Global Inhibition with Specific Activation: How p53 and MYC Redistribute the Transcriptome in the DNA Double-Strand Break Response.

In response to stresses, cells often halt normal cellular processes, yet stress-specific pathways must bypass such inhibition to generate effective responses. We investigated how cells redistribute global transcriptional activity in response to DNA damage. We show that an oscillatory increase of p53 levels in response to double-strand breaks drives a counter-oscillatory decrease of MYC levels. Using RNA sequencing (RNA-seq) of newly synthesized transcripts, we found that p53-mediated reduction of MYC suppressed general transcription, with the most highly expressed transcripts reduced to a greater extent. In contrast, upregulation of p53 targets was relatively unaffected by MYC suppression. Reducing MYC during the DNA damage response was important for cell-fate regulation, as counteracting MYC repression reduced cell-cycle arrest and elevated apoptosis. Our study shows that global inhibition with specific activation of transcriptional pathways is important for the proper response to DNA damage; this mechanism may be a general principle used in many stress responses.

CRISPR/Cas9-mediated gene knockout is insensitive to target copy number but is dependent on guide RNA potency and Cas9/sgRNA threshold expression level.

CRISPR/Cas9 is a powerful gene editing tool for gene knockout studies and functional genomic screens. Successful implementation of CRISPR often requires Cas9 to elicit efficient target knockout in a population of cells. In this study, we investigated the role of several key factors, including variation in target copy number, inherent potency of sgRNA guides, and expression level of Cas9 and sgRNA, in determining CRISPR knockout efficiency. Using isogenic, clonal cell lines with variable copy numbers of an EGFP transgene, we discovered that CRISPR knockout is relatively insensitive to target copy number, but is highly dependent on the potency of the sgRNA guide sequence. Kinetic analysis revealed that most target mutation occurs between 5 and 10 days following Cas9/sgRNA transduction, while sgRNAs with different potencies differ by their knockout time course and by their terminal-phase knockout efficiency. We showed that prolonged, low level expression of Cas9 and sgRNA often fails to elicit target mutation, particularly if the potency of the sgRNA is also low. Our findings provide new insights into the behavior of CRISPR/Cas9 in mammalian cells that could be used for future improvement of this platform.

Pancreatic cancers require autophagy for tumor growth.

Macroautophagy (autophagy) is a regulated catabolic pathway to degrade cellular organelles and macromolecules. The role of autophagy in cancer is complex and may differ depending on tumor type or context. Here we show that pancreatic cancers have a distinct dependence on autophagy. Pancreatic cancer primary tumors and cell lines show elevated autophagy under basal conditions. Genetic or pharmacologic inhibition of autophagy leads to increased reactive oxygen species, elevated DNA damage, and a metabolic defect leading to decreased mitochondrial oxidative phosphorylation. Together, these ultimately result in significant growth suppression of pancreatic cancer cells in vitro. Most importantly, inhibition of autophagy by genetic means or chloroquine treatment leads to robust tumor regression and prolonged survival in pancreatic cancer xenografts and genetic mouse models. These results suggest that, unlike in other cancers where autophagy inhibition may synergize with chemotherapy or targeted agents by preventing the up-regulation of autophagy as a reactive survival mechanism, autophagy is actually required for tumorigenic growth of pancreatic cancers de novo, and drugs that inactivate this process may have a unique clinical utility in treating pancreatic cancers and other malignancies with a similar dependence on autophagy. As chloroquine and its derivatives are potent inhibitors of autophagy and have been used safely in human patients for decades for a variety of purposes, these results are immediately translatable to the treatment of pancreatic cancer patients, and provide a much needed, novel vantage point of attack.

p53 and Pten control neural and glioma stem/progenitor cell renewal and differentiation.

Glioblastoma (GBM) is a highly lethal brain tumour presenting as one of two subtypes with distinct clinical histories and molecular profiles. The primary GBM subtype presents acutely as a high-grade disease that typically harbours mutations in EGFR, PTEN and INK4A/ARF (also known as CDKN2A), and the secondary GBM subtype evolves from the slow progression of a low-grade disease that classically possesses PDGF and TP53 events. Here we show that concomitant central nervous system (CNS)-specific deletion of p53 and Pten in the mouse CNS generates a penetrant acute-onset high-grade malignant glioma phenotype with notable clinical, pathological and molecular resemblance to primary GBM in humans. This genetic observation prompted TP53 and PTEN mutational analysis in human primary GBM, demonstrating unexpectedly frequent inactivating mutations of TP53 as well as the expected PTEN mutations. Integrated transcriptomic profiling, in silico promoter analysis and functional studies of murine neural stem cells (NSCs) established that dual, but not singular, inactivation of p53 and Pten promotes an undifferentiated state with high renewal potential and drives increased Myc protein levels and its associated signature. Functional studies validated increased Myc activity as a potent contributor to the impaired differentiation and enhanced renewal of NSCs doubly null for p53 and Pten (p53(-/-) Pten(-/-)) as well as tumour neurospheres (TNSs) derived from this model. Myc also serves to maintain robust tumorigenic potential of p53(-/-) Pten(-/-) TNSs. These murine modelling studies, together with confirmatory transcriptomic/promoter studies in human primary GBM, validate a pathogenetic role of a common tumour suppressor mutation profile in human primary GBM and establish Myc as an important target for cooperative actions of p53 and Pten in the regulation of normal and malignant stem/progenitor cell differentiation, self-renewal and tumorigenic potential.

Exceptional Response to Trastuzumab Deruxtecan in a Patient With Recurrent Ovarian Clear Cell Carcinoma With Human Epidermal Growth Factor Receptor 2 Expression.

Case report of a HER2-expressed ovarian clear cell carcinoma with exceptional response to trastuzumab deruxtecan.

Mig-6 controls EGFR trafficking and suppresses gliomagenesis.

Glioblastoma multiforme (GBM) is the most common and lethal primary brain cancer that is driven by aberrant signaling of growth factor receptors, particularly the epidermal growth factor receptor (EGFR). EGFR signaling is tightly regulated by receptor endocytosis and lysosome-mediated degradation, although the molecular mechanisms governing such regulation, particularly in the context of cancer, remain poorly delineated. Here, high-resolution genomic profiles of GBM identified a highly recurrent focal 1p36 deletion encompassing the putative tumor suppressor gene, Mig-6. We show that Mig-6 quells the malignant potential of GBM cells and dampens EGFR signaling by driving EGFR into late endosomes and lysosome-mediated degradation upon ligand stimulation. Mechanistically, this effect is mediated by the binding of Mig-6 to a SNARE protein STX8, a protein known to be required for late endosome trafficking. Thus, Mig-6 functions to ensure recruitment of internalized receptor to late endosomes and subsequently the lysosomal degradation compartment through its ability to specifically link EGFR and STX8 during ligand-stimulated EGFR trafficking. In GBM, the highly frequent loss of Mig-6 would therefore serve to sustain aberrant EGFR-mediated oncogenic signaling. Together, these data uncover a unique tumor suppression mechanism involving the regulation of receptor trafficking.

An omic and multidimensional spatial atlas from serial biopsies of an evolving metastatic breast cancer.

Mechanisms of therapeutic resistance and vulnerability evolve in metastatic cancers as tumor cells and extrinsic microenvironmental influences change during treatment. To support the development of methods for identifying these mechanisms in individual people, here we present an omic and multidimensional spatial (OMS) atlas generated from four serial biopsies of an individual with metastatic breast cancer during 3.5 years of therapy. This resource links detailed, longitudinal clinical metadata that includes treatment times and doses, anatomic imaging, and blood-based response measurements to clinical and exploratory analyses, which includes comprehensive DNA, RNA, and protein profiles; images of multiplexed immunostaining; and 2- and 3-dimensional scanning electron micrographs. These data report aspects of heterogeneity and evolution of the cancer genome, signaling pathways, immune microenvironment, cellular composition and organization, and ultrastructure. We present illustrative examples of how integrative analyses of these data reveal potential mechanisms of response and resistance and suggest novel therapeutic vulnerabilities.

Cholesterol Metabolism: A Potential Therapeutic Target in Glioblastoma.

Glioblastoma is a highly lethal adult brain tumor with no effective treatments. In this review, we discuss the potential to target cholesterol metabolism as a new strategy for treating glioblastomas. Twenty percent of cholesterol in the body is in the brain, yet the brain is unique among organs in that it has no access to dietary cholesterol and must synthesize it de novo. This suggests that therapies targeting cholesterol synthesis in brain tumors might render their effects without compromising cell viability in other organs. We will describe cholesterol synthesis and homeostatic feedback pathways in normal brain and brain tumors, as well as various strategies for targeting these pathways for therapeutic intervention.

LXRß controls glioblastoma cell growth, lipid balance, and immune modulation independently of ABCA1.

Cholesterol is a critical component of membranes and a precursor for hormones and other signaling molecules. Previously, we showed that unlike astrocytes, glioblastoma cells do not downregulate cholesterol synthesis when plated at high density. In this report, we show that high cell density induces ABCA1 expression in glioblastoma cells, enabling them to get rid of excess cholesterol generated by an activated cholesterol biosynthesis pathway. Because oxysterols are agonists for Liver X Receptors (LXRs), we investigated whether increased cholesterol activates LXRs to maintain cholesterol homeostasis in highly-dense glioblastoma cells. We observed that dense cells had increased oxysterols, which activated LXRß to upregulate ABCA1. Cells with CRISPR-mediated knockdown of LXRß, but not ABCA1, had decreased cell cycle progression and cell survival, and decreased feedback repression of the mevalonate pathway in densely-plated glioma cells. LXRß gene expression poorly correlates with ABCA1 in glioblastoma patients, and expression of each gene correlates with poor patient prognosis in different prognostic subtypes. Finally, gene expression and lipidomics analyses cells revealed that LXRß regulates the expression of immune response gene sets and lipids known to be involved in immune modulation. Thus, therapeutic targeting of LXRß in glioblastoma might be effective through diverse mechanisms.

Exploiting Radiation-Induced Signaling to Increase the Susceptibility of Resistant Cancer Cells to Targeted Drugs: AKT and mTOR Inhibitors as an Example.

Implementing targeted drug therapy in radio-oncologic treatment regimens has greatly improved the outcome of cancer patients. However, the efficacy of molecular targeted drugs such as inhibitory antibodies or small molecule inhibitors essentially depends on target expression and activity, which both can change during the course of treatment. Radiotherapy has previously been shown to activate prosurvival pathways, which can help tumor cells to adapt and thereby survive treatment. Therefore, we aimed to identify changes in signaling induced by radiation and evaluate the potential of targeting these changes with small molecules to increase the therapeutic efficacy on cancer cell survival. Analysis of "The Cancer Genome Atlas" database disclosed a significant overexpression of AKT1, AKT2, and MTOR genes in human prostate cancer samples compared with normal prostate gland tissue. Multifractionated radiation of three-dimensional-cultured prostate cancer cell lines with a dose of 2 Gy/day as a clinically relevant schedule resulted in an increased protein phosphorylation and enhanced protein-protein interaction between AKT and mTOR, whereas gene expression of AKT, MTOR, and related kinases was not altered by radiation. Similar results were found in a xenograft model of prostate cancer. Pharmacologic inhibition of mTOR/AKT signaling after activation by multifractionated radiation was more effective than treatment prior to radiotherapy. Taken together, our findings provide a proof-of-concept that targeting signaling molecules after activation by radiotherapy may be a novel and promising treatment strategy for cancers treated with multifractionated radiation regimens such as prostate cancer to increase the sensitivity of tumor cells to molecular targeted drugs. Mol Cancer Ther; 17(2); 355-67. ©2017 AACRSee all articles in this MCT Focus section, "Developmental Therapeutics in Radiation Oncology."

Disabled cell density sensing leads to dysregulated cholesterol synthesis in glioblastoma.

A hallmark of cellular transformation is the evasion of contact-dependent inhibition of growth. To find new therapeutic targets for glioblastoma, we looked for pathways that are inhibited by high cell density in astrocytes but not in glioma cells. Here we report that glioma cells have disabled the normal controls on cholesterol synthesis. At high cell density, astrocytes turn off cholesterol synthesis genes and have low cholesterol levels, but glioma cells keep this pathway on and maintain high cholesterol. Correspondingly, cholesterol pathway upregulation is associated with poor prognosis in glioblastoma patients. Densely-plated glioma cells increase oxygen consumption, aerobic glycolysis, and the pentose phosphate pathway to synthesize cholesterol, resulting in a decrease in reactive oxygen species, TCA cycle intermediates, and ATP. This constitutive cholesterol synthesis is controlled by the cell cycle, as it can be turned off by cyclin-dependent kinase inhibitors and it correlates with disabled cell cycle control though loss of p53 and RB. Finally, glioma cells, but not astrocytes, are sensitive to cholesterol synthesis inhibition downstream of the mevalonate pathway, suggesting that specifically targeting cholesterol synthesis might be an effective treatment for glioblastoma.

Phosphorylation by casein kinase I promotes the turnover of the Mdm2 oncoprotein via the SCF(beta-TRCP) ubiquitin ligase.

Mdm2 is the major negative regulator of the p53 pathway. Here, we report that Mdm2 is rapidly degraded after DNA damage and that phosphorylation of Mdm2 by casein kinase I (CKI) at multiple sites triggers its interaction with, and subsequent ubiquitination and destruction, by SCF(beta-TRCP). Inactivation of either beta-TRCP or CKI results in accumulation of Mdm2 and decreased p53 activity, and resistance to apoptosis induced by DNA damaging agents. Moreover, SCF(beta-TRCP)-dependent Mdm2 turnover also contributes to the control of repeated p53 pulses in response to persistent DNA damage. Our results provide insight into the signaling pathways controlling Mdm2 destruction and further suggest that compromised regulation of Mdm2 results in attenuated p53 activity, thereby facilitating tumor progression.

Malignant astrocytic glioma: genetics, biology, and paths to treatment.

Malignant astrocytic gliomas such as glioblastoma are the most common and lethal intracranial tumors. These cancers exhibit a relentless malignant progression characterized by widespread invasion throughout the brain, resistance to traditional and newer targeted therapeutic approaches, destruction of normal brain tissue, and certain death. The recent confluence of advances in stem cell biology, cell signaling, genome and computational science and genetic model systems have revolutionized our understanding of the mechanisms underlying the genetics, biology and clinical behavior of glioblastoma. This progress is fueling new opportunities for understanding the fundamental basis for development of this devastating disease and also novel therapies that, for the first time, portend meaningful clinical responses.

Coactivation of receptor tyrosine kinases affects the response of tumor cells to targeted therapies.

Targeted therapies that inhibit receptor tyrosine kinases (RTKs) and the downstream phosphatidylinositol 3-kinase (PI3K) signaling pathway have shown promising anticancer activity, but their efficacy in the brain tumor glioblastoma multiforme (GBM) and other solid tumors has been modest. We hypothesized that multiple RTKs are coactivated in these tumors and that redundant inputs drive and maintain downstream signaling, thereby limiting the efficacy of therapies targeting single RTKs. Tumor cell lines, xenotransplants, and primary tumors indeed show multiple concomitantly activated RTKs. Combinations of RTK inhibitors and/or RNA interference, but not single agents, decreased signaling, cell survival, and anchorage-independent growth even in glioma cells deficient in PTEN, a frequently inactivated inhibitor of PI3K. Thus, effective GBM therapy may require combined regimens targeting multiple RTKs.

Hdmx modulates the outcome of p53 activation in human tumor cells.

Tumors that express wild-type P53 provide a target for therapies designed to reactivate P53 function. This is supported by the potent activation of P53 in tumor cells by Nutlin, a cis-imidazoline that inhibits the Hdm2-P53 interaction. The efficacy of Hdm2.P53 antagonists could be compromised if they do not antagonize Hdmx, an Hdm2 homolog that inhibits P53 transactivation. We evaluated the role of Hdmx expression in sensitivity to Nutlin in a range of cancer cell lines. Nutlin reduced Hdmx levels in normal cells and some cancer cell lines, whereas other cancer cells were refractory to such down-regulation. Strikingly, Nutlin did not disrupt Hdmx.P53 complexes, and in cell lines where no Hdmx degradation occurred, Nutlin failed to induce apoptosis. shRNA-mediated reduction of Hdmx sensitized cells to apoptosis, but caspase-3 was neither required nor sufficient for Hdmx degradation or apoptosis. Our data imply that Hdmx is an important determinant of the outcome of P53 activation. Thus, targeting Hdmx may be a therapeutic strategy that complements drugs such as Nutlin.

A new twist in the feedback loop: stress-activated MDM2 destabilization is required for p53 activation.

The p53 tumor suppressor is a transcription factor that is activated by diverse genotoxic and cytotoxic stresses. Upon activation, p53 prevents the proliferation of genetically unstable cells by regulating the expression of genes that initiate cell cycle arrest, apoptosis, and DNA repair. Consequently, p53 must be kept inactive in unstressed cells as its inappropriate activation can cause premature senescence and death. p53 inhibition occurs primarily through the E3 ubiquitin ligase, MDM2. Because MDM2 is also a p53 target gene, stresses paradoxically activate p53 while simultaneously increasing MDM2 expression. Therefore, a challenge has been to explain how the abundant MDM2 is prevented from inhibiting p53, thus ensuring that p53 can execute an appropriate stress response. Here we discuss a new mechanism for p53 activation involving DNA damage-induced auto-degradation of MDM2. Our data reveal that DNA damage leads to the destabilization of MDM2, which correlates with p53 stabilization and target gene induction. Conversely, p53 levels and activity decrease when MDM2 returns to a more stable state later in the stress response. The destabilization of MDM2 is required for p53 activation, as blocking MDM2 degradation via proteasome inhibition prevents p53 transactivation in DNA-damaged cells by enabling MDM2 to bind and inhibit p53. MDM2 destabilization is controlled by DNA damage-activated post-translational modifications and by its own RING domain, implying a possible role for the RING domain-interacting protein, MDMX, in regulating MDM2 stability. We propose that accelerated degradation of MDM2 limits its binding to p53 during a stress response and enables p53 to accumulate and remain active, even as p53 transcriptionally activates more MDM2. Thus, the induction of MDM2 RNA by activated p53 may create a reserve of MDM2 that can inactivate p53 once the DNA damage stimulus has abated and MDM2 is restabilized. As many tumors inactivate wild type p53 through MDM2 overexpression, exploiting the pathways that trigger MDM2 auto-degradation may be an important new strategy for chemotherapeutic intervention.

Accelerated MDM2 auto-degradation induced by DNA-damage kinases is required for p53 activation.

p53 activation prevents the proliferation of genetically unstable cells. Conversely, p53 antagonism by its transcriptional target, the E3 ubiquitin ligase MDM2, is critical for the viability of unstressed, cycling cells. We demonstrate that MDM2 induces the degradation of p53 in both the nucleus and the cytoplasm. As p53 and MDM2 accumulate in the nuclei of stressed cells, we investigated mechanisms enabling p53 activation despite the high MDM2 levels generated during a DNA-damage response. We show that DNA damage destabilized MDM2 by a mechanism involving damage-activated kinases and MDM2 auto-ubiquitination. p53 was stable and transcriptionally active when MDM2 was unstable, but became unstable and inactive as the damage response waned and MDM2 stabilized. Importantly, blocking MDM2 destabilization in DNA-damaged cells prevented p53 target gene activation. Our data reveal that controlled MDM2 degradation is an important new step in p53 regulation.