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Immunohistochemical staining of tumors

Supplementary Figure 3

Mutant p53-associated genes

Tumors and Metastasis in KRas+/LA1;Dicer+/2SD mice

Dicer1 functions as a tumor suppressor in mouse models. In humans, somatic mutations are associated with many cancers in adults, and patients with DICER1 syndrome with DICER1 germline mutations are susceptible to childhood cancers. Dicer is phosphorylated by the ERK-MAP kinase pathway and because this pathway is activated in human cancers, we asked whether phosphorylated Dicer1 contributed to tumor development. In human endometrioid cancers, we discovered that phosphorylated DICER1 is significantly associated with invasive disease. To test a direct involvement of Dicer1 phosphorylation in tumor development, we studied mice with phosphomimetic alterations at the two conserved serines phosphorylated by ERK and discovered that a phosphomimetic Dicer1 drives tumor development and dissemination in two independent murine cancer models (KRas+/LA1 and p53+/−). Our findings demonstrate that phosphomimetic Dicer1 promotes tumor development and invasion.Significance:This work highlights the relevance of Dicer1 phosphorylation in mammalian tumor development and dissemination.

Supplementary Figures 3-4 from Tumor Cell Kill by c-MYC Depletion: Role of MYC-Regulated Genes that Control DNA Double-Strand Break Repair

XLSX file, 14K, Gene expression changes common to wild type MYC and all phosphorylation mutants (112 genes).

XLS file, 160K, GO Analysis of significant genes expression changes.

XLS file, 403K, Expression comparison between wild-type MYC and MYC phosphorylation mutants (from Venn diagram)

XLS file, 2439K, MYC ChIP-on-chip data in MCF10A cells.

Supplementary Figures 1-2 from Tumor Cell Kill by c-MYC Depletion: Role of MYC-Regulated Genes that Control DNA Double-Strand Break Repair

XLS file, 14K, Phosphopeptide analysis for ectopic MYC in 293Tv cells.

XLS file, 98K, GO analysis of common gene expression changes.

MYC regulates a myriad of genes controlling cell proliferation, metabolism, differentiation, and apoptosis. MYC also controls the expression of DNA double-strand break (DSB) repair genes and therefore may be a potential target for anticancer therapy to sensitize cancer cells to DNA damage or prevent genetic instability. In this report, we studied whether MYC binds to DSB repair gene promoters and modulates cell survival in response to DNA-damaging agents. Chromatin immunoprecipitation studies showed that MYC associates with several DSB repair gene promoters including Rad51, Rad51B, Rad51C, XRCC2, Rad50, BRCA1, BRCA2, DNA-PKcs, XRCC4, Ku70, and DNA ligase IV. Endogenous MYC protein expression was associated with increased RAD51 and KU70 protein expression of a panel of cancer cell lines of varying histopathology. Induction of MYC in G0-G1 and S-G2-M cells resulted in upregulation of Rad51 gene expression. MYC knockdown using small interfering RNA (siRNA) led to decreased RAD51 expression but minimal effects on homologous recombination based on a flow cytometry direct repeat green fluorescent protein assay. siRNA to MYC resulted in tumor cell kill in DU145 and H1299 cell lines in a manner independent of apoptosis. However, MYC-dependent changes in DSB repair protein expression were not sufficient to sensitize cells to mitomycin C or ionizing radiation, two agents selectively toxic to DSB repair–deficient cells. Our results suggest that anti-MYC agents may target cells to prevent genetic instability but would not lead to differential radiosensitization or chemosensitization. Cancer Res; 70(21); 8748–59. ©2010 AACR.

Supplementary Table 1 from Tumor Cell Kill by c-MYC Depletion: Role of MYC-Regulated Genes that Control DNA Double-Strand Break Repair

PDF file, 136K.

XLS file, 319K, Expression array analysis of MCF10A 3D cultures at 4 days.

XLS file, 3148K, Compiled expression array and ChIP-on-chip data.

XLSX file, 16K, Gene expression changes common to all gain-of-function MYC phosphorylation mutants (158 genes).

PDF file, 498K, Supplementary Figures S1-6 Supplementary Figure S1: Phosphorylation mutants increase Myc-induced transformation in MCF10A cells. Supplementary Figure S2: Phosphorylation mutants increase Myc-induced transformation in SH-EP cells. Supplementary Figure S3: Phosphorylation mutants do not effect MYC protein stability. Supplementary Figure S4: Serum starvation of MCF10A-GFP and MCF10A-MYC cells. Supplementary Figure S5: MYC protein expression through morphogenesis of MCF10A cells in 3D culture. Supplementary Figure S6: Characterization of day 4 acini for mRNA expression array analysis.

Supplementary Figure Legends 1-4 from Tumor Cell Kill by c-MYC Depletion: Role of MYC-Regulated Genes that Control DNA Double-Strand Break Repair

Pancreatic ductal adenocarcinoma (PDAC) is almost uniformly fatal and characterized by early metastasis. Oncogenic KRAS mutations prevail in 95% of PDAC tumors and co-occur with genetic alterations in the TP53 tumor suppressor in nearly 70% of patients. Most TP53 alterations are missense mutations that exhibit gain-of-function phenotypes that include increased invasiveness and metastasis, yet the extent of direct cooperation between KRAS effectors and mutant p53 remains largely undefined. We show that oncogenic KRAS effectors activate CREB1 to allow physical interactions with mutant p53 that hyperactivate multiple prometastatic transcriptional networks. Specifically, mutant p53 and CREB1 upregulate the prometastatic, pioneer transcription factor FOXA1, activating its transcriptional network while promoting WNT/β-catenin signaling, together driving PDAC metastasis. Pharmacologic CREB1 inhibition dramatically reduced FOXA1 and β-catenin expression and dampened PDAC metastasis, identifying a new therapeutic strategy to disrupt cooperation between oncogenic KRAS and mutant p53 to mitigate metastasis. Significance: Oncogenic KRAS and mutant p53 are the most commonly mutated oncogene and tumor suppressor gene in human cancers, yet direct interactions between these genetic drivers remain undefined. We identified a cooperative node between oncogenic KRAS effectors and mutant p53 that can be therapeutically targeted to undermine cooperation and mitigate metastasis. This article is highlighted in the In This Issue feature, p. 1861

A more comprehensive understanding of the molecular mechanisms underlying pancreatic diseases, including pancreatitis and cancer, is essential to improve clinical management. MEN1 has established roles in epigenetic regulation and tumor suppression in the endocrine pancreas; however, intriguing recent data suggest MEN1 may also function in the exocrine pancreas. Using physiologically relevant genetic mouse models, we provide direct evidence that Men1 is essential for exocrine pancreas homeostasis in response to inflammation and oncogenic stress. Men1 loss causes increased injury and impaired regeneration following acute caerulein-induced pancreatitis, leading to more severe damage, loss of the normal acinar compartment, and increased cytokeratin 19-positive metaplasias and immune cell infiltration. We further demonstrate the Men1 protein is stabilized in response to insult, and loss of Men1 is associated with the overexpression of proinflammatory Jund target genes, suggesting that loss of Men1-mediated repression of Jund activity is, at least in part, responsible for the impaired response. Finally, we demonstrate that Men1 loss significantly accelerates mutant Kras-dependent oncogenesis. Combined, this work establishes Men1 as an important mediator of pancreas homeostasis in vivo.

Background Epidermal growth factor receptor (EGFR) amplification and TP53 mutation are the two most common genetic alterations in glioblastoma multiforme (GBM). A comprehensive analysis of the TCGA GBM database revealed a subgroup with near mutual exclusivity of EGFR amplification and TP53 mutations indicative of a role of EGFR in regulating wild-type-p53 (wt-p53) function. The relationship between EGFR amplification and wt-p53 function remains undefined and this study describes the biological significance of this interaction in GBM. Methods Mass spectrometry was used to identify EGFR-dependent p53-interacting proteins. The p53 and DNA-dependent protein kinase catalytic subunit (DNA-PKcs) interaction was detected by co-immunoprecipitation. We used CRISPR-Cas9 gene editing to knockout EGFR and DNA-PKcs and the Edit-R CRIPSR-Cas9 system for conditional knockout of EGFR. ROS activity was measured with a CM-H2DCFDA probe, and real-time PCR was used to quantify expression of p53 target genes. Results Using glioma sphere-forming cells (GSCs), we identified, DNA-PKcs as a p53 interacting protein that functionally inhibits p53 activity. We demonstrate that EGFR knockdown increased wt-p53 transcriptional activity, which was associated with decreased binding between p53 and DNA-PKcs. We further show that inhibition of DNA-PKcs either by siRNA or an inhibitor (nedisertib) increased wt-p53 transcriptional activity, which was not enhanced further by EGFR knockdown, indicating that EGFR suppressed wt-p53 activity through DNA-PKcs binding with p53. Finally, using conditional EGFR-knockout GSCs, we show that depleting EGFR increased animal survival in mice transplanted with wt-p53 GSCs. Conclusion This study demonstrates that EGFR signaling inhibits wt-p53 function in GBM by promoting an interaction between p53 and DNA-PKcs.

Multiple Endocine Neoplasia Type 1 (MEN1) is associated with duodenopancreatic neuroendocrine tumors (dpNETs) and metastatic dpNET is the primary cause of disease-related mortality for this condition. Currently, there is a paucity of prognostic factors that can reliably identify patients with MEN1-related dpNETS who are at high risk of distant metastasis. Previously, we uncovered a blood-based polyamine metabolite signature that was associated with MEN1-dpNET disease progression. In the current study, we aimed to build upon our prior findings, by exploring the contributions of proteomics for identifying circulating protein markers associated with tumor progression. We performed in-depth proteomic analysis of serially collected plasmas from a genetically engineered mouse model of Men1-pNET, Men1fl/flPdx1-CreTg, and Men1fl/fl control mice to assess dynamic changes in the plasma proteome that associated with disease progression. Findings were compared to plasma proteomic profiles from a cohort of 56 patients with MEN1 (14 with distant metastasis dpNETs (cases) and 42 with either indolent dpNETs or no dpNETs (controls)). Analyses revealed 196 proteins related to oncogenic N-MYC, YAP1, POU5F1, and SMAD that were positively associated with pNET disease progression in Men1fl/flPdx1-CreTg mice. Similarly, 187 proteins were elevated in MEN1 patients with distant metastasis compared controls. Proteins with increased levels in metastatic cases included AMY2B, CELA3B, RNASE1, IGFBP2, CHI3L1, LYZ, TIMP1, LRG1, and COL18A1 previously associated with pancreatic cancer and other neuronal proteins. Cross-species intersection revealed 19 proteins including NDC80, DEF8, SPAG17, ATM, IMMT, DNAH6, DSP, CIT, HRG, CD79A, BDP1, SERPINA11, TARBP1, and SERPIND1 that were positively associated with disease progression in Men1fl/flPdx1-CreTg mice and human subjects. Our integrated analyses identified novel circulating protein features associated with disease progression in MEN1-related dpNET. Citation Format: Johannes F. Fahrmann, Amanda R. Wasylishen, Carolina R.C. Pieterman, Ehsan Irajizad, Jody Vykoukal, Ranran Wu, Jennifer D. Dennison, Christine B. Peterson, Guillermina Lozano, Hua Zhao, Kim-Ahn Do, Daniel M. Halperin, Sunita K. Agarwal, Jenny E. Blau, Jaydira D. Rivero, Naris Nilubol, Mary F. Walter, James M. Welch, Lee S. Weinstein, Menno R. Vriens, Rachel S. van Leeuwaarde, Mark J.C. van Treijen, Gerlof D. Valk, Nancy D. Perrier, Sam M Hanash, Hiroyuki Katayama. Blood-based proteomic signatures associated with MEN1-related duodenopancreatic neuroendocrine tumor progression. [abstract]. In: Proceedings of the AACR Special Conference: Precision Prevention, Early Detection, and Interception of Cancer; 2022 Nov 17-19; Austin, TX. Philadelphia (PA): AACR; Can Prev Res 2023;16(1 Suppl): Abstract nr P066.

Gastrointestinal (GI) syndrome is a serious side effect and dose-limiting toxicity observed in patients undergoing lower-abdominal radiotherapy. Previous mouse studies show that p53 gene dosage determines susceptibility to GI syndrome development. However, the translational relevance of p53 activity has not been addressed. Here, we used a knock-in mouse in which the p53–Mdm2 negative feedback loop is genetically disrupted. These mice retain biallelic p53 and thus, normal basal p53 levels and activity. However, due to the lack of p53-mediated Mdm2 transcription, irradiated Mdm2(P2/P2) mice exhibit enhanced acute p53 activity, which protects them from GI failure. Intestinal crypt cells residing in the +4 and higher positions exhibit decreased apoptosis, increased p21 expression, and hyperproliferation to reinstate intestinal integrity. Correspondingly, pharmacological augmentation of p53 activity in wild-type mice with an Mdm2 inhibitor protects against GI toxicity without affecting therapeutic outcome. Our results suggest that transient disruption of the p53–Mdm2 interaction to enhance p53 activity could be a viable prophylactic strategy for alleviating GI syndrome in patients undergoing radiotherapy.

Context Duodenopancreatic neuroendocrine tumors (dpNETs) frequently occur in patients with multiple endocrine neoplasia type 1 (MEN1), and metastatic dpNET is the primary cause of disease-related mortality. There is a need for biomarkers that can identify patients with MEN1-related dpNETs that are at high risk of developing distant metastasis. Polyamines have tumor-promoting roles in several cancer types. Objective We hypothesized that MEN1-dpNET–related disease progression is associated with elevated levels of circulating polyamines. Methods Through an international collaboration between The University of Texas MD Anderson Cancer Center, the National Institutes of Health, and the University Medical Center Utrecht, plasma polyamine levels were assessed using mass spectrometry in 84 patients with MEN1 (20 with distant metastatic dpNETs [patients] and 64 with either indolent dpNETs or no dpNETs [controls]). A mouse model of MEN1-pNET, Men1fl/flPdx1-CreTg, was used to test time-dependent changes in plasma polyamines associated with disease progression. Results A 3-marker plasma polyamine signature (3MP: N-acetylputrescine, acetylspermidine, and diacetylspermidine) distinguished patients with metastatic dpNETs from controls in an initial set of plasmas from the 3 participating centers. The fixed 3MP yielded an area under the curve of 0.84 (95% CI, 0.62-1.00) with 66.7% sensitivity at 95% specificity for distinguishing patients from controls in an independent test set from MDACC. In Men1fl/flPdx1-CreTg mice, the 3MP was elevated early and remained high during disease progression. Conclusion Our findings provide a basis for prospective testing of blood-based polyamines as a potential means for monitoring patients with MEN1 for harboring or developing aggressive disease.

Cell stress and DNA damage activate the tumor suppressor p53, triggering transcriptional activation of a myriad of target genes. The molecular, morphological, and physiological consequences of this activation remain poorly understood in vivo. We activated a p53 transcriptional program in mice by deletion of Mdm2, a gene that encodes the major p53 inhibitor. By overlaying tissue-specific RNA-sequencing data from pancreas, small intestine, ovary, kidney, and heart with existing p53 chromatin immunoprecipitation (ChIP) sequencing, we identified a large repertoire of tissue-specific p53 genes and a common p53 transcriptional signature of seven genes, which included Mdm2 but not p21. Global p53 activation caused a metaplastic phenotype in the pancreas that was missing in mice with acinar-specific p53 activation, suggesting non-cell-autonomous effects. The p53 cellular response at single-cell resolution in the intestine altered transcriptional cell state, leading to a proximal enterocyte population enriched for genes within oxidative phosphorylation pathways. In addition, a population of active CD8+ T cells was recruited. Combined, this study provides a comprehensive profile of the p53 transcriptional response in vivo, revealing both tissue-specific transcriptomes and a unique signature, which were integrated to induce both cell-autonomous and non-cell-autonomous responses and transcriptional plasticity.

Pancreatic ductal adenocarcinoma (PDAC) is almost uniformly fatal and characterized by early metastasis. Oncogenic KRAS mutations prevail in 95% of PDAC tumors and co-occur with genetic alterations in the TP53 tumor suppressor in nearly 70% of patients. Most TP53 alterations are missense mutations that exhibit gain-of-function phenotypes that include increased invasiveness and metastasis yet the extent of direct cooperation between KRAS effectors and mutant p53 remains largely undefined. We show that oncogenic KRAS effectors activate cyclic AMP responsive element binding protein 1 (CREB1) to allow physical interactions with mutant p53 that hyperactivate multiple pro-metastatic transcriptional networks. Specifically, mutant p53 and CREB1 upregulate the pro-metastatic, pioneer transcription factor, FOXA1, activating its transcriptional network while promoting WNT/β-catenin signaling, together driving PDAC metastasis. Pharmacologic CREB1 inhibition dramatically reduced FOXA1 and β-catenin expression and dampened PDAC metastasis, identifying a new therapeutic strategy to disrupt cooperation between oncogenic KRAS and mutant p53 to mitigate metastasis.

Daxx loss causes coordinate dysregulation of ERVs and protein-coding genes after physiologic stress and impairs tissue recovery., Tumor sequencing studies have emphasized the role of epigenetics and altered chromatin homeostasis in cancer. Mutations in DAXX, which encodes a chaperone for the histone 3.3 variant, occur in 25% of pancreatic neuroendocrine tumors (PanNETs). To advance our understanding of physiological functions of Daxx, we developed a conditional Daxx allele in mice. We demonstrate that Daxx loss is well tolerated in the pancreas but creates a permissive transcriptional state that cooperates with environmental stress (inflammation) and other genetic lesions (Men1 loss) to alter gene expression and cell state, impairing pancreas recovery from inflammatory stress in vivo. The transcriptional changes are associated with dysregulation of endogenous retroviral elements (ERVs), and dysregulation of endogenous genes near ERVs is also observed in human PanNETs with DAXX mutations. Our results reveal a physiologic function of DAXX, provide a mechanism associated with impaired tissue regeneration and tumorigenesis, and expand our understanding of ERV regulation in somatic cells.

p53 is a transcription factor that regulates expression of genes involved in cell cycle arrest, senescence, and apoptosis. TP53 harbors mutations that inactivate its transcriptional activity in roughly 30% of breast cancers, and these tumors are much more likely to undergo a pathological complete response to chemotherapy. Thus, the gene expression program activated by wild-type p53 contributes to a poor response. We used an in vivo genetic model system to comprehensively define the p53- and p21-dependent genes and pathways modulated in tumors following doxorubicin treatment. We identified genes differentially expressed in spontaneous mammary tumors harvested from treated MMTV-Wnt1 mice that respond poorly (Trp53+/+) or favorably (Trp53-null) and those that lack the critical senescence/arrest p53 target gene Cdkn1a. Trp53 wild-type tumors differentially expressed nearly 10-fold more genes than Trp53-null tumors after treatment. Pathway analyses showed that genes involved in cell cycle, senescence, and inflammation were enriched in treated Trp53 wild-type tumors; however, no genes/pathways were identified that adequately explain the superior cell death/tumor regression observed in Trp53-null tumors. Cdkn1a-null tumors that retained arrest capacity (responded poorly) and those that proliferated (responded well) after treatment had remarkably different gene regulation. For instance, Cdkn1a-null tumors that arrested upregulated Cdkn2a (p16), suggesting an alternative, p21-independent route to arrest. Live animal imaging of longitudinal gene expression of a senescence/inflammation gene reporter in Trp53+/+ tumors showed induction during and after chemotherapy treatment, while tumors were arrested, but expression rapidly diminished immediately upon relapse.

Increased levels of inhibitors of the p53 tumor suppressor such as Mdm2 and Mdm4 drive tumor development and thus serve as targets for therapeutic intervention. Recently, digestive organ expansion factor (Diexf) has been identified as a novel inhibitor of p53 in zebrafish. Here, we address the potential role of Diexf as a regulator of the p53 pathway in mammals by generating Diexf knockout mice. We demonstrate that, similar to Mdm2 and Mdm4, homozygous deletion of Diexf is embryonic lethal. However, unlike in Mdm2 and Mdm4 mice, loss of p53 does not rescue this phenotype. Moreover, Diexf heterozygous animals are not sensitive to sub-lethal ionizing radiation. Thus, we conclude that Diexf is an essential developmental gene in the mouse, but is not a significant regulator of the p53 pathway during development or in response to ionizing radiation.

Background Ewing sarcoma is a malignancy of primitive cells, possibly of mesenchymal origin. It is probable that genetic perturbations other than EWS-FLI1 cooperate with it to produce the tumor. Sequencing studies identified STAG2 mutations in approximately 15% of cases in humans. In the present study, we hypothesize that loss of Stag2 cooperates with EWS-FLI1 in generating sarcomas derived from murine mesenchymal stem cells (MSCs). Methods Mice bearing an inducible EWS-FLI1 transgene were crossed to p53−/− mice in pure C57/Bl6 background. MSCs were derived from the bone marrow of the mice. EWS-FLI1 induction and Stag2 knockdown were achieved in vitro by adenovirus-Cre and shRNA-bearing pGIPZ lentiviral infection, respectively. The cells were then treated with ionizing radiation to 10 Gy. Anchorage independent growth in vitro was assessed by soft agar assays. Cellular migration and invasion were evaluated by transwell assays. Cells were injected with Matrigel intramuscularly into C57/Bl6 mice to test for tumor formation. Results Primary murine MSCs with the genotype EWS-FLI1 p53−/− were resistant to transformation and did not form tumors in syngeneic mice without irradiation. Stag2 inhibition increased the efficiency and speed of sarcoma formation significantly in irradiated EWS-FLI1 p53−/− MSCs. The efficiency of tumor formation was 91% for cells in mice injected with Stag2-repressed cells and 22% for mice receiving cells without Stag2 inhibition (p Stag2 knockdown reduced survival of mice in Kaplan-Meier analysis (p Conclusion Loss of Stag2 has a synergistic effect with EWS-FLI1 in the production of sarcomas from murine MSCs, but the mechanism may not relate to increased proliferation or chromosomal instability. Primary murine MSCs are resistant to transformation, and the combination of p53 null mutation, EWS-FLI1, and Stag2 inhibition does not confer immediate conversion of MSCs to sarcomas. Irradiation is necessary in this model, suggesting that perturbations of other genes beside Stag2 and p53 are likely to be essential in the development of EWS-FLI1-driven sarcomas from MSCs.

Comprehensive analysis of the TCGA Glioblastoma database has identified 3 prognostically relevant subgroups (Proneural, Classical and Mesenchymal). Interestingly, it has also revealed a near mutual exclusivity of EGFR amplification and TP53 mutations. There are only a few tumors with both amplified EGFR and mutant TP53. We decided to investigate the functional relationship between EGFR and TP53 relative to tumorigenesis and tumor proliferation. We selected from our molecularly characterized GSC bank a series of EGFR amplified+TP53wt/TP53mut lines for this study. Here we report that EGFR amplification overrides the effect of p53wt but co-regulates tumorigenesis with TP53R175H mutation. We show that CRISPR EGFR knockout in TP53wt cells leads to complete loss of cell growth suggesting that EGFR is essential for cell survival, providing growth signal and suppression of p53wt function. We further show that EGFR induces physical binding between p53wt and DNA-PKcs, which suppresses p53wt by inhibiting p53 phosphorylation at S15. The knockdown of DNA-PKcs restores p53wt anti-tumor function. In contrast, GSC262 harboring TP53R175H mutation is the only GSC that survives after EGFR knockout, suggesting that this specific mutation has a gain of function (GOF) to sustain growth without EGFR. This is confirmed by dual knockout of EGFR and TP53R175H showing no surviving cells. Further investigation shows both EGFR and p53R175H induce tumor glycolysis, the major energy metabolism and heightened growth signal for this group. We further show that EGFR mediated suppression of p53R175H is through DNA-PKcs binding to p53R175H and reduced S15 phosphorylation. Taken together, this study reveals 1) EGFR amplification is essential for tumor growth in TP53wt GSCs; 2) novel crosstalk between EGFR and p53 through DNA-PKcs. 3) EGFR suppresses p53wt function by providing the energy required for tumor growth through inducing glycolysis, and 4) TP53R175H is a GOF mutation that sustains life without EGFR by inducing glycolysis.

Objectives Molecular characterization of sporadic pancreatic neuroendocrine tumors (PanNETs) demonstrates frequent alterations in MEN1. As the role of menin immunohistochemistry as a potential biomarker is being developed, knowledge of whether the pattern of menin expression is the same in primary tumors and distant metastases may help in patient care. Therefore, we compared patterns of menin expression in matched primary tumors and metastases. Methods We evaluated loss of menin nuclear expression by immunohistochemistry in 44 matched samples of primary and metastatic PanNETs and concordance in staining pattern between primary and metastatic tumors. Results Menin nuclear expression was lost in 18 (41%) of 44 primary tumors and 17 (39%) of 44 metastases. Concordant loss of menin expression was observed in 41 cases (93%); discordance was observed in 3 cases (7%; 95% confidence interval, 1.4%-18.7%), including 2 with loss in the primary tumor but not the metastasis. Conclusions The concordance of menin staining between primary tumor and metastasis in most cases suggests that menin loss is an early event in PanNET tumorigenesis. The discordant expression observed in a small subset may be a source of menin-directed therapy failure; thus, repeat assessment of metastases may be helpful for treatment selection.

Dicer1 functions as a tumor suppressor in mouse models. In humans, somatic mutations are associated with many cancers in adults, and patients with DICER1 syndrome with DICER1 germline mutations are susceptible to childhood cancers. Dicer is phosphorylated by the ERK-MAP kinase pathway and because this pathway is activated in human cancers, we asked whether phosphorylated Dicer1 contributed to tumor development. In human endometrioid cancers, we discovered that phosphorylated DICER1 is significantly associated with invasive disease. To test a direct involvement of Dicer1 phosphorylation in tumor development, we studied mice with phosphomimetic alterations at the two conserved serines phosphorylated by ERK and discovered that a phosphomimetic Dicer1 drives tumor development and dissemination in two independent murine cancer models (KRas+/LA1 and p53+/−). Our findings demonstrate that phosphomimetic Dicer1 promotes tumor development and invasion. Significance: This work highlights the relevance of Dicer1 phosphorylation in mammalian tumor development and dissemination.

Background: Multiple Endocrine Neoplasia Type 1 (MEN1) is a rare inherited autosomal dominant disease predisposing patients to endocrine tumors. MEN1 can be genetically diagnosed at an early age. Patients are prone to develop benign parathyroid tumors then multifocal duodenopancreatic neuroendocrine tumors (dpNETs), which have a penetrance of >80% by age 80. Although they can be identified at an early stage, preventive measures do not exist, and one fifth of patients develop distant metastases, which is the most significant cause of mortality. Currently, no biomarkers can reliably predict which patients with MEN1-related dpNETs are at high risk of developing metastatic disease. Polyamines are naturally occurring polycationic alkylamines that have been implicated to play functional roles in promoting neoplastic transformation and growth. We have previously demonstrated a plasma polyamine signature that associates with pancreatic cancer development and that also offers value for predicting future distant metastasis in patients with triple-negative breast cancer. We hypothesized that such a plasma polyamine signature may similarly associate with disease progression in subjects with MEN1-related dpNET. Methods: As part of an international collaboration, we measured plasma polyamine levels using mass spectrometry from 14 MEN1 patients with distant metastatic dpNET (cases), 28 MEN1 patients with indolent dpNETs without distant metastases (control-1), and 14 MEN1 patients without dpNETs (control-2). Five circulating plasma polyamines were quantified in this initial test set. A combination rule was developed using logistic regression models. Findings were validated in an independent set of plasma from 6 cases and 22 controls (n=13 control-1 and n=9 control-2). Results: Area under the Receiver Operating Characteristic Curve (AUC) of individual polyamines delineating cases from controls ranged from 0.50-0.75 in the test set. A polyamine signature consisting of 3 polyamines developed in the test set yielded an AUC of 0.84 (95% CI: 0.63-1.00) with 67% sensitivity at 95% specificity in the validation set for distinguishing cases from controls. The predictive performance of the polyamine signature for distinguishing cases from MEN1 patients with indolent dpNETS without distant metastasis or MEN1 patients without dpNETS in the validation set was 0.79 (95% CI: 0.53-1.00) and 0.91 (95% CI: 0.75-1.00) with respective resultant sensitivity at 95% specificity of 50% and 67%. Conclusion: Our findings reveal a plasma polyamine signature associated with disease progression in subjects with MEN1-related dpNETs. This polyamine signature may provide a potential breakthrough for predicting progression and distant metastasis in MEN1 patients. Further prospective studies are warranted.

The prognosis of metastatic osteosarcoma is dismal and a better understanding of the mechanisms underlying disease progression is essential to improve treatment options and patient outcomes. We previously demonstrated Pla2g16 overexpression in mouse osteosarcoma contributes to metastasis phenotypes and increased expression of PLA2G16 is associated with metastasis and poor prognosis in human tumors. To further examine the mechanisms through which PLA2G16 contributes to human osteosarcoma metastasis and explore the potential of PLA2G16 as a therapeutic target in osteosarcoma, we generated a panel of human osteosarcoma cell lines expressing different levels of PLA2G16. The functional analyses of these cell lines demonstrated high levels of PLA2G16 expression increased osteosarcoma cell migration, invasion, clonogenic survival, and anchorage-independent colony formation. Importantly, this activity was dependent on the phospholipase activity of PLA2G16. Additionally, PLA2G16 overexpression decreased the sensitivity of cells to a panel of chemotherapeutic agents. Analysis of downstream pathways revealed the pro-metastasis functions of PLA2G16 were mediated through the MAPK pathway, as knockdown of PLA2G16 decreased ERK1/2 phosphorylation and pharmacological inhibition of MEK significantly repressed PLA2G16 mediated cell migration and clonogenic survival. Furthermore, PLA2G16 overexpression promoted xenograft tumor growth in vivo, and these tumors exhibit increased ERK1/2 phosphorylation. Lastly, the expression of PLA2G16 is strongly correlated with the increased ERK1/2 phosphorylation in human osteosarcoma samples, and the combined lesions are associated with reduced overall and metastasis-free survival. Collectively, these results demonstrate increased PLA2G16 expression activates the MAPK pathway to enhance osteosarcoma metastasis and may be a novel therapeutic target for these cancers.

DICER1 gene alterations and decreased expression are associated with developmental disorders and diseases in humans. Oscillation of Dicer1 phosphorylation and dephosphorylation regulates its function during the oocyte-to-embryo transition in Caenorhabditis elegans . Dicer1 is also phosphorylated upon FGF stimulation at conserved serines in mouse embryonic fibroblasts and HEK293 cells. However, whether phosphorylation of Dicer1 has a role in mammalian development remains unknown. To investigate the consequence of constitutive phosphorylation, we generated phosphomimetic knock-in mouse models by replacing conserved serines 1712 and 1836 with aspartic acids individually or together. Dicer1 S1836D/S1836D mice display highly penetrant postnatal lethality, and the few survivors display accelerated aging and infertility. Homozygous dual-phosphomimetic Dicer1 augments these defects, alters metabolism-associated miRNAs, and causes a hypermetabolic phenotype. Thus, constitutive phosphorylation of Dicer1 results in multiple pathologic processes in mice, indicating that phosphorylation tightly regulates Dicer1 function and activity in mammals.

Epidermal growth factor receptor (EGFR) amplification/mutation and TP53 mutation are two most frequent genetic events that differentiate molecular subtypes of Glioblastoma. An extensive analysis of the Glioblastoma TCGA database showed a near exclusivity between p53 mutation and EGFR amplification in two groups of patients with a third group that has neither event. To understand the relationship between EFGR amplification and TP53 status in tumorigenesis, we identified a series of glioma stem cell (GSC) lines with EGFR amplification from our GSC bank at MD Anderson Cancer Center. The EGFR amplified group was further divided into two groups based on p53 status: one group with wild type TP53 and the second group with mutant p53. To elucidate the role of EGFR in regulating p53 function in gliomas, we used CRISPR gene editing system to knock out EGFR expression in amplified cells lines. EGFR knockout clones were isolated only from TP53 mutant GSCs, but not in TP53 wild type GSCs. Knocking down of EGFR expression by siRNA, showed that p53 activity was increased in wild type TP53 GSCs as demonstrated by increased p21/WAF1/CDKN1A and BCL2 Associated X (BAX) expression. However, direct binding between EGFR and wild type p53 was not detected in these GSCs. Using co-IP and mass spectrometry, we found that DNA-PK is the linker between EGFR and wild type p53. EGFR knock down decreased the binding between DNA-PK and wild type p53. Furthermore, EGFR knock down resulted in decreased sphere numbers and sizes in wild type TP53 GSCs but not in mutant GSCs. In conclusion, we propose that EGFR functions as the driver of tumorigenesis and progression in EGFR amplified TP53 wild type GSCs by inhibiting wild type p53 function through DNA-PK, while EGFR amplification and TP53 mutation co-regulate the tumorigenesis process in TP53 mutant GSCs.

p53(R172H/+) mice inherit a p53 mutation found in Li-Fraumeni syndrome and develop metastatic tumors at much higher frequency than p53(+/-) mice. To explore the mutant p53 metastatic phenotype, we used expression arrays to compare primary osteosarcomas from p53(R172H/+) mice with metastasis to osteosarcomas from p53(+/-) mice lacking metastasis. For this study, 213 genes were differentially expressed with a P value

The ability of Myc to promote cellular transformation is well established; however, a better understanding of the mechanisms through which Myc mediates tumorigenesis is essential for the development of therapeutic approaches to target this potent oncoprotein. Structure-function studies in rodent fibroblast cells have provided the basis for much of our current understanding of these mechanisms. To build on these approaches, we have characterized three novel human cell line models of Myc-dependent transformation: MCF10A, SH-EP Tet21/N-Myc, and LF1/TERT/LT/ST cells. We have also evaluated Myc family proteins (c-Myc and L-Myc), a naturally occurring isoform of Myc (MycS), and a set of N-terminal domain mutants (ΔMBII, W135E, T58A) for their ability to promote anchorage-independent growth in these models. Taken together, these results provide the field with three new human cell-based models to study Myc activity, highlight the importance of cellular context, and challenge the paradigm that the ability of Myc to promote tumorigenesis is exclusively MBII-dependent.

The iconic history of the Myc oncoprotein encompasses 3 decades of intense scientific discovery. There is no question that Myc has been a pioneer, advancing insight into the molecular basis of cancer as well as functioning as a critical control center for several diverse biological processes and regulatory mechanisms. This narrative chronicles the journey and milestones that have defined the understanding of Myc, and it provides an opportunity to consider future directions in this challenging yet rewarding field.

An improved synthesis of phenacyl methyl disulfide is described. It led to conditions that efficiently furnish the ketal of phenacyl mercaptan. The ketal of phenacyl mercaptan appears to be the first known α -mercapto ketal. Phenacyl mercaptan is smoothly prepared from its ketal under standard conditions.

The c-MYC transcription factor is a master regulator of many cellular processes and deregulation of this oncogene has been linked to more than 50% of all cancers. This deregulation can take many forms, including altered post-translational regulation. Here, using immunoprecipitation combined with mass spectrometry, we identified a MYC SUMOylation site (K326). Abrogation of signaling through this residue by substitution with arginine (K326R) has no obvious effects on MYC half-life, intracellular localization, transcriptional targets, nor on the biological effects of MYC overexpression in two different cell systems assessed for soft agar colony formation, proliferation, and apoptosis. While we have definitively demonstrated that MYC SUMOylation can occur on K326, future work will be needed to elucidate the mechanisms and biological significance of MYC regulation by SUMOylation.