. FOSL1 is a SMAD4-regulated gene that is necessary and sufficient to drive metastatic colonization.

Purpose: Rhabdoid tumors are devastating pediatric cancers in need of improved therapies. We sought to identify small molecules that exhibit in vitro and in vivo efficacy against preclinical models of rhabdoid tumor. Experimental Design: We screened eight rhabdoid tumor cell lines with 481 small molecules and compared their sensitivity with that of 879 other cancer cell lines. Genome-scale CRISPR–Cas9 inactivation screens in rhabdoid tumors were analyzed to confirm target vulnerabilities. Gene expression and CRISPR–Cas9 data were queried across cell lines and primary rhabdoid tumors to discover biomarkers of small-molecule sensitivity. Molecular correlates were validated by manipulating gene expression. Subcutaneous rhabdoid tumor xenografts were treated with the most effective drug to confirm in vitro results. Results: Small-molecule screening identified the protein-translation inhibitor homoharringtonine (HHT), an FDA-approved treatment for chronic myelogenous leukemia (CML), as the sole drug to which all rhabdoid tumor cell lines were selectively sensitive. Validation studies confirmed the sensitivity of rhabdoid tumor to HHT was comparable with that of CML cell lines. Low expression of the antiapoptotic gene BCL2L1, which encodes Bcl-XL, was the strongest predictor of HHT sensitivity, and HHT treatment consistently depleted Mcl-1, the synthetic-lethal antiapoptotic partner of Bcl-XL. Rhabdoid tumor cell lines and primary-tumor samples expressed low BCL2L1, and overexpression of BCL2L1 induced resistance to HHT in rhabdoid tumor cells. Furthermore, HHT treatment inhibited rhabdoid tumor cell line and patient-derived xenograft growth in vivo. Conclusions: Rhabdoid tumor cell lines and xenografts are highly sensitive to HHT, at least partially due to their low expression of BCL2L1. HHT may have therapeutic potential against rhabdoid tumors.

Malignant rhabdoid tumors (MRT) are aggressive cancers of early childhood that are largely resistant to traditional therapies. MRT exhibit a remarkably low mutation rate, with no recurrent mutations beyond the defining biallelic inactivating mutation in SMARCB1, a core subunit of the SWI/SNF (BAF) chromatin-remodeling complex. Thus, MRT do not display traditional oncogenic mutations that are amenable to targeted therapies, limiting their use for this disease. In order to nominate new drug targets for MRT, we screened MRT cell lines with large-scale RNAi, CRISPR-Cas9, and small-molecule libraries. The most significant vulnerabilities consistent across all three screens were MDM2 and MDM4, the major negative regulators of p53. We found that MRT cell lines are more sensitive than other p53 wild-type cancer cell lines to both idasanutlin (MDM2-specific) and ATSP-7041 (MDM2/4-dual) in vitro. Both inhibitors induced substantial activation of the p53 pathway in MRT cell lines, which responded with permanent apoptotic or senescent cell fate decisions. CRISPR-Cas9-mediated inactivation of TP53 caused a significant resistance to these compounds, confirming that on-target mechanisms were responsible for MRT sensitivity. We found that loss of SMARCB1 sensitizes MRT cells to idasanutlin by shifting the p53 response towards apoptosis. In MRT xenograft studies, both idasanutlin and ATSP-7041 slowed tumor growth. Most strikingly, an idasanutlin pulse of only five days was sufficient to induce sizable regression of all tumors, which remained complete and durable in 50% of mice. Finally, gene expression analysis of primary MRT predicts that, like cell lines and xenografts, MRT in patients are likely to be sensitive to MDM2 inhibition. Collectively, these studies describe a genetic link between SWI/SNF complex mutations and p53, while providing evidence to support the use of MDM2 and MDM2/4 inhibitors that have already entered clinical trials for the treatment of this devastating pediatric cancer. Citation Format: Thomas P. Howard, Taylor E. Arnoff, Melinda R. Song, Andrew O. Giacomelli, Xiaofeng Wang, Andrew L. Hong, Neekesh V. Dharia, Su Wang, Francisca Vazquez, Minh-Tam Pham, Ann M. Morgan, Franziska Wachter, Gregory H. Bird, Guillaume Kugener, Elaine M. Oberlick, Matthew G. Rees, Hong Tiv, Justin H. Hwang, Katherine H. Walsh, April Cook, John M. Krill-Burger, Aviad Tsherniak, Prafulla C. Gokhale, Peter J. Park, Kimberly Stegmaier, Loren D. Walensky, William C. Hahn, Charles W. Roberts. MDM2 and MDM4 are therapeutic vulnerabilities in malignant rhabdoid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2867.

The transcriptional regulator NPR1 mediates salicylic acid (SA)-induced plant immunity. NPR1 is also required for tolerance to high concentrations of SA. NPR1-interacting protein, NIMIN1, represses immune response by interacting with and negating NPR1. We tested the salicylic acid tolerance of transgenic plants overexpressing NIMIN1 and found that these plants displayed SA intolerance, similar to the npr1 mutant, due to sequestration of NPR1 by NIMIN1. Plants overexpressing mutated NIMIN1 that cannot interact with NPR1 showed no SA tolerance defect. Gene expression analysis showed that NPR1 is required for SA-stress induced as well as pathogen-induced NIMIN1 expression. These results indicate that over-accumulation of a negative regulator renders plants hypersensitive to SA by limiting NPR1 function. Furthermore, NPR1 activates negative regulators such as NIMIN1 for feedback inhibition of SA signaling to maintain immune homeostasis.