Context-dependent Tumor Suppressor Research Articles

YAP inhibits NF-κB signaling and ccRCC growth by opposing p65-ZHX2 cooperativity.

Hippo pathway functions as a tumor suppressor pathway by inhibiting the oncogenic potential of pathway effectors YAP/TAZ. However, YAP can also function as a context-dependent tumor suppressor in several types of cancer including clear cell renal cell carcinomas (ccRCC). Here we show that YAP blocks NF-κB signaling in ccRCC to inhibit cancer cell growth. Mechanistically, YAP inhibits the expression of ZHX2, a critical p65 co-factor in ccRCC. Furthermore, YAP competes with ZHX2 for binding to p65. Consequently, elevated nuclear YAP blocks the cooperativity between ZHX2 and p65, leading to diminished NF-κB target gene expression. Pharmacological inhibition of Hippo/MST1/2 blocked NF-κB transcriptional program and suppressed ccRCC cancer cell growth, which can be rescued by ZHX2/p65 overexpression. Our study uncovers a novel crosstalk between the Hippo and NF-κB pathways and its involvement in ccRCC growth inhibition, suggesting that targeting the Hippo pathway may provide a therapeutical opportunity for ccRCC treatment.

GLS and GLS2 Glutaminase Isoenzymes in the Antioxidant System of Cancer Cells.

A pathway frequently altered in cancer is glutaminolysis, whereby glutaminase (GA) catalyzes the main step as follows: the deamidation of glutamine to form glutamate and ammonium. There are two types of GA isozymes, named GLS and GLS2, which differ considerably in their expression patterns and can even perform opposing roles in cancer. GLS correlates with tumor growth and proliferation, while GLS2 can function as a context-dependent tumor suppressor. However, both isoenzymes have been described as essential molecules handling oxidant stress because of their involvement in glutathione production. We reviewed the literature to highlight the critical roles of GLS and GLS2 in restraining ROS and regulating both cellular signaling and metabolic stress due to their function as indirect antioxidant enzymes, as well as by modulating both reductive carboxylation and ferroptosis. Blocking GA activity appears to be a potential strategy in the dual activation of ferroptosis and inhibition of cancer cell growth in a ROS-mediated mechanism.

STAT1 mediated downregulation of the tumor suppressor gene PDCD4, is driven by the atypical cadherin FAT1, in glioblastoma

STAT1 (Signal Transducer and Activator of Transcription 1), belongs to the STAT protein family, essential for cytokine signaling. It has been reported to have either context dependent oncogenic or tumor suppressor roles in different tumors. Earlier, we demonstrated that Glioblastoma multiforme (GBMs) overexpressing FAT1, an atypical cadherin, had poorer outcomes. Overexpressed FAT1 promotes pro-tumorigenic inflammation, migration/invasion by downregulating tumor suppressor gene, PDCD4. Here, we demonstrate that STAT1 is a novel mediator downstream to FAT1, in downregulating PDCD4 in GBMs. In-silico analysis of GBM databases as well as q-PCR analysis in resected GBM tumors showed positive correlation between STAT1 and FAT1 mRNA levels. Kaplan-Meier analysis showed poorer survival of GBM patients having high FAT1 and STAT1 expression. SiRNA-mediated knockdown of FAT1 decreased STAT1 and increased PDCD4 expression in glioblastoma cells (LN229 and U87MG). Knockdown of STAT1 alone resulted in increased PDCD4 expression. In silico analysis of the PDCD4 promoter revealed four putative STAT1 binding sites (Site1-Site4). ChIP assay confirmed the binding of STAT1 to site1. ChIP-PCR revealed decrease in the binding of STAT1 on the PDCD4 promoter after FAT1 knockdown. Site directed mutagenesis of Site1 resulted in increased PDCD4 luciferase activity, substantiating STAT1 mediated PDCD4 inhibition. EMSA confirmed STAT1 binding to the Site 1 sequence. STAT1 knockdown led to decreased expression of pro-inflammatory cytokines and EMT markers, and reduced migration/invasion of GBM cells. This study therefore identifies STAT1 as a novel downstream mediator of FAT1, promoting pro-tumorigenic activity in GBM, by suppressing PDCD4 expression.

Abstract 6281: Context dependent role of glucocorticoids in breast cancer

Abstract Glucocorticoids bind to the transcription factor glucocorticoid receptor (GR) and regulate genes in response to cellular metabolism, inflammation, and stress. Although GR has been extensively evaluated in inflammation and metabolic diseases, its role as a therapeutic target in cancer has not been thoroughly studied. Results from studies with another member of the steroid receptor family, androgen receptor (AR), demonstrated that the AR is an oncogene in estrogen receptor (ER)-negative breast cancer, but a tumor suppressor in ER-positive breast cancer. We expected a comparable mechanism with GR in different breast cancer subtypes. In this study, we evaluated the role of GR in various breast cancer subtypes and discerned the mechanism of action. Six breast cancer cell lines and seven patient-derived xenografts (PDXs) were characterized by Western blot for their expression of GR, ER and AR. Proliferation assays were performed in cell lines with a dose-response of a synthetic glucocorticoid, dexamethasone. Subsequently, we performed xenograft studies in cell line xenografts (CDXs) and PDXs. GR was ubiquitously expressed in various cell lines and PDX models. Dexamethasone reduced cell proliferation in ER-positive models, while it increased the proliferation in ER-negative cell lines, suggesting that the GR might have a mechanism of action that is similar to AR. GR antagonist, mifepristone, reversed the effects of dexamethasone. CDX studies in an ER-positive cell line, MCF7 suggested that dexamethasone (10 mpk) inhibited the tumor growth significantly that was better than the standard of care fulvestrant. This result was confirmed in an ER positive PDX model HCI013. Study by Obradović et al, indicates that glucocorticoids have an oncogenic role and furthermore promote breast cancer metastasis in TNBC model thereby validating the subtype and context dependent role of the glucocorticoid receptor. We are currently exploring the mechanism of GR’s context-dependent tumor suppressor and oncogenic role in distinct breast cancer subtypes. Citation Format: Wendy Effah, Suriyan Ponnusamy, Sarah Asemota, Yekta Khosrosereski, Ramesh Narayanan. Context dependent role of glucocorticoids in breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6281.

KLF4 is an epigenetically modulated, context-dependent tumor suppressor.

The epigenetic layer of regulation has become increasingly relevant in the research focused on tumor suppressors. KLF4 is a well-described zinc-finger transcription factor, mainly known for its role in the acquisition of cell pluripotency. Here we report and describe the most relevant epigenetic regulation mechanisms that affect KLF4 expression in tumors. CpG island methylation emerges as the most common mechanism in several tumors including lung adenocarcinoma, hepatocellular carcinoma, non-Hodgkin lymphomas, among others. Further layers of regulation represented by histone methylation and acetylation and by non-coding RNAs are described. Overall, KLF4 emerges as a crucial target in the fight against cancer.

Emerging Roles of SIRT5 in Metabolism, Cancer, and SARS-CoV-2 Infection.

Sirtuin 5 (SIRT5) is a predominantly mitochondrial enzyme catalyzing the removal of glutaryl, succinyl, malonyl, and acetyl groups from lysine residues through a NAD+-dependent deacylase mechanism. SIRT5 is an important regulator of cellular homeostasis and modulates the activity of proteins involved in different metabolic pathways such as glycolysis, tricarboxylic acid (TCA) cycle, fatty acid oxidation, electron transport chain, generation of ketone bodies, nitrogenous waste management, and reactive oxygen species (ROS) detoxification. SIRT5 controls a wide range of aspects of myocardial energy metabolism and plays critical roles in heart physiology and stress responses. Moreover, SIRT5 has a protective function in the context of neurodegenerative diseases, while it acts as a context-dependent tumor promoter or suppressor. In addition, current research has demonstrated that SIRT5 is implicated in the SARS-CoV-2 infection, although opposing conclusions have been drawn in different studies. Here, we review the current knowledge on SIRT5 molecular actions under both healthy and diseased settings, as well as its functional effects on metabolic targets. Finally, we revise the potential of SIRT5 as a therapeutic target and provide an overview of the currently reported SIRT5 modulators, which include both activators and inhibitors.

FAT1 expression in T-cell acute lymphoblastic leukemia (T-ALL) modulates proliferation and WNT signaling

FAT atypical cadherin 1 (FAT1), a transmembrane protein, is frequently mutated in various cancer types and has been described as context-dependent tumor suppressor or oncogene. The FAT1 gene is mutated in 12–16% of T-cell acute leukemia (T-ALL) and aberrantly expressed in about 54% of T-ALL cases contrasted with absent expression in normal T-cells. Here, we characterized FAT1 expression and profiled the methylation status from T-ALL patients. In our T-ALL cohort, 53% of patient samples were FAT1 positive (FAT1pos) compared to only 16% FAT1 positivity in early T-ALL patient samples. Aberrant expression of FAT1 was strongly associated with FAT1 promotor hypomethylation, yet a subset, mainly consisting of TLX1-driven T-ALL patient samples showed methylation-independent high FAT1 expression. Genes correlating with FAT1 expression revealed enrichment in WNT signaling genes representing the most enriched single pathway. FAT1 knockdown or knockout led to impaired proliferation and downregulation of WNT pathway target genes (CCND1, MYC, LEF1), while FAT1 overexpressing conveyed a proliferative advantage. To conclude, we characterized a subtype pattern of FAT1 gene expression in adult T-ALL patients correlating with promotor methylation status. FAT1 dependent proliferation and WNT signaling discloses an impact on deeper understanding of T-ALL leukemogenesis as a fundament for prospective therapeutic strategies.

YAP antagonizes TEAD-mediated AR signaling and prostate cancer growth.

Hippo signaling restricts tumor growth by inhibiting the oncogenic potential of YAP/TAZ-TEAD transcriptional complex. Here, we uncover a context-dependent tumor suppressor function of YAP in androgen receptor (AR) positive prostate cancer (PCa) and show that YAP impedes AR+ PCa growth by antagonizing TEAD-mediated AR signaling. TEAD forms a complex with AR to enhance its promoter/enhancer occupancy and transcriptional activity. YAP and AR compete for TEAD binding and consequently, elevated YAP in the nucleus disrupts AR-TEAD interaction and prevents TEAD from promoting AR signaling. Pharmacological inhibition of MST1/2 or LATS1/2, or transgenic activation of YAP suppressed the growth of PCa expressing therapy resistant AR splicing variants. Our study uncovers an unanticipated crosstalk between Hippo and AR signaling pathways, reveals an antagonistic relationship between YAP and TEAD in AR+ PCa, and suggests that targeting the Hippo signaling pathway may provide a therapeutical opportunity to treat PCa driven by therapy resistant AR variants.

Computational modeling implicates protein scaffolding in p38 regulation of Akt

Cells process environmental cues by activating intracellular signaling pathways with numerous interconnections and opportunities for cross-regulation. We employed a systems biology approach to investigate intersections of kinase p38, a context-dependent tumor suppressor or promoter, with Akt and ERK, two kinases known to promote cell survival, proliferation, and drug resistance in cancer. Using live, single cell microscopy, multiplexed fluorescent reporters of p38, Akt, and ERK activities, and a custom automated image-processing pipeline, we detected marked heterogeneity of signaling outputs in breast cancer cells stimulated with chemokine CXCL12 or epidermal growth factor (EGF). Basal activity of p38 correlated inversely with amplitude of Akt and ERK activation in response to either ligand. Remarkably, small molecule inhibitors of p38 immediately decreased basal activities of Akt and ERK but increased the proportion of cells with high amplitude ligand-induced activation of Akt signaling. To identify mechanisms underlying cross-talk of p38 with Akt signaling, we developed a computational model incorporating subcellular compartmentalization of signaling molecules by scaffold proteins. Dynamics of this model revealed that subcellular scaffolding of Akt accounted for observed regulation by p38. The model also predicted that differences in the amount of scaffold protein in a subcellular compartment captured the observed single cell heterogeneity in signaling. Finally, our model predicted that reduction in kinase signaling can be accomplished by both scaffolding and direct kinase inhibition. However, scaffolding inhibition can potentiate future kinase activity by redistribution of pathway components, potentially amplifying oncogenic signaling. These studies reveal how computational modeling can decipher mechanisms of cross-talk between the p38 and Akt signaling pathways and point to scaffold proteins as central regulators of signaling dynamics and amplitude.

Therapeutic Potential and Activity Modulation of the Protein Lysine Deacylase Sirtuin 5.

Sirtiun 5 (SIRT5) is a NAD+-dependent protein lysine deacylase primarily located in mitochondria. SIRT5 displays an affinity for negatively charged acyl groups and mainly catalyzes lysine deglutarylation, desuccinylation, and demalonylation while possessing weak deacetylase activity. SIRT5 substrates play crucial roles in metabolism and reactive oxygen species (ROS) detoxification, and SIRT5 activity is protective in neuronal and cardiac physiology. Moreover, SIRT5 exhibits a dichotomous role in cancer, acting as context-dependent tumor promoter or suppressor. Given its multifaceted activity, SIRT5 is a promising target in the design of activators or inhibitors that might act as therapeutics in many pathologies, including cancer, cardiovascular disorders, and neurodegeneration. To date, few cellular-active peptide-based SIRT5 inhibitors (SIRT5i) have been described, and potent and selective small-molecule SIRT5i have yet to be discovered. In this perspective, we provide an outline of SIRT5’s roles in different biological settings and describe SIRT5 modulators in terms of their mode of action, pharmacological activity, and structure–activity relationships.

Abstract 814: Identification of ERBB4 mutant alleles that function as tumor drivers

Abstract Introduction: ERBB4 (HER4) is a member of the ERBB family of receptor tyrosine kinases, which includes the epidermal growth factor receptor (EGFR/ERBB1/HER1), ERBB2 (Neu/HER2), and ERBB3 (HER3). EGFR and ERBB2 are well-validated targets in a variety of solid tumors. In contrast, the roles that ERBB4 plays in human tumors are less well defined. Nonetheless, it is accepted that ERBB4 homodimers function as tumor suppressors and ERBB4 heterodimers function as oncogenes. Moreover, our in silico analyses of The Cancer Genome Atlas - Skin Cutaneous Melanoma dataset suggest that ERBB4 mutant alleles function as drivers of BRAF WT metastatic melanomas. Furthermore, ERBB2-ERBB4 heterodimers are associated with elevated tumor proliferation in pediatric ependymomas. Thus, here we describe model systems that we are using to identify ERBB4 gain-of-function (GOF) mutant alleles that stimulate the oncogenic activity of ERBB4-ERBB2 or ERBB4-EGFR heterodimers, as well as ERBB4 loss-of-function (LOF) mutant alleles that disrupt the tumor suppressor activity of ERBB4 homodimers. Experimental Procedures: In the 32D mouse myeloid cell line, ERBB4 homodimers inhibit cell proliferation, whereas ERBB4-ERBB4 heterodimers stimulate cell proliferation. Moreover, we have obtained seven human melanoma cell lines that possess WT BRAF. In these model systems we will express known GOF ERBB4 mutants (Y285C, D595V, Q646C, D931Y, K935I), known LOF ERBB4 mutants (V673I, K751M, G802dup, D861Y, R927Q, R1275W, Y1056F, Ct-b) and ERBB4 mutants found in melanoma samples (R106C, E452K, R711C, P759L, D813N, D861N, R992C). We will evaluate the effects of these ERBB4 mutants on cell proliferation, anchorage-independence, and motility. Data Summary: We anticipate that we will identify GOF ERBB4 mutant alleles that stimulate the oncogenic activity of ERBB4 heterodimers and LOF ERBB4 mutant alleles that disrupt the tumor suppressor activity of ERBB4 homodimers. By evaluating these mutants in the context of human melanoma cell lines, we anticipate that we will identify ERBB4 mutant alleles that function as bona fide tumor drivers. Conclusion: ERBB4 appears to be important to the genesis and/or progression of several types of cancer. However, the fact that ERBB4 functions as a context-dependent oncogene and tumor suppressor gene poses a unique challenge to the identification of driver mutant alleles. The development of the model systems that we present here is an invaluable step forward in the study of ERBB4 as a driver of cancer genesis and/or progression. Citation Format: Lauren Lucas, Vipasha Dwivedi, Joely Burgess, Connor Kelley, Elizabeth Knerr, Jessica Markham, Stephen Miller, David Riese. Identification of ERBB4 mutant alleles that function as tumor drivers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 814.

Abstract 5932: Targeting BRAF WT metastatic melanomas: Identifying ERBB4 mutations as biomarkers for novel combinatorial treatment strategies

Abstract Introduction: The treatment of metastatic melanoma (MM) has been dramatically improved through the use of BRAF and MEK inhibitors in patients whose tumors harbor activating mutations in BRAF. However, these mutations occur in only ~50% of MMs and a targeted therapy(ies) has yet to be identified for those MMs that contain wild-type (WT) BRAF. Using The Cancer Genome Atlas metastatic melanoma (TCGA-SKCM) dataset, we have discovered that 50% of BRAF WT melanomas harbor a missense mutation in ERBB4, which encodes a member of the epidermal growth factor receptor (EGFR/ErbB1) family of receptor tyrosine kinases. Hence, our goal is to evaluate whether ERBB4 mutant alleles function as biomarkers and targets for therapeutic intervention in BRAF WT MMs. Experimental Procedures/Data Summary: Of the 469 genomes in the TCGA-SKCM dataset, 70 harbor at least one missense mutation in ERBB4, resulting in a total of 76 unique ERBB4 missense mutations. These mutations do not appear to be random occurrences, but instead appear to be the result of selection. For example, the incidence of ERBB4 mutant alleles is greatly reduced in melanomas that possess BRAF driver mutations and/or driver events in PI3K pathway genes. In contrast, the incidence of ERBB4 mutant alleles is markedly elevated in melanomas that possess genetic alterations that result in elevated RAS signaling. Indeed, melanomas that possess an ERBB4 mutant allele AND a genetic alteration that results in elevated RAS signaling are associated with a marked decrease in survival. Taken together, these data indicate that ERBB4 mutant alleles cause elevated PI3K signaling, which cooperates with elevated RAS signaling to drive the genesis and/or progression of BRAF WT MMs. The existence of 76 unique ERBB4 mutations in the TCGA-SKCM dataset highlights one of the challenges in identifying the ERBB4 mutant alleles that function as bona fide BRAF WT MM drivers. The other challenge is that ERBB4 functions as a context-dependent oncogene and tumor suppressor gene. Hence, we will describe the ongoing development and validation of innovative, positive-selection screens for gain-of-function ERBB4 mutant alleles and for loss-of-function ERBB4 mutant alleles from the library of ERBB4 mutant alleles found in the TCGA-SKCM dataset. The gain-of-function ERBB4 mutant alleles would enhance ERBB4 signaling in contexts in which ERBB4 functions as an MM oncogene, whereas the loss-of-function ERBB4 mutant alleles would disrupt ERBB4 signaling in contexts in which ERBB4 functions as an MM tumor suppressor gene. Conclusions: In silico analyses of the TCGA-SKCM dataset indicate that ERBB4 mutant alleles function as drivers of BRAF WT MM. We will present our progress in developing and validating positive selection strategies to identify which of the 76 ERBB4 mutant alleles found in the TCGA-SKCM dataset function as bona fide drivers of BRAF WT MMs. Citation Format: Lauren Lucas, Richard Cullum, Damien Waits, Taraswi Ghosh, Laura Cook, David Kaufmann, Jessica Markham, Cristina Rael, Kenneth Halanych, Allan David, David Riese. Targeting BRAF WT metastatic melanomas: Identifying ERBB4 mutations as biomarkers for novel combinatorial treatment strategies [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5932.

TRPS1 acts as a context-dependent regulator of mammary epithelial cell growth/differentiation and breast cancer development.

The GATA-type zinc finger transcription factor TRPS1 has been implicated in breast cancer. However, its precise role remains unclear, as both amplifications and inactivating mutations in TRPS1 have been reported. Here, we used in vitro and in vivo loss-of-function approaches to dissect the role of TRPS1 in mammary gland development and invasive lobular breast carcinoma, which is hallmarked by functional loss of E-cadherin. We show that TRPS1 is essential in mammary epithelial cells, since TRPS1-mediated suppression of interferon signaling promotes in vitro proliferation and lactogenic differentiation. Similarly, TRPS1 expression is indispensable for proliferation of mammary organoids and in vivo survival of luminal epithelial cells during mammary gland development. However, the consequences of TRPS1 loss are dependent on E-cadherin status, as combined inactivation of E-cadherin and TRPS1 causes persistent proliferation of mammary organoids and accelerated mammary tumor formation in mice. Together, our results demonstrate that TRPS1 can function as a context-dependent tumor suppressor in breast cancer, while being essential for growth and differentiation of normal mammary epithelial cells.

Innate Immune Signaling Suppresses Acute Leukemia By Modifying MYC Oncogenic Activity

Individuals with clonal hematopoiesis of indeterminant potential (CHIP) are healthy, however they are at an increased risk of developing hematopoietic malignancies. The most frequent mutations in CHIP target DNMT3A and TET2, also are observed in acute myeloid leukemia (AML), myeloproliferative neoplasms (MPN), and myelodysplastic syndromes (MDS). These findings indicate that additional alterations are needed for the transition from a pre-leukemic stage to frank leukemia, although the identity of such molecular events remains uncharacterized. To identify cellular states that cooperate with Tet2 loss, we used in vivo RNAi screening and identified the ubiquitin ligase TRAF6 required for malignant transformation of pre-leukemic TET2-deficient hematopoietic stem/progenitor cell (HSPC). Importantly, TRAF6 expression is significantly reduced in 25-50% of AML and MPN patients as compared to healthy controls. Furthermore, TET2 mutations are more strongly correlated with lower expression of TRAF6 as compared to patients with higher TRAF6 expression in certain subsets of AML. To evaluate the consequences of TRAF6 deletion on TET2-deficienct pre-leukemic cells, we generated mice in which TRAF6 and TET2 are conditionally deleted in hematopoietic cells (VavCre;Traf6fl/fl;Tet2fl/fl[DKO]). Traf6KO mice developed a lethal phenotype with signs of MPN, including lymphopenia, neutrophilia, and increased hemoglobin levels; however, this disease was not transplantable. In striking contrast, deletion of TRAF6 in the context of TET2-deficient HSPC resulted in a rapid, penetrant, aggressive, and transplantable MPN/AML. To firmly establish that TRAF6 exhibits tumor suppressor functions, we determined whether physiological levels of TRAF6 overexpression could prevent malignant transformation. Overexpression of TRAF6 in FLT3-ITD mice inhibited malignant myeloid cell expansion in FLT3-ITD mice, and rescued the survival of the animals. To uncover the molecular basis of TRAF6's tumor suppressor function, we performed gene expression profiling and proteomic characterization of TRAF6 ubiquitination substrates in leukemic cells. RNA-sequencing of HSPC revealed that deletion of TRAF6 resulted in a significant overexpression of MYC regulated genes in pre-leukemic HSPC. In support of these findings, the proteomic screen along with extensive in vitro validation experiments identified MYC as a substrate of TRAF6. Unlike the majority of reported ubiquitin-dependent post-translational modifications of MYC, we found that ubiquitination of MYC on Lysine (K) 148 by TRAF6 does not affect its protein stability but rather antagonizes acetylation of MYC on the same lysine and thus suppresses MYC oncogenic activity. We extended these observations to investigate whether inflammatory signaling via Toll-like receptors (TLRs) can antagonize MYC function and suppress leukemic cells. Stimulation of TLRs on leukemic cells resulted in TRAF6-dependent ubiquitination of MYC at K148, which coincided with repositioning of MYC off of its target gene promoters and enhancers, and ultimately in the suppression of leukemic cell viability. Our results demonstrate that TRAF6 functions as a tumor suppressor via its ubiquitination activity that antagonizes K148 acetylation leading to a decrease of MYC transcriptional activity without affecting its protein abundance. Our findings identify TRAF6 as a novel, context-dependent tumor suppressor in myeloid neoplasms, and suggest that innate immune signaling via TLR/TRAF6 could explain why some of the clonal hematopoiesis patients develop AML and others do not. Disclosures Lowe: Blueprint Medicines: Consultancy, Equity Ownership; PMV Pharmaceuticals: Consultancy, Equity Ownership; Petra Pharmaceuticals: Consultancy, Equity Ownership; Constellation Pharma: Consultancy, Equity Ownership; Mirimus: Consultancy, Equity Ownership; ORIC pharmaceuticals: Consultancy, Equity Ownership; Faeth Therapeutics: Consultancy, Equity Ownership. Starczynowski:Kurome Therapeutics: Consultancy.

Abstract B68: Targeted inhibition of Tgfβr2 reduces IL-6 production from cancer-associated fibroblasts, suppresses Stat3 activation in pancreatic cancer cells and reverses immunosuppression

Abstract Pancreatic cancer is characterized by dense stroma consisting of myofibroblasts, abundant extracellular matrix (ECM) and inflammatory/immune cells. Elevated levels of transforming growth factor β (TGFβ), a prominent driver of stromal accumulation, is a negative prognostic indicator for pancreatic cancer patients. However, inhibition of TGFβ has been challenging due to the context-dependent tumor suppressor function of TGFβ in pancreatic cancer. We reported previously that neutralizing the activity of murine TGFβ receptor 2 (Tgfβr2) using a monoclonal antibody (2G8), has potent antitumor activity in orthotopic human pancreatic tumor xenografts. The findings in xenografts highlight that TGFβ induction of stromal cell function is critical for its impact on tumor cell behavior and PDA progression and supports that inhibition of TGFβ-dependent effector functions on stromal cells has potential efficacy in PDA. Herein we demonstrate that inhibition of murine Tgfβr2 reduces IL-6 production from cancer-associated fibroblasts (CAFs), resulting in a reduction of Stat3 activation in tumor cells and reversion of the immunosuppressive landscape of murine models of PDA. Moreover, the reduction of stromal IL-6 results in activation of NK cells. In summary, we found TGFβ induced IL-6 secretion from CAFs in PDA contributes to tumorigenesis and immunosuppression and can be abrogated by inhibition of stromal Tgfβr2. Citation Format: Huocong Huang, Moriah Hagopian, Wenting Du, Yuqing Zhang, Kyla E. Driscoll, Rolf A. Brekken. Targeted inhibition of Tgfβr2 reduces IL-6 production from cancer-associated fibroblasts, suppresses Stat3 activation in pancreatic cancer cells and reverses immunosuppression [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2017 Oct 1-4; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2018;6(9 Suppl):Abstract nr B68.

Abstract A52: Arid1a exerts context-dependent oncogenic and tumor suppressor functions in liver cancer

Abstract ARID1A, an ATP-dependent SWI/SNF chromatin remodeling complex component, is hypothesized to be one of the most commonly mutated tumor suppressors in human cancer. Using mouse liver cancer models, we sought to elucidate the functional consequences of ARID1A gain and loss. Surprisingly, mice with liver-specific Arid1a deficiency were resistant to chemical- and MYC-induced liver carcinogenesis, while ARID1A overexpression accelerated tumorigenesis when combined with a variety of oncogenes, indicating an important role during tumor initiation. Mechanistically, Arid1a promoted tumor initiation by increasing cytochrome P450-mediated oxidative stress. In human HCCs, late ARID1A loss has been observed upon metastatic spread, suggesting that deleterious mutations do occur after tumor initiation. Indeed, both homozygous and heterozygous loss after liver tumor formation in mice were sufficient to drive progression and metastasis. Intriguingly, the Arid1a haploinsufficient state was sufficient to globally increase chromatin occupancy and downregulate metastasis suppressor genes within tumors. In summary, ARID1A can play both tumor-suppressive and oncogenic roles in liver cancer in a temporally dependent manner, explaining the expression and mutation patterns seen in human HCC. Citation Format: Xuxu Sun, Sam Wang, Shuyuan Zhang, Hao Zhu. Arid1a exerts context-dependent oncogenic and tumor suppressor functions in liver cancer [abstract]. In: Proceedings of the AACR Special Conference: Advances in Modeling Cancer in Mice: Technology, Biology, and Beyond; 2017 Sep 24-27; Orlando, Florida. Philadelphia (PA): AACR; Cancer Res 2018;78(10 Suppl):Abstract nr A52.

Off and back-on again: a tumor suppressor's tale.

Tumor suppressor genes play critical roles orchestrating anti-cancer programs that are both context dependent and mechanistically diverse. Beyond canonical tumor suppressive programs that control cell division, cell death, and genome stability, unexpected tumor suppressor gene activities that regulate metabolism, immune surveillance, the epigenetic landscape, and others have recently emerged. This diversity underscores the important roles these genes play in maintaining cellular homeostasis to suppress cancer initiation and progression, but also highlights a tremendous challenge in discerning precise context-specific programs of tumor suppression controlled by a given tumor suppressor. Fortunately, the rapid sophistication of genetically engineered mouse models of cancer has begun to shed light on these context-dependent tumor suppressor activities. By using techniques that not only toggle ‘off’ tumor suppressor genes in nascent tumors, but also facilitate the timely restoration of gene function ‘back-on again’ in disease specific contexts, precise mechanisms of tumor suppression can be revealed in an unbiased manner. This review discusses the development and implementation of genetic systems designed to toggle tumor suppressor genes off and back-on again and their potential to uncover the tumor suppressor’s tale.

Abstract 2524: MiR-124 suppresses p62 and p65/NFkB to regulate autophagy, inflammation and cell death in KRAS mutant mesenchymal NSCLC cells

Abstract Background KRAS mutant non-small cell lung cancers (NSCLC) are molecularly and histologically diverse. Epithelial-like cells are more KRAS dependent, whereas mesenchymal-like cells are less KRAS dependent. These two subtypes are designated KE (epithelial) and KM (mesenchymal), respectively. A KE versus KM subtype transcriptional signature reveals specific modes of KRAS dependent survival signaling in the KE subtype. This KRAS dependency signature is significantly enriched with predicted microRNA (miRNA) target genes of miR-205 and miR-34b/c. MiRNAs can function as tumor suppressors by coordinately regulating multiple oncogenic signaling pathways. The role of deregulated miRNA function in mediating the survival of KM versus KE NSCLC cells has not been investigated to date. Methods Differential miRNA expression in KRAS mutant cell lines was determined using Taqman low-density qPCR arrays (TLDA). Functional miRNA reconstitution experiments of downregulated miRNAs were performed in a panel of KM cell lines. Effects on apoptosis and autophagy were performed by Western blotting, immunofluorescence and live cell microscopy and caspase assays. The molecular targets of miR-124 were computationally identified by TargetScan or miRWalk and experimentally verified using 3’UTR luciferase-based assays. Functional rescue of miRNA-dependent cell viability defects was determined by ectopic predicted target gene expression. Results Comparison of KE to KM cells yielded a KE-KM miRNA subtype classifier/signature. This signature revealed a number of silenced or suppressed miRNAs in KM cell lines, including members of the miR-200 family. MiR-200 and miR-205 reconstitution in KM cells modulated epithelial plasticity by Zeb1 protein suppression and increased E-cadherin levels. Reconstitution of miR-124, miR-625 and miR-518-3p in KM cells caused pronounced loss of cell viability. Furthermore, miR-124 caused autolysosome maturation defects. We identified SQSTM1/p62, TRAF6 and RELA/p65 as key predicted targets of miR-124. MiR-124 reconstitution in KM cells caused decreased p62, TRAF6 and p65 protein levels. The effect of miR-124 on p62 expression was verified using a Luciferase-p62-3’UTR reporter construct. Overexpression of p62 in KM cells rescued the cell viability defects caused by miR-124. Conclusion These studies implicate miR-124 as a context-dependent tumor suppressor miRNA in KM subtype cells. MiR-124 directly suppresses expression of SQSTM1/p62 to promote defects in autolysosome maturation. In parallel, miR-124 suppresses RELA/ p65 and in some cases, TRAF6, to alter expression levels of several cytokines. Thus, miR-124 coordinately regulates autophagy and inflammation to disrupt the finely-tuned balance between pro and anti-inflammatory signals, resulting in cytotoxic effects in a specific subtype of mesenchymal-like KRAS mutant NSCLC cells. Citation Format: Anita K. Mehta, Kevin Hua, William Whipple, Mihn-Thuy Nguyen, Rushika M. Perera, Johanns Haybaeck, Joanne Weidhass, Jeffrey Settleman, Anurag Singh. MiR-124 suppresses p62 and p65/NFkB to regulate autophagy, inflammation and cell death in KRAS mutant mesenchymal NSCLC cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2524. doi:10.1158/1538-7445.AM2017-2524

Sox2 Suppresses Gastric Tumorigenesis in Mice

Sox2 expression marks gastric stem and progenitor cells, raising important questions regarding the genes regulated by Sox2 and the role of Sox2 itself during stomach homeostasis and disease. By using ChIP-seq analysis, we have found that the majority of Sox2 targets in gastric epithelial cells are tissue specific and related to functions such as endoderm development, Wnt signaling, and gastric cancer. Unexpectedly, we found that Sox2 itself is dispensable for gastric stem cell and epithelial self-renewal, yet Sox2(+) cells are highly susceptible to tumorigenesis in an Apc/Wnt-driven mouse model. Moreover, Sox2 loss enhances, rather than impairs, tumor formation in Apc-deficient gastric cells invivo and invitro by inducing Tcf/Lef-dependent transcription and upregulating intestinal metaplasia-associated genes, providing a mechanistic basis for the observed phenotype. Together, these data identify Sox2 as a context-dependent tumor suppressor protein that is dispensable for normal tissue regeneration but restrains stomach adenoma formation through modulation of Wnt-responsive and intestinal genes.

Abstract PL07-03: The multifaceted role of Notch signaling in tumor/microenvironment interaction

Abstract Notch receptors participate in a highly conserved signaling pathway with critical developmental roles in multicellular animals. Based on studies involving developing and adult organisms, it is evident that the outcome of Notch signaling is not fixed but varies widely with dose and context, allowing Notch to either drive or modulate a large number of different biologic processes. This complexity extends to cancer. The first mammalian Notch receptor to be identified, Notch1, was found due to its rearrangement in a rare subset of T cell acute lymphoblastic leukemia (T-ALL), immediately indicating a role for the pathway in oncogenesis. Since this time, mechanistic work in animal models and cancer genome sequencing studies have greatly expanded the scope of the proposed roles for Notch signaling in cancer to the point where it is now reasonable to think that Notch impacts all of the Weinberg/Hanahan “Hallmarks of Cancer”. Some of these roles stem from cell autonomous mutations that constitutively activate oncogenic Notch signaling in tumors such as T-ALL and triple negative breast cancer, or that inactivate Notch in tumors such as squamous cell carcinomas of the skin and head and neck, confirming that Notch can act as a context dependent oncogene or tumor suppressor. In addition, recent data suggest that certain classes of Notch mutations are selected for their ability to upregulate signaling in a microenvironment-dependent fashion, a type of gene-environment interaction that is highly specific for particular cancers, such as chronic lymphocytic leukemia and marginal zone B cell lymphoma. Additional roles for Notch involving the stromal compartment in cancers, particularly angiogenesis and host immunity, also are coming into focus. This talk will touch on a number of these emerging relationships, which have implications for ongoing attempts to effectively target the Notch pathway in patients with a broad spectrum of different cancers. Citation Format: Jon C. Aster. The multifaceted role of Notch signaling in tumor/microenvironment interaction. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr PL07-03.