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Adrenocortical carcinoma (ACC) is a rare cancer in which tissue-specific differentiation is paradoxically associated with dismal outcomes. The differentiated ACC subtype CIMP-high is prevalent, incurable, and routinely fatal. CIMP-high ACC possess abnormal DNA methylation and frequent β-catenin activating mutations. Here, we demonstrated that ACC differentiation is maintained by a balance between nuclear, tissue-specific β-catenin-containing complexes and the epigenome. On chromatin, β-catenin bound master adrenal transcription factor SF1 and hijacked the adrenocortical super-enhancer landscape to maintain differentiation in CIMP-high ACC; off chromatin, β-catenin bound histone methyltransferase EZH2. SF1/β-catenin and EZH2/β-catenin complexes present in normal adrenals persisted through all phases of ACC evolution. Pharmacologic EZH2 inhibition in CIMP-high ACC expelled SF1/β-catenin from chromatin and favored EZH2/β-catenin assembly, erasing differentiation and restraining cancer growth in vitro and in vivo. These studies illustrate how tissue-specific programs shape oncogene selection, surreptitiously encoding targetable therapeutic vulnerabilities.

Tumor heterogeneity is a major driver of cancer progression. In epithelial-derived malignancies, carcinoma-associated fibroblasts (CAFs) contribute to tumor heterogeneity by depositing extracellular matrix (ECM) proteins that dynamically remodel the tumor microenvironment (TME). Ewing sarcomas (EwS) are histologically monomorphous, mesenchyme-derived tumors that are devoid of CAFs. Here we identify a previously uncharacterized subpopulation of transcriptionally distinct EwS tumor cells that deposit pro-tumorigenic ECM. Single cell analyses revealed that these CAF-like cells differ from bulk EwS cells by their upregulation of a matrisome-rich gene signature that is normally repressed by EWS::FLI1, the oncogenic fusion transcription factor that underlies EwS pathogenesis. Further, our studies showed that ECM-depositing tumor cells express the cell surface marker CD73, allowing for their isolation ex vivo and detection in situ. Spatial profiling of tumor xenografts and patient biopsies demonstrated that CD73+EwS cells and tumor cell-derived ECM are prevalent along tumor borders and invasive fronts. Importantly, despite loss of EWS::FLI1-mediated gene repression, CD73+EwS cells retain expression of EWS::FLI1 and the fusion-activated gene signature, as well as tumorigenic and proliferative capacities. Thus, EwS tumor cells can be reprogrammed to adopt CAF-like properties and these transcriptionally and phenotypically distinct cell subpopulations contribute to tumor heterogeneity by remodeling the TME.

GSK-LSD1, Doxorubicin and AraC have no effect on KDM1A and KDM1B expression

Protein expression of ER-stress response genes following treatment with SP-2509

Supplementary Figure S1. Real-time cell proliferation analysis of shATF4, MI-503 treatment, and dox-inducible shMEN1 knockdown in Ewing sarcoma cells.

Targeted KDM1A inhibition and EWS/FLI knockdown induces similar transcriptional signatures

KCNA5 promoter methylation in diverse cancer types

Hypersensitive SP-2509 cell lines display similar transcriptomic profiles

Cell line source/culture conditions, primer/shRNA sequences, antibodies utilized in this study and additional methods

To show that MRTF-A localization was dependent on F-actin dynamics, we stimulated SK-Mel-19 cells with 20% FBS to induce stress fiber formation which induced MRTF-A nuclear localization. We also treated SK-Mel-147 cells with latrunculin B which inhibits F-actin formation and MRTF-A relocated to the cytosol.

Validation of KDM1A knockdown in Ewing sarcoma cells

Multi-agent chemotherapeutic regimes remain the cornerstone treatment for Ewing sarcoma, the second most common bone malignancy diagnosed in pediatric and young adolescent populations. We have reached a therapeutic ceiling with conventional cytotoxic agents, highlighting the need to adopt novel approaches that specifically target the drivers of Ewing sarcoma oncogenesis. As KDM1A/lysine-specific demethylase 1 (LSD1) is highly expressed in Ewing sarcoma cell lines and tumors, with elevated expression levels associated with worse overall survival (P = 0.033), this study has examined biomarkers of sensitivity and mechanisms of cytotoxicity to targeted KDM1A inhibition using SP-2509 (reversible KDM1A inhibitor). We report, that innate resistance to SP-2509 was not observed in our Ewing sarcoma cell line cohort (n = 17; IC50 range, 81 –1,593 nmol/L), in contrast resistance to the next-generation KDM1A irreversible inhibitor GSK-LSD1 was observed across multiple cell lines (IC50 > 300 μmol/L). Although TP53/STAG2/CDKN2A status and basal KDM1A mRNA and protein levels did not correlate with SP-2509 response, induction of KDM1B following SP-2509 treatment was strongly associated with SP-2509 hypersensitivity. We show that the transcriptional profile driven by SP-2509 strongly mirrors KDM1A genetic depletion. Mechanistically, RNA-seq analysis revealed that SP-2509 imparts robust apoptosis through engagement of the endoplasmic reticulum stress pathway. In addition, ETS1/HIST1H2BM were specifically induced/repressed, respectively following SP-2509 treatment only in our hypersensitive cell lines. Together, our findings provide key insights into the mechanisms of SP-2509 cytotoxicity as well as biomarkers that can be used to predict KDM1A inhibitor sensitivity in Ewing sarcoma. Mol Cancer Ther; 17(9); 1902–16. ©2018 AACR.

shRNA sequences, plasmid information, western blot antibodies

Ewing sarcomas are driven by EWS–ETS fusions, most commonly EWS-FLI1, which promotes widespread metabolic reprogramming, including activation of serine biosynthesis. We previously reported that serine biosynthesis is also activated in Ewing sarcoma by the scaffolding protein menin through as yet undefined mechanisms. Here, we investigated whether EWS-FLI1 and/or menin orchestrate serine biosynthesis via modulation of ATF4, a stress-response gene that acts as a master transcriptional regulator of serine biosynthesis in other tumors. Our results show that in Ewing sarcoma, ATF4 levels are high and that ATF4 modulates transcription of core serine synthesis pathway (SSP) genes. Inhibition of either EWS-FLI1 or menin leads to loss of ATF4, and this is associated with diminished expression of SSP transcripts and proteins. We identified and validated an EWS–FLI1 binding site at the ATF4 promoter, indicating that the fusion can directly activate ATF4 transcription. In contrast, our results suggest that menin-dependent regulation of ATF4 is mediated by transcriptional and post-transcriptional mechanisms. Importantly, our data also reveal that the downregulation of SSP genes that occurs in the context of EWS-FLI1 or menin loss is indicative of broader inhibition of ATF4-dependent transcription. Moreover, we find that menin inhibition similarly leads to loss of ATF4 and the ATF4-dependent transcriptional signature in MLL-rearranged B-cell acute lymphoblastic leukemia, extending our findings to another cancer in which menin serves an oncogenic role.Implications:These studies provide new insights into metabolic reprogramming in Ewing sarcoma and also uncover a previously undescribed role for menin in the regulation of ATF4.

Primer Sequences

The Cancer Genome Atlas (TCGA) cutaneous melanoma dataset was stratified into quartiles based upon expression of indicated genes. Kaplan-Meier plots were generated from the highest (grey) and lowest (black) expressing quartiles. Survival curves were analyzed with the log-rank test with a cutoff of P < 0.05 as statistically significant.

Cytotoxic effects of the irreversible KDM1A inhibitors GSK-LSD1 and Tranylcypromine

Purpose:Propagation of Ewing sarcoma requires precise regulation of EWS::FLI1 transcriptional activity. Determining the mechanisms of fusion regulation will advance our understanding of tumor progression. Here we investigated whether HOXD13, a developmental transcription factor that promotes Ewing sarcoma metastatic phenotypes, influences EWS::FLI1 transcriptional activity.Experimental Design:Existing tumor and cell line datasets were used to define EWS::FLI1 binding sites and transcriptional targets. Chromatin immunoprecipitation and CRISPR interference were employed to identify enhancers. CUT&RUN and RNA sequencing defined binding sites and transcriptional targets of HOXD13. Transcriptional states were investigated using bulk and single-cell transcriptomic data from cell lines, patient-derived xenografts, and patient tumors. Mesenchymal phenotypes were assessed by gene set enrichment, flow cytometry, and migration assays.Results:We found that EWS::FLI1 creates a de novo GGAA microsatellite enhancer in a developmentally conserved regulatory region of the HOXD locus. Knockdown of HOXD13 led to widespread changes in expression of developmental gene programs and EWS::FLI1 targets. HOXD13 binding was enriched at established EWS::FLI1 binding sites where it influenced expression of EWS::FLI1-activated genes. More strikingly, HOXD13 bound and activated EWS::FLI1-repressed genes, leading to adoption of mesenchymal and migratory cell states that are normally suppressed by the fusion. Single-cell analysis confirmed that direct transcriptional antagonism between HOXD13-mediated gene activation and EWS::FLI1-dependent gene repression defines the state of Ewing sarcoma cells along a mesenchymal axis.Conclusions:Ewing sarcoma tumors are comprised of tumor cells that exist along a mesenchymal transcriptional continuum. The identity of cells along this continuum is, in large part, determined by the competing activities of EWS::FLI1 and HOXD13.See related commentary by Weiss and Bailey, p. 4360

Supplementary Table from EWS::FLI1 and HOXD13 Control Tumor Cell Plasticity in Ewing Sarcoma

Supplementary Figure from EWS::FLI1 and HOXD13 Control Tumor Cell Plasticity in Ewing Sarcoma

Supplemental table S1. RNA-seq differentially expressed genes modulated by Wnt3a (Table S1A) or Wnt3a+RSPO2 (Table S1B).

Purpose: We report a novel analytic method, named intercohort co-analysis or Ican, which aids in the discovery of genes with predictive value for the progression or outcome of diseases from small-size cohorts. We tested this premise in Ewing's sarcoma (ES), a highly metastatic cancer of bone and soft tissues that lacks validated molecular metastasis and prognostic indicators.Experimental Design: To uncover genes significantly expressed in ES patient subsets, we first determined a nonarbitrary gene expression significance cutoff based on expression levels in validated expressing and nonexpressing tissues. We next searched for genes that were consistently significantly expressed in several ES cohort and cell line datasets. Significantly expressed genes were independently validated by quantitative reverse transcription-PCR in an additional ES cohort.Results: Analysis of ES cohorts revealed marked intercohort gene expression variability. After filtering out the intercohort variability, CXCR4 and CXCR7 were found to be consistently associated with specific ES subsets. Pairwise analyses showed CXCR4 to correlate with ES metastases, and CXCR4 and CXCR7 to patient survival, but not with several other clinicopathological variables.Conclusion: Ican is a powerful novel method to identifying genes consistently associated with particular disease states in cancers for which large cohorts are not available, currently the case of most cancers. We report for the first time that high CXCR4 expression preferentially associates with metastatic ES, and that of CXCR7 with poor patient survival. Clin Cancer Res; 16(14); 3769–78. ©2010 AACR.

Rhabdomyosarcoma, consisting of alveolar (aRMS) and embryonal (eRMS) subtypes, is the most common type of sarcoma in children. Currently, there are no targeted drug therapies available for rhabdomyosarcoma. In searching for new molecular therapeutic targets, we carried out genome-wide small interfering RNA (siRNA) library screens targeting human phosphatases (n = 206) and kinases (n = 691) initially against an aRMS cell line, RH30. Sixteen phosphatases and 50 kinases were identified based on growth inhibition after 72 hours. Inhibiting polo-like kinase 1 (PLK1) had the most remarkable impact on growth inhibition (∼80%) and apoptosis on all three rhabdomyosarcoma cell lines tested, namely, RH30, CW9019 (aRMS), and RD (eRMS), whereas there was no effect on normal muscle cells. The loss of PLK1 expression and subsequent growth inhibition correlated with decreased p-CDC25C and Cyclin B1. Increased expression of WEE 1 was also noted. The induction of apoptosis after PLK1 silencing was confirmed by increased p-H2AX, propidium iodide uptake, and chromatin condensation, as well as caspase-3 and poly(ADP-ribose) polymerase cleavage. Pediatric Ewing's sarcoma (TC-32), neuroblastoma (IMR32 and KCNR), and glioblastoma (SF188) models were also highly sensitive to PLK1 inhibition. Finally, based on cDNA microarray analyses, PLK1 mRNA was overexpressed (>1.5 fold) in 10 of 10 rhabdomyosarcoma cell lines and in 47% and 51% of primary aRMS (17 of 36 samples) and eRMS (21 of 41 samples) tumors, respectively, compared with normal muscles. Similarly, pediatric Ewing's sarcoma, neuroblastoma, and osteosarcoma tumors expressed high PLK1. We conclude that PLK1 could be a promising therapeutic target for the treatment of a wide range of pediatric solid tumors including rhabdomyosarcoma. [Mol Cancer Ther 2009;8(11):3024–35]

Ewing sarcomas are characterized by the presence of EWS/ETS fusion genes in the absence of other recurrent genetic alterations and mechanisms of tumor heterogeneity that contribute to disease progression remain unclear. Mutations in the Wnt/β-catenin pathway are rare in Ewing sarcoma but the Wnt pathway modulator LGR5 is often highly expressed, suggesting a potential role for the axis in tumor pathogenesis. We evaluated β-catenin and LGR5 expression in Ewing sarcoma cell lines and tumors and noted marked intra- and inter-tumor heterogeneity. Tumors with evidence of active Wnt/β-catenin signaling were associated with increased incidence of tumor relapse and worse overall survival. Paradoxically, RNA sequencing revealed a marked antagonism of EWS/ETS transcriptional activity in Wnt/β-catenin–activated tumor cells. Consistent with this, Wnt/β-catenin–activated cells displayed a phenotype that was reminiscent of Ewing sarcoma cells with partial EWS/ETS loss of function. Specifically, activation of Wnt/β-catenin induced alterations to the actin cytoskeleton, acquisition of a migratory phenotype, and upregulation of EWS/ETS–repressed genes. Notably, activation of Wnt/β-catenin signaling led to marked induction of tenascin C (TNC), an established promoter of cancer metastasis, and an EWS/ETS–repressed target gene. Loss of TNC function in Ewing sarcoma cells profoundly inhibited their migratory and metastatic potential. Our studies reveal that heterogeneous activation of Wnt/β-catenin signaling in subpopulations of tumor cells contributes to phenotypic heterogeneity and disease progression in Ewing sarcoma. Significantly, this is mediated, at least in part, by inhibition of EWS/ETS fusion protein function that results in derepression of metastasis-associated gene programs. Cancer Res; 76(17); 5040–53. ©2016 AACR.

Supplementary Figure 2 from Small interfering RNA library screen of human kinases and phosphatases identifies polo-like kinase 1 as a promising new target for the treatment of pediatric rhabdomyosarcomas

Supplementary Figure 1 from Small interfering RNA library screen of human kinases and phosphatases identifies polo-like kinase 1 as a promising new target for the treatment of pediatric rhabdomyosarcomas

Detailed methods and reagents lists for lentiviral transduction, AQUA, in situ hybridization and microscopy.

Supplementary Data from Intercohort Gene Expression Co-Analysis Reveals Chemokine Receptors as Prognostic Indicators in Ewing's Sarcoma

Supplemental table S2. qRT-PCR primer sequences.

Legends for supplementary Figures S1 through S5

Supplemental Figure S1. RNAseq identifies a Ewing sarcoma-specific Wnt/beta-catenin transcriptional response Supplemental Figure S2. GSEA shows inverse relationship between Wnt/beta-catenin and EWS/ETS transcriptional targets Supplemental Figure S3. EWS/FLI1 inhibits Wnt-activation and Wnt-stimulated cells activate microtubule genes Supplemental Figure S4. Activation of Wnt/beta-catenin results in cell spreading and increased tumor cell size Supplemental Figure S5. TNC is repressed by EWS/FLI1 and high- level expression of TNC promotes anchorage-independent growth

Supplementary Tables S1-S4. Identification of HEY1 regulated genes downstream of EWS-FLI1 silencing in Ewing sarcoma (S1); Combined immunohistochemical staining results for samples from patients with metastases at diagnosis on TMA1 and 2 according to site of metastasis (S2); Clinical characteristics and SIRT1 staining results of primary tumors from 43 ES patients treated at St. Jude Children´s Hospital between 1979 and 1987 using vincristine, doxorubicin, and cyclophosphamide-based regimens, with subsequent addition of ifosfamide and etoposide (S3); Materials used in the study (S4).

Supplementary Table 1 from BMI-1 Promotes Ewing Sarcoma Tumorigenicity Independent of CDKN2A Repression

Deregulation of the polycomb group gene BMI-1 is implicated in the pathogenesis of many human cancers. In this study, we have investigated if the Ewing sarcoma family of tumors (ESFT) expresses BMI-1 and whether it functions as an oncogene in this highly aggressive group of bone and soft tissue tumors. Our data show that BMI-1 is highly expressed by ESFT cells and that, although it does not significantly affect proliferation or survival, BMI-1 actively promotes anchorage-independent growth in vitro and tumorigenicity in vivo. Moreover, we find that BMI-1 promotes the tumorigenicity of both p16 wild-type and p16-null cell lines, demonstrating that the mechanism of BMI-1 oncogenic function in ESFT is, at least in part, independent of CDKN2A repression. Expression profiling studies of ESFT cells following BMI-1 knockdown reveal that BMI-1 regulates the expression of hundreds of downstream target genes including, in particular, genes involved in both differentiation and development as well as cell-cell and cell-matrix adhesion. Gain and loss of function assays confirm that BMI-1 represses the expression of the adhesion-associated basement membrane protein nidogen 1. In addition, although BMI-1 promotes ESFT adhesion, nidogen 1 inhibits cellular adhesion in vitro. Together, these data support a pivotal role for BMI-1 ESFT pathogenesis and suggest that its oncogenic function in these tumors is in part mediated through modulation of adhesion pathways. [Cancer Res 2008;68(16):6507–15]

Supplementary Figures 1-4 from BMI-1 Promotes Ewing Sarcoma Tumorigenicity Independent of CDKN2A Repression

Supplementary Figure Legends. Legend for Supplementary Figures S1-S5.

Gene Lists

Supplementary Figures S1-S5. MDM4 modulation by HEY1 occurs downstream of TP53 (S1); Modulation of SIRT1 and TP53 induction upon ectopic expression of Flag-tagged HEY1 in the B-cell malignancy cell lines "697" and NALM6, and in primary keratinocytes (S2); The degree of SIRT1 modulation depends on HEY1 levels (S3); Flow-diagram of samples analysed on tissue micro arrays (TMA1, TMA2, TMA3), and FFPE sections used in this study (S4); Kaplan-Meier survival estimates for patients analysed for SIRT1 expression on TMA3 (S5).

Supplementary Figures 1-2 from Reversible Kinetic Analysis of Myc Targets In vivo Provides Novel Insights into Myc-Mediated Tumorigenesis

Deregulated expression of the Myc transcription factor is a frequent causal mutation in human cancer. Thousands of putative Myc target genes have been identified in in vitro studies, indicating that Myc exerts highly pleiotropic effects within cells and tissues. However, the complexity and diversity of Myc gene targets has confounded attempts at identifying which of these genes are the critical targets mediating Myc-driven tumorigenesis in vivo. Acute activation of Myc in a reversibly switchable transgenic model of Myc-mediated β cell tumorigenesis induces rapid tumor onset, whereas subsequent Myc deactivation triggers equally rapid tumor regression. Thus, sustained Myc activity is required for tumor maintenance. We have used this reversibly switchable kinetic tumor model in combination with high-density oligonucleotide microarrays to develop an unbiased strategy for identifying candidate Myc-regulated genes responsible for maintenance of Myc-dependent tumors. Consistent with known Myc functions, some Myc-regulated genes are involved in cell growth, cycle, and proliferation. In addition, however, many Myc-regulated genes are specific to β cells, indicating that a significant component of Myc action is cell type specific. Finally, we identify a very restricted cadre of genes with expression that is inversely regulated upon Myc activation-induced tumor progression and deactivation-induced tumor regression. By definition, such genes are candidates for tumor maintenance functions. Combining reversibly switchable, transgenic models of tumor formation and regression with genomic profiling offers a novel strategy with which to deconvolute the complexities of oncogenic signaling pathways in vivo. (Cancer Res 2006; 66(9): 4591-601)

The EWSR1/FLI1 gene fusion is the most common rearrangement leading to cell transformation in Ewing sarcoma (ES). Previous studies have indicated that expression at the cellular level is heterogeneous, and that levels of expression may oscillate, conferring different cellular characteristics. In ES the role of EWSR1/FLI1 in regulating subpopulation dynamics is currently unknown.We used siRNA to transiently suppress EWSR1/FLI1 expression and followed population dynamics using both single cell expression profiling, CyTOF and functional assays to define characteristics of exponentially growing ES cells and of ES cells in which EWSR1/FLI1 had been downregulated. Novel transcriptional states with distinct features were assigned using random forest feature selection in combination with machine learning. Cells isolated from ES xenografts in immune-deficient mice were interrogated to determine whether characteristics of specific subpopulations of cells in vitro could be identified. Stem-like characteristics were assessed by primary and secondary spheroid formation in vitro, and invasion/motility was determined for each identified subpopulation. Autophagy was determined by expression profiling, cell sorting and immunohistochemical staining.We defined a workflow to study EWSR1/FLI1 driven transcriptional states and phenotypes. We tracked EWSR1/FLI1 dependent proliferative activity over time to discover sources of intra-tumoral diversity. Single-cell RNA profiling was used to compare expression profiles in exponentially growing populations (si-Control) or in two dormant populations (D1, D2) in which EWSR1/FLI1 had been suppressed. Three distinct transcriptional states were uncovered contributing to ES intra-heterogeneity. Our predictive model identified ~1% cells in a dormant-like state and ~ 2-4% cells with stem-like and neural stem-like features in an exponentially proliferating ES cell line and in ES xenografts. Following EWSR1/FLI1 knockdown, cells re-entering the proliferative cycle exhibited greater stem-like properties, whereas for those cells remaining quiescent, FAM134B-dependent dormancy may provide a survival mechanism.We show that time-dependent changes induced by suppression of oncogenic EWSR1/FLI1 expression induces dormancy, with different subpopulation dynamics. Cells re-entering the proliferative cycle show enhanced stem-like characteristics, whereas those remaining dormant for prolonged periods appear to survive through autophagy. Cells with these characteristics identified in exponentially growing cell populations and in tumor xenografts may confer drug resistance and could potentially contribute to metastasis.

Purpose: Propagation of Ewing sarcoma requires precise regulation of EWS::FLI1 transcriptional activity. Determining the mechanisms of fusion regulation will advance our understanding of tumor progression. Here we investigated whether HOXD13, a developmental transcription factor that promotes Ewing sarcoma metastatic phenotypes, influences EWS::FLI1 transcriptional activity. Experimental Design: Existing tumor and cell line datasets were used to define EWS::FLI1 binding sites and transcriptional targets. Chromatin immunoprecipitation and CRISPR interference were employed to identify enhancers. CUT&RUN and RNA sequencing defined binding sites and transcriptional targets of HOXD13. Transcriptional states were investigated using bulk and single-cell transcriptomic data from cell lines, patient-derived xenografts, and patient tumors. Mesenchymal phenotypes were assessed by gene set enrichment, flow cytometry, and migration assays. Results: We found that EWS::FLI1 creates a de novo GGAA microsatellite enhancer in a developmentally conserved regulatory region of the HOXD locus. Knockdown of HOXD13 led to widespread changes in expression of developmental gene programs and EWS::FLI1 targets. HOXD13 binding was enriched at established EWS::FLI1 binding sites where it influenced expression of EWS::FLI1-activated genes. More strikingly, HOXD13 bound and activated EWS::FLI1-repressed genes, leading to adoption of mesenchymal and migratory cell states that are normally suppressed by the fusion. Single-cell analysis confirmed that direct transcriptional antagonism between HOXD13-mediated gene activation and EWS::FLI1-dependent gene repression defines the state of Ewing sarcoma cells along a mesenchymal axis. Conclusions: Ewing sarcoma tumors are comprised of tumor cells that exist along a mesenchymal transcriptional continuum. The identity of cells along this continuum is, in large part, determined by the competing activities of EWS::FLI1 and HOXD13. See related commentary by Weiss and Bailey, p. 4360

Primary bone sarcomas, including osteosarcoma (OS) and Ewing sarcoma (ES), are aggressive tumors with peak incidence in childhood and adolescence. The intense standard treatment for these patients consists of combined surgery and/or radiation and maximal doses of chemotherapy; a regimen that has not seen improvement in decades. Like other tumor types, ES and OS are characterized by dysregulated cellular metabolism and a rewiring of metabolic pathways to support the biosynthetic demands of malignant growth. Not only are cancer cells characterized by Warburg metabolism, or aerobic glycolysis, but emerging work has revealed a dependence on amino acid metabolism. Aside from incorporation into proteins, amino acids serve critical functions in redox balance, energy homeostasis, and epigenetic maintenance. In this review, we summarize current studies describing the amino acid metabolic requirements of primary bone sarcomas, focusing on OS and ES, and compare these dependencies in the normal bone and malignant tumor contexts. We also examine insights that can be gleaned from other cancers to better understand differential metabolic susceptibilities between primary and metastatic tumor microenvironments. Lastly, we discuss potential metabolic vulnerabilities that may be exploited therapeutically and provide better-targeted treatments to improve the current standard of care.

Adrenocortical carcinoma (ACC) is a rare cancer in which tissue-specific differentiation is paradoxically associated with dismal outcomes. The differentiated ACC subtype CIMP-high is prevalent, incurable, and routinely fatal. CIMP-high ACC possess abnormal DNA methylation and frequent β-catenin activating mutations. Here, we demonstrate that ACC differentiation is maintained by a balance between nuclear, tissue-specific β-catenin-containing complexes and the epigenome. On chromatin, β-catenin binds master adrenal transcription factor SF1 and hijacks the adrenocortical super-enhancer landscape to maintain differentiation. Off chromatin, β-catenin binds histone methyltransferase EZH2, which is redistributed by the CIMP-high DNA methylation signature. SF1/β-catenin and EZH2/β-catenin complexes exist in normal adrenals and are selected for through all phases of ACC evolution. Pharmacologic EZH2 inhibition in CIMP-high ACC favors EZH2/β-catenin assembly and purges SF1/β-catenin from chromatin, erasing differentiation and restraining cancer growth in vitro and in vivo. Our studies illustrate how tissue-specific programs shape oncogene selection, surreptitiously encoding targetable therapeutic vulnerabilities.

Glioblastoma (GBM) is an aggressive primary brain cancer; with a 5-year survival of ~5%. Challenges that hamper GBM therapeutic efficacy include: (i) tumor heterogeneity, (ii) treatment resistance, (iii) immunosuppressive tumor microenvironment (TME) and (iv) the blood-brain barrier (BBB). The C-X-C Motif Chemokine Ligand-12/ C-X-C Motif Chemokine Receptor-4 (CXCL12/CXCR4) signaling pathway is activated in GBM and is associated with tumor progression. Although the CXCR4 antagonist (AMD3100) has been proposed as an attractive anti-GBM therapeutic target, it’s poor pharmacokinetic properties, and unfavorable bioavailability have hampered its clinical implementation. Thus, we developed synthetic protein nanoparticles (SPNPs) coated with the transcytotic peptide iRGD (AMD3100-SPNPs) to target the CXCL2/CXCR4 pathway in GBM via systemic delivery. We showed that AMD3100-SPNPs block CXCL12/CXCR4 signaling in three mouse and human GBM cell cultures in vitro and in a GBM mouse model in vivo. This results in (i) inhibition of GBM proliferation, (ii) reduced infiltration of CXCR4(+) monocytic myeloid derived suppressor cells (M-MDSCs) into the TME, (iii) restoration of BBB integrity, and (iv) induction of immunogenic cell death (ICD), sensitizing the tumor to radiotherapy, and leading to anti-GBM immunity. Additionally, we showed that combining AMD3100-SPNPs with radiation led to long term survival; with ~60% of GBM tumor bearing mice remaining tumor free, after rechallenging with a second GBM in the contralateral hemisphere. This was due to a sustained anti-GBM immunological memory response that prevented tumor recurrence without additional treatment. In view of the potent ICD induction and reprogrammed tumor microenvironment, this SPNP-mediated strategy has a significant clinical translation applicability.