Oncogenic Effector Research Articles

USP39 phase separates into the nucleolus and drives lung adenocarcinoma progression by promoting GLI1 expression

BackgroundIntracellular membraneless organelles formed by liquid-liquid phase separation (LLPS) function in diverse physiological processes and have been linked to tumor-promoting properties. The nucleolus is one of the largest membraneless organelle formed through LLPS. Deubiquitylating enzymes (DUBs) emerge as novel therapeutic targets against human cancers. However, the nucleolar phase separation of DUBs and association with lung cancer development have remained incompletely investigated till now.MethodsGFP-USP39 fusion proteins were analyzed for LLPS properties using immunofluorescence, fluorescence recovery after photobleaching (FRAP) and in vitro LLPS assays. Intrinsically-disordered regions of USP39 were analyzed by PhaSepDB database. Transcriptomic profiling, Western blot, RT-PCR and luciferase reporter assays were conducted to identify targets regulated by USP39. The effects of USP39 depletion on tumor progression were tested using doxycycline-inducible USP39 knockdown and rescue lung adenocarcinoma cells both in vitro and in vivo by performing MTT, colony formation, EdU staining, transwell and tumor xenograft model experiments.ResultsUSP39 phase separates into nucleoli depending upon its N-terminal disordered region with amino acid residues 1-103. Lung cancer cell growth and migration were dramatically inhibited by USP39 knockdown, which was rescued by exogenous USP39 complementation. Moreover, knockdown of USP39 reduced oncogenic transcription effector GLI1 levels. Finally, USP39 downregulation restricted the formation of lung cancer xenografts in nude mice.ConclusionsUSP39 undergoes LLPS in the nucleolus and promotes tumor progression by regulating GLI1 expression. Downregulation of USP39 effectively suppressed lung cancer growth, and therefore targeting USP39 provides novel therapeutic strategy to treat lung cancer.

Rac1 GTPase Regulates the βTrCP-Mediated Proteolysis of YAP Independently of the LATS1/2 Kinases.

Background: Oncogenic mutations in the KRAS gene are detected in >90% of pancreatic cancers (PC). In genetically engineered mouse models of PC, oncogenic KRAS drives the formation of precursor lesions and their progression to invasive PC. The Yes-associated Protein (YAP) is a transcriptional coactivator required for transformation by the RAS oncogenes and the development of PC. In Ras-driven tumors, YAP can also substitute for oncogenic KRAS to drive tumor survival after the repression of the oncogene. Ras oncoproteins exert their transforming properties through their downstream effectors, including the PI3K kinase, Rac1 GTPase, and MAPK pathways. Methods: To identify Ras effectors that regulate YAP, YAP levels were measured in PC cells exposed to inhibitors of oncogenic K-Ras and its effectors. Results: In PC cells, the inhibition of Rac1 leads to a time-dependent decline in YAP protein, which could be blocked by proteosome inhibitor MG132. This YAP degradation after Rac1 inhibition was observed in a range of cell lines using different Rac1 inhibitors, Rac1 siRNA, or expression of dominant negative Rac1T17N mutant. Several E3 ubiquitin ligases, including SCFβTrCP, regulate YAP protein stability. To be recognized by this ligase, the βTrCP degron of YAP (amino acid 383-388) requires its phosphorylation by casein kinase 1 at Ser384 and Ser387, but these events must first be primed by the phosphorylation of Ser381 by LATS1/2. Using Flag-tagged mutants of YAP, we show that YAP degradation after Rac1 inhibition requires the integrity of this degron and is blocked by the silencing of βTrCP1/2 and by the inhibition of casein kinase 1. Unexpectedly, YAP degradation after Rac1 inhibition was still observed after the silencing of LATS1/2 or in cells carrying a LATS1/2 double knockout. Conclusions: These results reveal Rac1 as an oncogenic KRAS effector that contributes to YAP stabilization in PC cells. They also show that this regulation of YAP by Rac1 requires the SCFβTrCP ligase but occurs independently of the LATS1/2 kinases.

Guanine nucleotide exchange factors for Rho GTPases (RhoGEFs) as oncogenic effectors and strategic therapeutic targets in metastatic cancer

Metastatic cancer cells dynamically adjust their shape to adhere, invade, migrate, and expand to generate secondary tumors. Inherent to these processes is the constant assembly and disassembly of cytoskeletal supramolecular structures. The subcellular places where cytoskeletal polymers are built and reorganized are defined by the activation of Rho GTPases. These molecular switches directly respond to signaling cascades integrated by Rho guanine nucleotide exchange factors (RhoGEFs), which are sophisticated multidomain proteins that control morphological behavior of cancer and stromal cells in response to cell-cell interactions, tumor-secreted factors and actions of oncogenic proteins within the tumor microenvironment. Stromal cells, including fibroblasts, immune and endothelial cells, and even projections of neuronal cells, adjust their shapes and move into growing tumoral masses, building tumor-induced structures that eventually serve as metastatic routes. Here we review the role of RhoGEFs in metastatic cancer. They are highly diverse proteins with common catalytic modules that select among a variety of homologous Rho GTPases enabling them to load GTP, acquiring an active conformation that stimulates effectors controlling actin cytoskeleton remodeling. Therefore, due to their strategic position in oncogenic signaling cascades, and their structural diversity flanking common catalytic modules, RhoGEFs possess unique characteristics that make them conceptual targets of antimetastatic precision therapies. Preclinical proof of concept, demonstrating the antimetastatic effect of inhibiting either expression or activity of βPix (ARHGEF7), P-Rex1, Vav1, ARHGEF17, and Dock1, among others, is emerging.

The Myc Family and the Metastasis Suppressor NDRG1: Targeting Key Molecular Interactions with Innovative Therapeutics.

Cancer is a leading cause of death worldwide, resulting in ∼10 million deaths in 2020. Major oncogenic effectors are the Myc proto-oncogene family, which consists of three members including c-Myc, N-Myc, and L-Myc. As a pertinent example of the role of the Myc family in tumorigenesis, amplification of MYCN in childhood neuroblastoma strongly correlates with poor patient prognosis. Complexes between Myc oncoproteins and their partners such as hypoxia-inducible factor-1α and Myc-associated protein X (MAX) result in proliferation arrest and pro-proliferative effects, respectively. Interactions with other proteins are also important for N-Myc activity. For instance, the enhancer of zest homolog 2 (EZH2) binds directly to N-Myc to stabilize it by acting as a competitor against the ubiquitin ligase, SCFFBXW7, which prevents proteasomal degradation. Heat shock protein 90 may also be involved in N-Myc stabilization since it binds to EZH2 and prevents its degradation. N-Myc downstream-regulated gene 1 (NDRG1) is downregulated by N-Myc and participates in the regulation of cellular proliferation via associating with other proteins, such as glycogen synthase kinase-3β and low-density lipoprotein receptor-related protein 6. These molecular interactions provide a better understanding of the biologic roles of N-Myc and NDRG1, which can be potentially used as therapeutic targets. In addition to directly targeting these proteins, disrupting their key interactions may also be a promising strategy for anti-cancer drug development. This review examines the interactions between the Myc proteins and other molecules, with a special focus on the relationship between N-Myc and NDRG1 and possible therapeutic interventions. SIGNIFICANCE STATEMENT: Neuroblastoma is one of the most common childhood solid tumors, with a dismal five-year survival rate. This problem makes it imperative to discover new and more effective therapeutics. The molecular interactions between major oncogenic drivers of the Myc family and other key proteins; for example, the metastasis suppressor, NDRG1, may potentially be used as targets for anti-neuroblastoma drug development. In addition to directly targeting these proteins, disrupting their key molecular interactions may also be promising for drug discovery.

Abstract 1720: Examining mechanistic underpinnings of chemoresistance in triple negative breast cancer

Abstract Background: Triple negative breast cancer (TNBC) is not only the most aggressive subtype of breast cancer, but it does also not have many targeted therapeutic options due to the lack of hormone receptor expression and enrichment of HER2. TNBCs are also more prone to the development of chemoresistance and metastatic progression, which are the main obstacles to reducing TNBC-related mortality. The objective of this study is to identify targetable key node (s) that contribute to the development of chemoresistance in TNBC. Method: Differentially expressed genes in TNBC patients with or without relapse were analyzed to select functionally important genes in chemoresistant TNBC. TRIM29 was selected based on survival analysis. Carboplatin-resistant TNBC cells were established to explore the phenotypic and molecular differences. Various growth and migration assays were used to explore the phenotype of chemoresistant TNBC cells. The expression of TRIM29 and related pathways were assessed by immunoblotting and immunofluorescence analysis. Cells with TRIM29-knockout (KO) were developed by the CRISPR system. Result: Chemoresistant TNBC cells overexpress TRIM29. Exhibiting the functional importance, overexpression of TRIM29 in MDAMB231 confers resistance to carboplatin. A stable knockout of TRIM29 in carboplatin-resistant cells results in an improved response to carboplatin. The breast tumor xenograft model revealed that TRIM29-KO in chemoresistant TNBC formed significantly less dense tumor in mice compared to control. Mechanistically, an enhanced expression of β-catenin was seen in chemoresistant as well as TRIM29-overexpressing cells. TRIM29-KO in carboplatin-resistant cell line results in a drastic decrease in β-catenin level. Combination treatment of carboplatin and β-catenin-inhibitor resulted in an enhanced growth inhibition in chemoresistant TNBC cells. RNA-Seq analysis revealed that there are 24 candidate genes which are overexpressed in chemoresistant TNBC cells but downregulated in TRIM29-KO chemoresistant cells, thereby demanding further investigation. Conclusion: Our findings implicate TRIM29 enrichment as an important node in chemoresistant TNBC that may concomitantly modulate β-catenin as downstream oncogenic effectors. Citation Format: Qitong Wu, Sumit Siddharth, Deepak Verma, Sheetal Parida, Dipali Sharma. Examining mechanistic underpinnings of chemoresistance in triple negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1720.

FA-HA-Amygdalin@Fe2O3 and/or γ-Rays Affecting SIRT1 Regulation of YAP/TAZ-p53 Signaling and Modulates Tumorigenicity of MDA-MB231 or MCF-7 Cancer Cells.

Breast cancer (BC) has a complex and heterogeneous etiology, and the emergence of resistance to conventional chemo-and radiotherapy results in unsatisfactory outcomes during BC treatment. Targeted nanomedicines have tremendous therapeutic potential in BC treatment over their free drug counterparts. Hence, this study aimed to evaluate the newly fabricated pH-sensitive multifunctional FAHA- Amygdalin@Fe2O3 nano-core-shell composite (AF nanocomposite) and/or γ-radiation for effective localized BC therapy. The physicochemical properties of nanoparticles were examined, including stability, selectivity, responsive release to pH, cellular uptake, and anticancer efficacy. MCF-7 and MDA-MB-231 cells were treated with AF at the determined IC50 doses and/or exposed to γ-irradiation (RT) or were kept untreated as controls. The antitumor efficacy of AF was proposed via assessing anti-proliferative effects, cell cycle distribution, apoptosis, and determination of the oncogenic effectors. In a bio-relevant medium, AF nanoparticles demonstrated extended-release characteristics that were amenable to acidic pH and showed apparent selectivity towards BC cells. The bioassays revealed that the HA and FA-functionalized AF markedly hindered cancer cell growth and enhanced radiotherapy (RT) through inducing cell cycle arrest (pre-G1 and G2/M) and increasing apoptosis, as well as reducing the tumorigenicity of BCs by inhibiting Silent information regulation factor 1 (SIRT1) and restoring p53 expression, deactivating the Yes-associated protein (YAP)/ Transcriptional coactivator with PDZ-binding motif (TAZ) signaling axis, and interfering with the tumor growth factor- β(TGF- β)/SMAD3 and HIF-1α/VEGF signaling hub while up-regulating SMAD7 protein expression. Collectively, the novel AF alone or prior RT abrogated BC tumorigenicity.

A cell-based bioluminescence reporter assay of human Sonic Hedgehog protein autoprocessing to identify inhibitors and activators

The Sonic Hedgehog (SHh) precursor protein undergoes biosynthetic autoprocessing to cleave off and covalently attach cholesterol to the SHh signaling ligand, a vital morphogen and oncogenic effector protein. Autoprocessing is self-catalyzed by SHhC, the SHh precursor's C-terminal enzymatic domain. A method to screen for small molecule regulators of this process may be of therapeutic value. Here, we describe the development and validation of the first cellular reporter to monitor human SHhC autoprocessing noninvasively in high-throughput compatible plates. The assay couples intracellular SHhC autoprocessing using endogenous cholesterol to the extracellular secretion of the bioluminescent nanoluciferase enzyme. We developed a WT SHhC reporter line for evaluating potential autoprocessing inhibitors by concentration response-dependent suppression of extracellular bioluminescence. Additionally, a conditional mutant SHhC (D46A) reporter line was developed for identifying potential autoprocessing activators by a concentration response-dependent gain of extracellular bioluminescence. The D46A mutation removes a conserved general base that is critical for the activation of the cholesterol substrate. Inducibility of the D46A reporter was established using a synthetic sterol, 2-α carboxy cholestanol, designed to bypass the defect through intramolecular general base catalysis. To facilitate direct nanoluciferase detection in the cell culture media of 1536-well plates, we designed a novel anionic phosphonylated coelenterazine, CLZ-2P, as the nanoluciferase substrate. This new reporter system offers a long-awaited resource for small molecule discovery for cancer and for developmental disorders where SHh ligand biosynthesis is dysregulated.

CSIG-11. HYPOXIA SIGNALING IN ACVR1-MUTANT DIFFUSE INTRINSIC PONTINE GLIOMA

Abstract Diffuse intrinsic pontine glioma (DIPG) is a devastating glial tumor of the brainstem primary occurring in the pediatric population. The majority of DIPG tumors (60-80%) carry lysine- to methionine point mutation in histone H3, resulting in a global reduction of H3K27 trimethylation and subsequently a unique epigenetic landscape. Moreover, a large subset of the DIPG tumors carry activating mutations in ACVR1 – the gene encoding for the BMP receptor ALK2, suggesting an oncogenic role of signaling from this receptor. Importantly potential mechanisms of ACVR1-driven oncogenesis, remain poorly understood. Similar ACVR1 mutations have been described in the rare disorder Fibrodysplasia Ossificans Progressiva (FOP), and evidence from FOP models have suggested several links between hypoxia signaling, BMP signaling, and FOP pathogenesis thus opening up for evaluation of hypoxia signaling as oncogenic effector in ACVR1mutant DIPG. Using cultured DIPG cells and adult glioma cells respectively we have tested the activity of the hypoxia pathway using a hypoxia-responsive elements (HRE)-luciferase reporter assay. Intriguingly, DIPG but not glioma cells overexpressing a mutant ACVR1 construct significantly elevated hypoxia signaling at hypoxia. Similar trends are seen when assaying neuronal stem cell cultures harboring Histone 3.1 mutations along with activated ACVR1/ALK2 signaling. Furthermore, BMP ligand stimulation resulted in a dose-dependent increase of hypoxia signaling in DIPG cells. Through querying clinical gene expression data, we found elevated hypoxia signature scores in pediatric gliomas with mutant ACVR1 as compared to those with wildtype ACVR1. Finally, ACVR1 mutations frequently co-occur with Histone 3.3 and 3.1 mutations, and the hypoxia signature score was significantly higher than baseline only in tumors also harboring these Histone 3.3 or 3.1 mutations. Together, our data suggest that hypoxia signaling is elevated downstream of ACVR1 specifically in pediatric gliomas and prompts for evaluation of hypoxia signaling as potential therapeutical target in DIPG.

Abstract 3717: KMT2D loss cooperates with PTCH loss for medulloblastoma genesis

Abstract We have previously reported that the histone H3 lysine 4 methyltransferase KMT2D (also called MLL4, MLL2, and ALR; a transcriptional coactivator) is required for neuronal differentiation of human neuron-committed NT2/D1 embryonal carcinoma stem cells. Notably, we have shown that brain-specific knockout of Kmt2d alone induces spontaneous medulloblastoma (MB) in mice and that Kmt2d loss in the brain highly upregulates several oncogenic signaling programs and downregulates tumor suppressor genes. Consistent with this, our analysis of several publicly available databases indicates that Kmt2d is the most frequently mutated epigenetic modifier (8%‒10%) in MB. In this study, we sought to assess whether Kmt2d loss cooperates with another oncogenic event in MB genesis. Particularly, PTCH (also known as PTCH1), a potent MB-signaling suppressor, is one of the most frequently mutated genes in MB, and KMT2D mutations co-occur with PTCH mutations more than with many other mutations in MB. Therefore, we determined the effect of Kmt2d loss on of Ptch+/--driven MB genesis by generating and analyzing Nestin-Cre Kmt2dfl/+ Ptch+/- mice. Our results showed that heterozygous loss of Kmt2d strongly increased the genesis and incidence of Ptch+/--driven MB. Supporting this, immunohistochemistry staining of Ki-67 (a proliferation marker) showed that heterozygous loss of Kmt2d in Ptch+/--driven MB highly increased the proliferation of MB cells. Our transcriptomic analysis showed that heterozygous Kmt2d loss upregulated tumor-promoting programs, including G-protein-coupled receptor signaling and oxidation-reduction process. In line with this, heterozygous Kmt2d loss upregulated oncogenic kinases (e.g., phospho-AKT1 and phospho-ERK) that may be downstream of G-protein-coupled receptor signaling. Mechanistically, our results indicate that these tumor-promoting programs are repressed, at least in part, by the transcription-repressive and tumor-suppressive factor (s) NCOR2, whose expression is activated by KMT2D. Our results would be the first to provide molecular insights into how co-losses of an epigenetic modifier and an MB-signaling suppressor cooperate for MB genesis. Our new mouse model would be helpful in future preclinical therapeutic experiments for MB treatment. Our findings suggest that targeting oncogenic effectors downstream of KMT2D loss can be considered a potential therapeutic strategy for MB treatment. Citation Format: Shilpa S. Dhar, Calena J. Brown, Ali S. Rizvi, Janak R. Abraham, Roy V. Sillitoe, Kaifu Chen, Min Gyu Lee. KMT2D loss cooperates with PTCH loss for medulloblastoma genesis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3717.

TBIO-08. The molecular basis for rational targeting of FGFR-driven growth and invasiveness in pediatric brain tumors

Abstract The oncogenic activation of receptor tyrosine kinases (RTK) promotes growth, survival and dissemination in pediatric tumors including glioma, ependymoma and medulloblastoma (MB). Direct targeting of either the RTK or of downstream kinases can effectively block tumor promoting pathway functions. However, emergence of resistance is common. We hypothesized that alternative interference strategies that target protein-protein interactions (PPIs) instead of enzymatic activities could overcome the emergence of resistance. We characterized the molecular interactions downstream of the FGFR that regulate relevant growth and invasion-promoting mechanisms in MB cells, to identify potentially druggable PPIs. We found that the FRS2 protein is an essential up-stream effector of FGFR signaling towards invasiveness. Using a proteomics approach, we furthermore identified the Striatin 3 protein as a novel oncogenic effector of the FGFR pathway downstream of FRS2, as it integrates antagonistic growth and invasion signals downstream of FGFR. Mechanistically, Striatin 3 interacts with the Ser/Thr kinase MAP4K4, couples it to the protein phosphatase 2A, and thereby inactivates growth repressing activities of MAP4K4. In parallel, Striatin 3 enables MAP4K4-mediated phosphorylation of PKC-theta and VASP, which combined are necessary to promote tissue invasion. To selectively repress pro-invasive FGFR functions, we identified and functionally validated small molecule ligands of FRS2, that prevent FRS2 activation and downstream signaling. We demonstrate efficacy of these compounds in inhibiting invasion and growth promoting activities in vitro and in vivo, and identified potential off-target activities of the ligand using a proteome-wide interaction analysis. We propose inhibition of FRS2 by a small molecular PTB domain ligand as a strategy to repress FGF signaling in FGFR-driven tumors. The development of this ligand, and the de novo design of functional analogs thereof bear promise for further pre-clinical evaluation of these structures as anti-growth promoting and anti-metastatic therapeutics applicable to FGFR-driven tumors.

Precise tumor immune rewiring via synthetic CRISPRa circuits gated by concurrent gain/loss of transcription factors

Reinvigoration of antitumor immunity has recently become the central theme for the development of cancer therapies. Nevertheless, the precise delivery of immunotherapeutic activities to the tumors remains challenging. Here, we explore a synthetic gene circuit-based strategy for specific tumor identification, and for subsequently engaging immune activation. By design, these circuits are assembled from two interactive modules, i.e., an oncogenic TF-driven CRISPRa effector, and a corresponding p53-inducible off-switch (NOT gate), which jointly execute an AND-NOT logic for accurate tumor targeting. In particular, two forms of the NOT gate are developed, via the use of an inhibitory sgRNA or an anti-CRISPR protein, with the second form showing a superior performance in gating CRISPRa by p53 loss. Functionally, the optimized AND-NOT logic circuit can empower a highly specific and effective tumor recognition/immune rewiring axis, leading to therapeutic effects in vivo. Taken together, our work presents an adaptable strategy for the development of precisely delivered immunotherapy.

EZH2 depletion potentiates MYC degradation inhibiting neuroblastoma and small cell carcinoma tumor formation

Efforts to therapeutically target EZH2 have generally focused on inhibition of its methyltransferase activity, although it remains less clear whether this is the central mechanism whereby EZH2 promotes cancer. In the current study, we show that EZH2 directly interacts with both MYC family oncoproteins, MYC and MYCN, and promotes their stabilization in a methyltransferase-independent manner. By competing against the SCFFBW7 ubiquitin ligase to bind MYC and MYCN, EZH2 counteracts FBW7-mediated MYC(N) polyubiquitination and proteasomal degradation. Depletion, but not enzymatic inhibition, of EZH2 induces robust MYC(N) degradation and inhibits tumor cell growth in MYC(N) driven neuroblastoma and small cell lung carcinoma. Here, we demonstrate the MYC family proteins as global EZH2 oncogenic effectors and EZH2 pharmacologic degraders as potential MYC(N) targeted cancer therapeutics, pointing out that MYC(N) driven cancers may develop inherent resistance to the canonical EZH2 enzymatic inhibitors currently in clinical development.

3186 – TRANSCRIPTOMIC ANALYSIS OF SYNTHETIC HUMAN CORD BLOOD LEUKEMIAS REVEALS DEVELOPMENT STAGE-SPECIFIC DEPENDENCE ON N-MYC

Descriptive sequencing of human leukemia samples has revealed the diversity and combinatorial complexity of oncogenic effector genes which can occur within all or just a subset of tumor cells. With the aim of deconvoluting the individual contributions of T-cell acute lymphoblastic leukemia (T-ALL) oncogenes, we employed our recently developed synthetic model of human T-ALL (Kusakabe et al, 2019) to perform a systematic analysis of transcriptional signatures associated with each of the four oncogenes used in our model. We identified synthetic leukemias to cluster based on two gene signatures which resembled distinct stages in T-cell development and had marked variation in MYCN expression, with its highest expression found on the more "immature" cluster. All synthetic leukemias carried activated NOTCH1, which is best known for upregulating MYC, but not MYCN. By analyzing publicly available RNA sequencing datasets, we found MYCN to be highly expressed in early T-cell progenitors (ETPs) and T-ALLs of more immature phenotype. To assess oncogene "addiction" to MYCN, cell lines and primary synthetic leukemias were transduced with shRNAs or sgRNAs to knock-down (KD) or knock-out (KO) MYCN, respectively. By both approaches, bulk cell growth and/or clonogenic activity were reduced in KD/KO cells as compared to controls, supporting their functional dependence on MYCN. MYCN overexpression resulted in increased clonogenic activity and differentiation delay of NOTCH1-transduced cells in vitro but was still insufficient to generate leukemia in vivo. We are currently exploring other genes with similar expression patterns to MYCN to identify upstream/downstream mediators of MYCN dependence in this context. Our results emphasize stage-specific oncogene dependencies and identify alternate therapeutic targets in T-ALLs with high MYCN expression.

Mitochondrial genome and its regulator TFAM modulates head and neck tumourigenesis through intracellular metabolic reprogramming and activation of oncogenic effectors

Mitochondrial transcriptional factor A (TFAM) acts as a key regulatory to control mitochondrial DNA (mtDNA); the impact of TFAM and mtDNA in modulating carcinogenesis is controversial. Current study aims to define TFAM mediated regulations in head and neck cancer (HNC). Multifaceted analyses in HNC cells genetically manipulated for TFAM were performed. Clinical associations of TFAM and mtDNA encoded Electron Transport Chain (ETC) genes in regulating HNC tumourigenesis were also examined in HNC specimens. At cellular level, TFAM silencing led to an enhanced cell growth, motility and chemoresistance whereas enforced TFAM expression significantly reversed these phenotypic changes. These TFAM mediated cellular changes resulted from (1) metabolic reprogramming by directing metabolism towards aerobic glycolysis, based on the detection of less respiratory capacity in accompany with greater lactate production; and/or (2) enhanced ERK1/2-Akt-mTORC-S6 signalling activity in response to TFAM induced mtDNA perturbance. Clinical impacts of TFAM and mtDNA were further defined in carcinogen-induced mouse tongue cancer and clinical human HNC tissues; as the results showed that TFAM and mtDNA expression were significantly dropped in tumour compared with their normal counterparts and negatively correlated with disease progression. Collectively, our data uncovered a tumour-suppressing role of TFAM and mtDNA in determining HNC oncogenicity and potentially paved the way for development of TFAM/mtDNA based scheme for HNC diagnosis.

Abstract 2419: Two-tiered inhibition of TGFBR2 signaling via ITD-1 and miR-149-3p targets CD44High glioma stem cells and non-stem-like GBM cells

Abstract The remarkable heterogeneity of glioblastoma (GBM), its therapy resistance and high recurrence rate present formidable challenges for effective treatment. A critical component of GBM malignancy derives from the heterogeneous population of glioma stem-like cells (GSCs) that are especially efficient in promoting tumorigenicity and chemoradiation resistance. Previous findings by our lab and others have established Oct4 and Sox2 transcription factors as key drivers of the GSC phenotype. Development of effective GBM therapeutics requires a deeper knowledge of the genetic and epigenetic drivers of GSCs, particularly the Oct4/Sox2-mediated mechanisms that promote and maintain more tumorigenic and resistant GSC subsets. In GBM, TGF-β has well-established oncogenic functions and is tied to multiple phenotypic aspects of GSCs. However, little has been done to elucidate the relevance of TGF-β type II receptor (TGFBR2) in GSCs and GBM as a whole. Here, we show that TGFBR2 is a direct transcriptional target of Oct4 and Sox2 and is upregulated in both newly diagnosed and recurrent GBM. Multidimensional analysis identified overactivation of canonical TGF-β signaling in Oct4/Sox2-expressing GSCs. Furthermore, transcriptional targets of Smad2/3 induced by Oct4 and Sox2 are upregulated in TGFBR2High clinical GBM samples, a subpopulation also shown to associate with poor patient outcome. Notably, scRNA-Seq analysis revealed enrichment of TGFBR2 in CD44High, but not CD133/SSEA-1High, GSCs, a subset known to be highly proliferative and resistant to standard therapy. Likewise, a strong correlation between TGFBR2 and CD44 expression was found in clinical GBM samples. Despite promising results in preclinical studies, inhibition of TGF-β signaling through ligand or receptor targeting has been ineffective against clinical GBM, due to multiple potential reasons including activation of compensatory signaling pathways. Therefore, we apply a multidimensional approach, incorporating direct inhibition of both TGFBR2 and its oncogenic downstream effectors, in an effort to produce a more robust response and minimize the potential for compensatory resistance. Bioinformatic analysis predicted miR-149-3p to target a collection of Oct4/Sox2-induced Smad2/3-regulated transcripts, most of which, including CD44, have well-established oncogenic function and/or overexpression in GBM. Transient expression of miR-149-3p reduced self-renewal capacity of GSC spheres, a necessary ability for in vivo tumor propagation. Furthermore, miR-149-3p enhanced the growth-inhibitory effects of ITD-1, a small molecule TGFBR2 inhibitor, in both GSCs and non-stem-like GBM cells. These findings demonstrate that Oct4/Sox2-induced TGFBR2 signaling is clinically relevant and has the potential to be effectively combated using a carefully structured two-tiered therapeutic approach in GBM. Citation Format: Amanda L. Johnson, Jack Korleski, Hernando Lopez-Bertoni, John Laterra. Two-tiered inhibition of TGFBR2 signaling via ITD-1 and miR-149-3p targets CD44High glioma stem cells and non-stem-like GBM cells [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 2419.

Abstract 2417: Mutant p53 and oncogenic KRAS converge on CREB1 to drive pancreatic cancer metastasis

Abstract 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 p53 alterations are missense mutations that exhibit gain-of-function phenotypes that include increased invasiveness and metastasis. However, the extent of direct cooperation or synergism between KRAS effectors and mutant p53 remain undefined. To study how KRAS effectors and mutant p53 cooperate to drive PDAC development, we developed a novel somatic model that expresses mutant KRAS and p53 only in pancreas tumor cells, leaving the tumor microenvironment and immune system intact. PDAC genetically engineered mouse models (GEMMs) were generated by crossing a conditional oncogenic KRasG12D (K) allele with a conditional null p53 allele (Pfl) or a novel, conditional knock-in mutant p53R172H allele (Pwm) that selectively expresses mutant p53 in tumor cells while preserving wildtype p53 expression in all other animal cells. The incidence of metastatic lesions in the liver and lungs of KPwmC mice was over 2-fold greater (p=.03) relative to KPflC mice. Through orthogonal analyses of this model and human PDAC samples, we show that oncogenic KRAS effectors phosphorylate and activate cyclic AMP responsive element binding protein 1 (CREB1), allowing physical interactions with mutant p53 to transcriptionally upregulate the pro-metastatic transcription factor, FOXA1, which enhances β-catenin stabilization and activity. Targeting KRAS and mutant p53 cooperativity through pharmacologic inhibition of CREB1 dramatically dampens FOXA1 expression and PDAC metastasis. Our findings demonstrate a direct, mechanistic link between key oncogenic KRAS signaling elements and mutant p53 that result in a broad, multiplexed activation of cancer-associated transcriptional networks. Moreover, we identify CREB1 as a viable therapeutic strategy to undermine oncogenic KRAS and mutant p53 cooperation to mitigate PDAC metastasis. Citation Format: Michael Paul Kim, Xinqun Li, Jenying Deng, Yun Zhang, Bingbing Dai, Kendra Allton, Tara Hughes, Christian Siangco, Jithesh Augustine, Yaan Kang, Joy M. McDaniel, Shunbin Xiong, Eugene Koay, Florencia McAllister, Christopher Bristow, Timothy Heffernan, Anirban Maitra, Bin Liu, Michelle Barton, Amanda Wasylischen, Jason Fleming, Guillermina lozano. Mutant p53 and oncogenic KRAS converge on CREB1 to drive pancreatic cancer metastasis [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 2417.

Parallel Rap1>RalGEF>Ral and Ras signals sculpt the C. elegans nervous system

Ras is the most commonly mutated oncogene in humans and uses three oncogenic effectors: Raf, PI3K, and RalGEF activation of Ral. Understanding the importance of RalGEF>Ral signaling in cancer is hampered by the paucity of knowledge about their function in animal development, particularly in cell movements. We found that mutations that disrupt function of RalGEF or Ral enhance migration phenotypes of mutants for genes with established roles in cell migration. We used as a model the migration of the canal associated neurons (CANs), and validated our results in HSN cell migration, neurite guidance, and general animal locomotion. These functions of RalGEF and Ral are specific to their control of Ral signaling output rather than other published functions of these proteins. In this capacity Ral functions cell autonomously as a permissive developmental signal. In contrast, we observed Ras, the canonical activator of RalGEF>Ral signaling in cancer, to function as an instructive signal. Furthermore, we unexpectedly identified a function for the close Ras relative, Rap1, consistent with activation of RalGEF>Ral. These studies define functions of RalGEF>Ral, Rap1 and Ras signaling in morphogenetic processes that fashion the nervous system. We have also defined a model for studying how small GTPases partner with downstream effectors. Taken together, this analysis defines novel molecules and relationships in signaling networks that control cell movements during development of the nervous system.

A signalling cascade for Ral

ABSTRACT Ras is the most mutated oncoprotein in cancer. Among the three oncogenic effectors of Ras – Raf, PI3 Kinase and RalGEF>Ral – signalling through RalGEF>Ral (Ras-like) is by far the least well understood. A variety of signals and binding partners have been defined for Ral, yet we know little of how Ral functions in vivo. This review focuses on previous research in Drosophila that defined a function for Ral in apoptosis and established indirect relationships among Ral, the CNH-domain MAP4 Kinase misshapen, and the JNK MAP kinase basket. Most of the described signalling components are not essential in C. elegans, facilitating subsequent analysis using developmental patterning of the C. elegans vulval precursor cells (VPCs). The functions of two paralogous CNH-domain MAP4 Kinases were defined relative to Ras>Raf, Notch and Ras>RalGEF>Ral signalling in VPCs. MIG-15, the nematode ortholog of misshapen, antagonizes both the Ral-dependent and Ras>Raf-dependent developmental outcomes. In contrast, paralogous GCK-2, the C. elegans ortholog of Drosophila happyhour, propagates the 2°-promoting signal of Ral. Manipulations via CRISPR of Ral signalling through GCK-2 coupled with genetic epistasis delineated a Ras>RalGEF>Ral>Exo84>GCK-2>MAP3KMLK−1> p38PMK−1 cascade. Thus, genetic analysis using invertebrate experimental organisms defined a cascade from Ras to p38 MAP kinase.

Oncogenic KRAS Recruits an Expansive Transcriptional Network through Mutant p53 to Drive Pancreatic Cancer Metastasis.

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.

Combinatorial ETS1-dependent control of oncogenic NOTCH1 enhancers in T-cell leukemia.

Notch activation is highly prevalent among cancers, in particular T-cell acute lymphoblastic leukemia (T-ALL). However, the use of pan-Notch inhibitors to treat cancers has been hampered by adverse effects, particularly intestinal toxicities. To circumvent this barrier in T-ALL, we aimed to inhibit ETS1, a developmentally important T-cell transcription factor previously shown to co-bind Notch response elements. Using complementary genetic approaches in mouse models, we show that ablation of Ets1 leads to strong Notch-mediated suppressive effects on T-cell development and leukemogenesis, but milder intestinal effects than pan-Notch inhibitors. Mechanistically, genome-wide chromatin profiling studies demonstrate that Ets1 inactivation impairs recruitment of multiple Notch-associated factors and Notch-dependent activation of transcriptional elements controlling major Notch-driven oncogenic effector pathways. These results uncover previously unrecognized hierarchical heterogeneity of Notch-controlled genes and points to Ets1-mediated enucleation of Notch-Rbpj transcriptional complexes as a target for developing specific anti-Notch therapies in T-ALL that circumvent the barriers of pan-Notch inhibition.