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The therapeutic efficacy of temozolomide (TMZ) is hindered by inherent and acquired resistance. Biomarkers such as MGMT expression and MMR proficiency are used as predictors of response. However, not all MGMTlow/−ve/MMRproficient patients benefit from TMZ treatment, indicating a need for additional patient selection criteria. We explored the role of ATR in mediating TMZ resistance and whether ATR inhibitors (ATRi) could reverse this resistance in multiple cancer lines. We observed that only 31% of MGMTlow/−ve/MMRproficient patient-derived and established cancer lines are sensitive to TMZ at clinically relevant concentrations. TMZ treatment resulted in DNA damage signaling in both sensitive and resistant lines, but prolonged G2/M arrest and cell death were exclusive to sensitive models. Inhibition of ATR but not ATM, sensitized the majority of resistant models to TMZ and resulted in measurable DNA damage and persistent growth inhibition. Also, compromised homologous recombination (HR) via RAD51 or BRCA1 loss only conferred sensitivity to TMZ when combined with an ATRi. Furthermore, low REV3L mRNA expression correlated with sensitivity to the TMZ and ATRi combination in vitro and in vivo. This suggests that HR defects and low REV3L levels could be useful selection criteria for enhanced clinical efficacy of an ATRi plus TMZ combination.

Green tea catechins are associated with a delay in aging. We have designed the current study to investigate the impact and to unveil the target of the most abundant green tea catechins, epigallocatechin gallate (EGCG) and epicatechin gallate (ECG). Experiments were performed in Caenorhabditis elegans to analyze cellular metabolism, ROS homeostasis, stress resistance, physical exercise capacity, health- and lifespan, and the underlying signaling pathways. Besides, we examined the impact of EGCG and ECG in isolated murine mitochondria. A concentration of 2.5 μM EGCG and ECG enhanced health- and lifespan as well as stress resistance in C. elegans. Catechins hampered mitochondrial respiration in C. elegans after 6–12 h and the activity of complex I in isolated rodent mitochondria. The impaired mitochondrial respiration was accompanied by a transient drop in ATP production and a temporary increase in ROS levels in C. elegans. After 24 h, mitochondrial respiration and ATP levels got restored, and ROS levels even dropped below control conditions. The lifespan increases induced by EGCG and ECG were dependent on AAK-2/AMPK and SIR-2.1/SIRT1, as well as on PMK-1/p38 MAPK, SKN-1/NRF2, and DAF-16/FOXO. Long-term effects included significantly diminished fat content and enhanced SOD and CAT activities, required for the positive impact of catechins on lifespan. In summary, complex I inhibition by EGCG and ECG induced a transient drop in cellular ATP levels and a temporary ROS burst, resulting in SKN-1 and DAF-16 activation. Through adaptative responses, catechins reduced fat content, enhanced ROS defense, and improved healthspan in the long term., Aging, 13 (19), ISSN:1945-4589

The major adaptive response to hypoxia involves hypoxia-inducible factor HIF-1α which is regulated by von Hippel Lindau (VHL) E3 ligase. We previously observed a stabilization of HIF-1α by cyclin-dependent kinases CDK1 and CDK4/6 that is independent of VHL, hypoxia or p53, and found that CDK4/6 inhibitors destabilize HIF-1α under normoxia and hypoxia. To further investigate the molecular mechanism of HIF-1α destabilization by CDK1 or CDK4/6 inhibitors, we performed a proteomic screen on immunoprecipitated HIF-1α from hypoxic colorectal cancer cells that were either untreated or treated with CDK1 inhibitor Ro3306 and CDK4/6 inhibitor palbociclib. Our proteomics screen identified a number of candidates that were enriched in palbociclib-treated hypoxic cells including SMAD specific E3 ubiquitin protein ligase 2 (Smurf2). We also identified a HIF-1α peptide that appeared to be differentially phosphorylated after palbociclib treatment. Gene knockdown of SMURF2 increased basal expression of HIF-1α even in the presence of Ro3306 or two different CDK4/6 inhibitors, palbociclib and abemaciclib. Overexpression of Smurf2 inhibited expression of HIF-1α and enhanced HIF-1α ubiquitination in normoxia. Proteasome inhibitor MG-132 partially rescued HIF-1α expression when Smurf2 was overexpressed. Smurf2 overexpression also inhibited HIF-1α expression level in two other cell lines, SW480 and VHL-deficient RCC4. Overexpression of SMURF2 mRNA is correlated with improved disease-free survival and overall survival in clear cell renal cell cancer. Our results unravel a previously unknown mechanism involving Smurf2 for HIF-1α destabilization in CDK4/6 inhibitor-treated cells, thereby shedding light on VHL-independent HIF-1α regulation.

Senescent cells, which arise due to damage-associated signals, are apoptosis-resistant and can express a pro-inflammatory, tissue-destructive senescence-associated secretory phenotype (SASP). We recently reported that a component of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) surface protein, S1, can amplify the SASP of senescent cultured human cells and that a related mouse β-coronavirus, mouse hepatitis virus (MHV), increases SASP factors and senescent cell burden in infected mice. Here, we show that SARS-CoV-2 induces senescence in human non-senescent cells and exacerbates the SASP in human senescent cells through Toll-like receptor-3 (TLR-3). TLR-3, which senses viral RNA, was increased in human senescent compared to non-senescent cells. Notably, genetically or pharmacologically inhibiting TLR-3 prevented senescence induction and SASP amplification by SARS-CoV-2 or Spike pseudotyped virus. While an artificial TLR-3 agonist alone was not sufficient to induce senescence, it amplified the SASP in senescent human cells. Consistent with these findings, lung p16INK4a+ senescent cell burden was higher in patients who died from acute SARS-CoV-2 infection than other causes. Our results suggest that induction of cellular senescence and SASP amplification through TLR-3 contribute to SARS-CoV-2 morbidity, indicating that clinical trials of senolytics and/or SASP/TLR-3 inhibitors for alleviating acute and long-term SARS-CoV-2 sequelae are warranted.

Background Telomerase/telomere-targeting therapy is a potentially promising approach for cancer treatment because even transient telomere dysfunction can induce chromosomal instability (CIN) and may be a barrier to tumor growth. Method: We recently developed a dual-HAC (Human Artificial Chromosome) assay that enables identification and ranking of compounds that induce CIN as a result of telomere dysfunction. This assay is based on the use of two isogenic HT1080 cell lines, one carrying a linear HAC (containing telomeres) and the other carrying a circular HAC (lacking telomeres). Disruption of telomeres in response to drug treatment results in specific destabilization of the linear HAC. Results In this study, we used the dual-HAC assay for the analysis of the platinum-derived G4 ligand Pt-tpy and five of its derivatives: Pt-cpym, Pt-vpym, Pt-ttpy, Pt(PA)-tpy, and Pt-BisQ. Our analysis revealed four compounds, Pt-tpy, Pt-ttpy, Pt-vpym and Pt-cpym, that induce a specific loss of a linear but not a circular HAC. Increased CIN after treatment by these compounds correlates with the induction of double-stranded breaks (DSBs) predominantly localized at telomeres and reflecting telomere-associated DNA damage. Analysis of the mitotic phenotypes induced by these drugs revealed an elevated rate of chromatin bridges (CBs) in late mitosis and cytokinesis. Conclusions These terpyridine platinum-derived G4 ligands are promising compounds for cancer treatment.

Cellular senescence is linked to chronic age-related diseases including atherosclerosis, diabetes, and neurodegeneration. Compared to proliferating cells, senescent cells express distinct subsets of proteins. In this study, we used cultured human diploid fibroblasts rendered senescent through replicative exhaustion or ionizing radiation to identify proteins differentially expressed during senescence. We identified acid ceramidase (ASAH1), a lysosomal enzyme that cleaves ceramide into sphingosine and fatty acid, as being highly elevated in senescent cells. This increase in ASAH1 levels in senescent cells was associated with a rise in the levels of ASAH1 mRNA and a robust increase in ASAH1 protein stability. Furthermore, silencing ASAH1 in pre-senescent fibroblasts decreased the levels of senescence proteins p16, p21, and p53, and reduced the activity of the senescence-associated β-galactosidase. Interestingly, depletion of ASAH1 in pre-senescent cells sensitized these cells to the senolytics Dasatinib and Quercetin (D+Q). Together, our study indicates that ASAH1 promotes senescence, protects senescent cells, and confers resistance against senolytic drugs. Given that inhibiting ASAH1 sensitizes cells towards senolysis, this enzyme represents an attractive therapeutic target in interventions aimed at eliminating senescent cells.

GRSF1 is a mitochondrial RNA-binding protein important for maintaining mitochondrial function. We found that GRSF1 is highly expressed in cultured skeletal myoblasts differentiating into myotubes. To understand the physiological function of GRSF1 in vivo, we generated mice in which GRSF1 was specifically ablated in skeletal muscle. The conditional knockout mice (Grsf1cKO) appeared normal until 7-9 months of age. Importantly, however, a reduction of muscle endurance compared to wild-type controls was observed in 16- to 18-month old Grsf1cKO mice. Transcriptomic analysis revealed more than 200 mRNAs differentially expressed in Grsf1cKO muscle at this age. Notably, mRNAs encoding proteins involved in mitochondrial function, inflammation, and ion transport, including Mgarp, Cxcl10, Nfkb2, and Sln mRNAs, were significantly elevated in aged Grsf1cKO muscle. Our findings suggest that GRSF1 deficiency exacerbates the functional decline of aged skeletal muscle, likely through multiple downstream effector proteins.

Senescent cells produce chronic inflammation that contributes to the diseases and debilities of aging. How this process is orchestrated in epithelial cells, the origin of human carcinomas, is poorly understood. We used human normal oral keratinocytes (NOKs) to elucidate senescence programs in a prototype primary mucosal epithelial cell that senesces spontaneously. While NOKs exhibit several typical facets of senescence, they also display distinct characteristics. These include expression of p21WAF1/CIP1 at early passages, making this common marker of senescence unreliable in NOKs. Transcriptome analysis by RNA-seq revealed specific commonalities with and differences from cancer cells, explicating the tumor avoidance role of senescence. Repression of DNA repair genes that correlated with downregulation of E2F1 mRNA and protein was observed for two donors; a divergent result was seen for the third. Using proteomic profiling of soluble (non-vesicular) and extracellular vesicle (EV) associated secretions, we propose additions to the senescence associated secretory phenotype, including HSP60, which localizes to the surface of EVs. Finally, EVs from senescent NOKs activate interferon pathway signaling in THP-1 monocytes in a STING-dependent manner and associate with mitochondrial and nuclear DNA. Our results highlight senescence changes in epithelial cells and how they might contribute to chronic inflammation and age-related diseases.

Cellular senescence of endothelial cells causes vascular dysfunction, promotes atherosclerosis, and contributes to the development of age-related vascular diseases. Sirtuin 6 (SIRT6), a conserved NAD+-dependent protein deacetylase, has beneficial effects against aging, despite the fact that its functional mechanisms are largely uncharacterized. Here, we show that SIRT6 protects endothelial cells from senescence. SIRT6 expression is progressively decreased during both oxidative stress-induced senescence and replicative senescence. SIRT6 deficiency leads to endothelial dysfunction, growth arrest, and premature senescence. Using genetically engineered endothelial cell-specific SIRT6 knockout mice, we also show that down-regulation of SIRT6 expression in endothelial cells exacerbates vascular aging. Expression microarray analysis demonstrated that SIRT6 modulates the expression of multiple genes involved in cell cycle regulation. Specifically, SIRT6 appears to regulate the expression of forkhead box M1 (FOXM1), a critical transcription factor for cell cycle progression and senescence. Overexpression of FOXM1 ameliorates SIRT6 deficiency-induced endothelial cell senescence. In this work, we demonstrate the role of SIRT6 as an anti-aging factor in the vasculature. These data may provide the basis for future novel therapeutic approaches against age-related vascular disorders.

Foxg1 plays important roles in regeneration of hair cell (HC) in the cochlea of neonatal mouse. Here, we used Sox9-CreER to knock down Foxg1 in supporting cells (SCs) in the utricle in order to investigate the role of Foxg1 in HC regeneration in the utricle. We found Sox9 an ideal marker of utricle SCs and bred Sox9CreER/+Foxg1loxp/loxp mice to conditionally knock down Foxg1 in utricular SCs. Conditional knockdown (cKD) of Foxg1 in SCs at postnatal day one (P01) led to increased number of HCs at P08. These regenerated HCs had normal characteristics, and could survive to at least P30. Lineage tracing showed that a significant portion of newly regenerated HCs originated from SCs in Foxg1 cKD mice compared to the mice subjected to the same treatment, which suggested SCs trans-differentiate into HCs in the Foxg1 cKD mouse utricle. After neomycin treatment in vitro, more HCs were observed in Foxg1 cKD mice utricle compared to the control group. Together, these results suggest that Foxg1 cKD in utricular SCs may promote HC regeneration by inducing trans-differentiation of SCs. This research therefore provides theoretical basis for the effects of Foxg1 in trans-differentiation of SCs and regeneration of HCs in the mouse utricle.

Severe/critical patients with coronavirus disease 2019 (COVID-19) have become the central issue in the current global pandemic due to their high mortality rate. However, the relationship between antibody response and clinical outcomes has not been well described in this group. We conducted a single-center, retrospective, cohort study to investigate the relationship between serum immunoglobulin G (IgG) and IgM and clinical outcomes in severe/critical patients with COVID-19. Seventy-nine severe/critical patients with COVID-19 admitted in Wuhan Asia General Hospital in Wuhan, China during January 22, 2020 to March 6, 2020 were included. Serum antibodies were measured at day 25 (SD, 7) post illness onset. The median IgG titer was 113 (IQR 81-167) AU/ml, and IgM titer was 50 (IQR, 23-105) AU/ml. Patients whose IgM titer ≥ 50 AU/ml had higher in-hospital mortality (p=0.026). IgM titer ≥ 50 AU/ml was also correlated with higher incidences of Acute Respiratory Distress Syndrome (ARDS) and sepsis shock. Antibody remeasurements were performed in 42 patients, where IgM titer declined significantly in survivors (p=0.031). Serum IgM titer changes according to the COVID-19 progression. The severe/critical patients with COVID-19 have a higher risk of clinical adverse events when IgM titer ≥ 50 AU/ml. Further decreasing of IgM could imply a better outcome in severe/critical cases.

When transcribed DNA is damaged, the transcription and DNA repair machineries must interact to ensure successful DNA repair. The mechanisms of this interaction in the context of chromatin are still being elucidated. Here we show that the SIRT6 protein enhances non-homologous end joining (NHEJ) DNA repair by transiently repressing transcription. Specifically, SIRT6 mono-ADP ribosylates the lysine demethylase JHDM1A/KDM2A leading to rapid displacement of KDM2A from chromatin, resulting in increased H3K36me2 levels. Furthermore, we found that through HP1α binding, H3K36me2 promotes subsequent H3K9 tri-methylation. This results in transient suppression of transcription initiation by RNA polymerase II and recruitment of NHEJ factors to DNA double-stranded breaks (DSBs). These data reveal a mechanism where SIRT6 mediates a crosstalk between transcription and DNA repair machineries to promote DNA repair. SIRT6 functions in multiple pathways related to aging, and its novel function coordinating DNA repair and transcription is yet another way by which SIRT6 promotes genome stability and longevity.

Humanin is a member of a new family of peptides that are encoded by short open reading frames within the mitochondrial genome. It is conserved in animals and is both neuroprotective and cytoprotective. Here we report that in C. elegans the overexpression of humanin is sufficient to increase lifespan, dependent on daf-16/Foxo. Humanin transgenic mice have many phenotypes that overlap with the worm phenotypes and, similar to exogenous humanin treatment, have increased protection against toxic insults. Treating middle-aged mice twice weekly with the potent humanin analogue HNG, humanin improves metabolic healthspan parameters and reduces inflammatory markers. In multiple species, humanin levels generally decline with age, but here we show that levels are surprisingly stable in the naked mole-rat, a model of negligible senescence. Furthermore, in children of centenarians, who are more likely to become centenarians themselves, circulating humanin levels are much greater than age-matched control subjects. Further linking humanin to healthspan, we observe that humanin levels are decreased in human diseases such as Alzheimer's disease and MELAS (Mitochondrial Encephalopathy, Lactic Acidosis, and Stroke-like episodes). Together, these studies are the first to demonstrate that humanin is linked to improved healthspan and increased lifespan.

Endothelial dysfunction, with increased endothelin-1 (ET-1) synthesis, and sarcopenia, characterized by the loss of muscular mass and strength, are two aging–related conditions. However, a relationship between them has not been already established. The aim of this study was to determine whether ET-1 induces senescence and fibrosis in cultured murine myoblasts, which could be involved in the development of sarcopenia related to aging. For this purpose, myoblasts were incubated with ET-1 to assess cellular senescence, analyzed by senescence associated β-galactosidase activity and p16 expression; and fibrosis, assessed by fibronectin expression. ET-1 induced myoblast senescence and fibrosis through ETA receptor. The use of antioxidants and several antagonists revealed that ET-1 effect on senescence and fibrosis depended on ROS production and activation of PI3K-AKT-GSK pathway. To stress the in vivo relevance of these results, circulating ET-1, muscular strength, muscular fibrosis and p16 expression were measured in male C57Bl6 mice from 5-18-24-months-old. Old mice shown high levels of ET-1 correlated with muscular fibrosis, muscular p16 expression and loss of muscle strength. In conclusion, ET-1 promotes fibrosis and senescence in cultured myoblasts, similar results were found in old mice, suggesting a potential role for ET-1 in the development of sarcopenia related to aging.

Oxidatively-induced DNA damage, widely accepted as a key player in the onset of cancer, is predominantly repaired by base excision repair (BER). BER is initiated by DNA glycosylases, which locate and remove damaged bases from DNA. NTHL1 is a bifunctional DNA glycosylase in mammalian cells that predominantly removes oxidized pyrimidines. In this study, we investigated a germline variant in the N-terminal domain of NTHL1, R33K. Expression of NTHL1 R33K in human MCF10A cells resulted in increased proliferation and anchorage-independent growth compared to NTHL1 WT-expressing cells. However, wt-NTHL1 and R33K-NTHL1 exhibited similar substrate specificity, excision kinetics, and enzyme turnover in vitro and in vivo. The results of this study indicate an important function of R33 in BER that is disrupted by the R33K mutation. Furthermore, the cellular transformation induced by R33K-NTHL1 expression suggests that humans harboring this germline variant may be at increased risk for cancer incidence.

Altered adipose tissue may contribute to the longevity of Snell dwarf and growth hormone receptor (GHR) knock-out mice. We report here that white (WAT) and brown (BAT) fat have elevated UCP1 in both kinds of mice, and that adipocytes in WAT depots turn beige/brown. These imply increased thermogenesis and are expected to lead to improved glucose control. Both kinds of long-lived mice show lower levels of inflammatory M1 macrophages and higher levels of anti-inflammatory M2 macrophages in BAT and WAT, with correspondingly lower levels of TNFα, IL-6, and MCP1. Experiments with mice with tissue-specific disruption of GHR showed that these adipocyte and macrophage changes were not due to hepatic IGF1 production nor to direct GH effects on adipocytes, but instead reflect GH effects on muscle. Muscles deprived of GH signals, either globally (GKO) or in muscle only (MKO), produce higher levels of circulating irisin and its precursor FNDC5. The data thus suggest that the changes in adipose tissue differentiation and inflammatory status seen in long-lived mutant mice reflect interruption of GH-dependent irisin inhibition, with consequential effects on metabolism and thermogenesis.

Cellular senescence, triggered by sublethal damage, is characterized by indefinite growth arrest, altered gene expression patterns, and a senescence-associated secretory phenotype. While the accumulation of senescent cells during aging decreases tissue function and promotes many age-related diseases, at present there is no universal marker to detect senescent cells in tissues. Cyclin-dependent kinase inhibitors 2A (p16/CDKN2A) and 1A (p21/CDKN1A) can identify senescent cells, but few studies have examined the numbers of cells expressing these markers in different organs as a function of age. Here, we investigated systematically p16- and p21-positive cells in tissue arrays designed to include normal organs from persons across a broad spectrum of ages. Increased numbers of p21-positive and p16-positive cells with donor age were found in skin (epidermis), pancreas, and kidney, while p16-expressing cells increased in brain cortex, liver, spleen and intestine (colon), and p21-expressing cells increased in skin (dermis). The numbers of cells expressing p16 or p21 in lung did not change with age, and muscle did not appear to have p21- or p16-positive cells. In summary, different organs display different levels of the senescent proteins p16 and p21 as a function of age across the human life span.

// Aaditya Khatri 1 , Jing Jin Gu 1 , Courtney M. McKernan 1 , Xia Xu 1 and Ann Marie Pendergast 1 1 Department of Pharmacology and Cancer Biology, Duke University Medical Center, Durham, NC, USA Correspondence to: Ann Marie Pendergast, email: ann.pendergast@duke.edu Keywords : lung adenocarcinoma; docetaxel; ABL kinases; differentiation; YAP1 Received: January 05, 2019 Accepted: February 15, 2019 Published: March 08, 2019 Abstract Lung cancer is the leading cause of cancer mortality in the United States, with an overall five-year survival rate of ~16%. Non-small cell lung cancer (NSCLC) accounts for ~80% of all lung cancer cases, and the majority (40%) of these are adenocarcinomas. Loss of function point mutations in TP53 (46%) and activating mutations in KRAS (33%) are the most common mutations in human lung adenocarcinomas. Because neither of these genetic alterations are clinically actionable, chemotherapy remains the mainstay of treatment in patients with oncogenic KRAS driver mutations. However, chemoresistance to genotoxic agents such as docetaxel remains a major clinical challenge facing lung cancer patients. Here we show that ABL kinase allosteric inhibitors can be effectively used for the treatment of Kras G12D/+ ; p53 -/- lung adenocarcinomas in an autochthonous mouse model. Unexpectedly, we found that treatment of tumor-bearing mice with an ABL allosteric inhibitor promoted differentiation of lung adenocarcinomas from poorly differentiated tumors expressing basal cell markers to tumors expressing terminal differentiation markers in vivo , which rendered lung adenocarcinomas susceptible to chemotherapy. These findings uncover a novel therapeutic approach for the treatment of lung adenocarcinomas with poor response to chemotherapy.

PTEN is a tumor suppressor that is highly mutated in a variety of human cancers. Recent studies have suggested a link between PTEN loss and deficiency in the non-homologous end-joining (NHEJ) pathway of DNA double strand break (DSB) repair. As a means to achieve synthetic lethality in this context, we tested the effect of 3E10, a cell-penetrating autoantibody that inhibits RAD51, a key factor in the alternative pathway of DSB repair, homology dependent repair (HDR). We report here that treatment of PTEN-deficient glioma cells with 3E10 leads to an accumulation of DNA damage causing decreased proliferation and increased cell death compared to isogenic PTEN proficient controls. Similarly, 3E10 was synthetically lethal to a series of PTEN-deficient, patient-derived primary melanoma cell populations. Further, 3E10 was found to synergize with a small molecule inhibitor of the ataxia telangiectasia and Rad3-related (ATR) protein, a DNA damage checkpoint kinase, in both PTEN-deficient glioma cells and primary melanoma cells. These results point to a targeted synthetic lethal strategy to treat PTEN-deficient cancers through a combination designed to disrupt both DNA repair and DNA damage checkpoint signaling.

Cancer stem cells (CSCs), which play important roles in tumor initiation and progression, are resistant to many types of therapies. However, the regulatory mechanisms underlying CSC-specific properties, including self-renewal, are poorly understood. Here, we found that LATS1/2, the core Hippo pathway-kinases, were highly expressed in the oral squamous cell carcinoma line SAS, which exhibits high capacity of CSCs, and that depletion of these kinases prevented SAS cells from forming spheres under serum-free conditions. Detailed examination of the expression and activation of LATS kinases and related proteins over a time course of sphere formation revealed that LATS1/2 were more highly expressed and markedly activated before initiation of self-renewal. Moreover, TAZ, SNAIL, CHK1/2, and Aurora-A were expressed in hierarchical, oscillating patterns during sphere formation, suggesting that the process consists of four sequential steps. Our results indicate that LATS1/2 trigger self-renewal of CSCs by regulating the Hippo pathway, the EMT, and cell division.

// David K. Edwards V 1,* , David Tyler Sweeney 2,* , Hibery Ho 2 , Christopher A. Eide 2 , Angela Rofelty 2 , Anupriya Agarwal 2 , Selina Qiuying Liu 2 , Alexey V. Danilov 2 , Patrice Lee 3 , David Chantry 3 , Shannon K. McWeeney 4 , Brian J. Druker 2,5 , Jeffrey W. Tyner 1 , Stephen E. Spurgeon 2,** and Marc M. Loriaux 2,** 1 Department of Cell, Developmental and Cancer Biology, Oregon Health & Science University, Portland, OR, USA 2 Division of Hematology & Medical Oncology, Oregon Health & Science University Knight Cancer Institute, Portland, OR, USA 3 Array BioPharma, Boulder, CO, USA 4 Department of Bioinformatics and Computational Biology, Oregon Health & Science University, Portland, OR, USA 5 Howard Hughes Medical Institute, Oregon Health & Science University, Knight Cancer Institute, Portland, OR, USA * These authors have contributed equally to this work ** These authors have contributed equally to this work Correspondence to: Marc M. Loriaux, email: // Keywords : colony-stimulating factor 1 receptor; chronic lymphocytic leukemia; tumor-associated macrophages; tumor microenvironment; small-molecule inhibitors Received: September 26, 2017 Accepted: March 01, 2018 Published: May 15, 2018 Abstract In many malignancies, the tumor microenvironment includes CSF1R-expressing supportive monocyte/macrophages that promote tumor cell survival. For chronic lymphocytic leukemia (CLL), these supportive monocyte/macrophages are known as nurse-like cells (NLCs), although the potential effectiveness of selective small-molecule inhibitors of CSF1R against CLL is understudied. Here, we demonstrate the preclinical activity of two inhibitors of CSF1R, GW-2580 and ARRY-382, in primary CLL patient samples. We observed at least 25% of CLL samples showed sub-micromolar sensitivity to CSF1R inhibitors. This sensitivity was observed in samples with varying genetic and clinical backgrounds, although higher white cell count and monocyte cell percentage was associated with increased sensitivity. Depleting CD14-expressing monocytes preferentially decreased viability in samples sensitive to CSF1R inhibitors, and treating samples with CSF1R inhibitors eliminated the presence of NLCs in long-term culture conditions. These results indicate that CSF1R small-molecule inhibitors target CD14-expressing monocytes in the CLL microenvironment, thereby depriving leukemia cells of extrinsic support signals. In addition, significant synergy was observed combining CSF1R inhibitors with idelalisib or ibrutinib, two current CLL therapies that disrupt tumor cell intrinsic B-cell receptor signaling. These findings support the concept of simultaneously targeting supportive NLCs and CLL cells and demonstrate the potential clinical utility of this combination.

Transforming growth factor-β (TGFβ) is an important driver of tumor growth via intrinsic and extrinsic mechanisms, and is therefore an attractive target for developing cancer therapeutics. Using preclinical models, we characterized the anti-tumor activity of a small molecule inhibitor of TGFβ receptor I (TGFβRI), galunisertib (LY2157299 monohydrate). Galunisertib demonstrated potent and selective inhibition of TGFβRI with corresponding inhibition of downstream signaling via inhibition of SMAD phosphorylation (pSMAD). Galunisertib also inhibited TGFβ-induced pSMAD in vivo, which enabled a pharmacokinetic/pharmacodynamic profile in Calu6 and EMT6-LM2 tumors. Galunisertib demonstrated anti-tumor activity including inhibition of tumor cell migration and mesenchymal phenotype, reversal of TGFβ-mediated immune-suppression, and tumor growth delay. A concentration-effect relationship was established with a dosing schedule to achieve the optimal level of target modulation. Finally, a rat model demonstrated a correlation between galunisertib-dependent inhibition of pSMAD in tumor tissues and in PBMCs, supporting the use of PBMCs for assessing pharmacodynamic effects. Galunisertib has been tested in several clinical studies with evidence of anti-tumor activity observed in subsets of patients. Here, we demonstrate that galunisertib inhibits a number of TGFβ-dependent functions leading to anti-tumor activity. The enhanced understanding of galunisertib provides rationale for further informed clinical development of TGFβ pathway inhibitors.

Reprogramming of mitochondrial functions sustains tumor growth and may provide therapeutic opportunities. Here, we targeted the protein folding environment in mitochondria by coupling a purine-based inhibitor of the molecular chaperone Heat Shock Protein-90 (Hsp90), PU-H71 to the mitochondrial-targeting moiety, triphenylphosphonium (TPP). Binding of PU-H71-TPP to ADP-Hsp90, Hsp90 co-chaperone complex or mitochondrial Hsp90 homolog, TRAP1 involved hydrogen bonds, π-π stacking, cation-π contacts and hydrophobic interactions with the surrounding amino acids in the active site. PU-H71-TPP selectively accumulated in mitochondria of tumor cells (17-fold increase in mitochondria/cytosol ratio), whereas unmodified PU-H71 showed minimal mitochondrial localization. Treatment of tumor cells with PU-H71-TPP dissipated mitochondrial membrane potential, inhibited oxidative phosphorylation in sensitive cell types, and reduced ATP production, resulting in apoptosis and tumor cell killing. Unmodified PU-H71 had no effect. Bioinformatics analysis identified a “mitochondrial Hsp90” signature in Acute Myeloid Leukemia (AML), which correlates with worse disease outcome. Accordingly, inhibition of mitochondrial Hsp90s killed primary and cultured AML cells, with minimal effects on normal peripheral blood mononuclear cells. These data demonstrate that directing Hsp90 inhibitors with different chemical scaffolds to mitochondria is feasible and confers improved anticancer activity. A potential “addiction” to mitochondrial Hsp90s may provide a new therapeutic target in AML.

SLPI has been implicated in the progression and metastasis of certain cancers. However, the effects of SLPI seem to be tumor-specific and the mechanisms remain poorly defined. Here, we demonstrate that highly metastatic, triple-negative breast cancer (TNBC) 4T1 cells secreted more SLPI compared to their non-metastatic counterparts. Furthermore, SLPI secretion directly correlated with spontaneous lung metastasis from 4T1 tumors orthotopically implanted in mice. Consistent with our experimental results, we also found that higher SLPI expression levels correlate with worse clinical outcome in basal/TNBC patients. Using high-throughput screening we identified a novel compound, C74, which significantly inhibits SLPI secretion. C74 administration in our mouse model slows the growth of primary 4T1 tumors and inhibits their dissemination to the lung. We also discovered that SLPI physically interacts with the retinoblastoma tumor suppressor protein (Rb) and releases FoxM1 from the Rb-FoxM1 complex, which may activate FoxM1 target genes involved in breast cancer metastasis.

// Ourania N. Kostopoulou 1,* , Vanessa Wilhelmi 1,* , Sina Raiss 1 , Sharan Ananthaseshan 1 , Mikael S. Lindstrom 2 , Jiri Bartek 2 and Cecilia Soderberg-Naucler 1 1 Department of Medicine, Center for Molecular Medicine L8:03, Karolinska University Hospital, Stockholm, Sweden 2 Department of Medical Biochemistry and Biophysics, Science For Life Laboratory, Division of Genome Biology, Karolinska Institute, Solna, Sweden * These authors have contributed equally to this study Correspondence to: Cecilia Soderberg-Naucler, email: // Jiri Bartek, email: // Keywords : cytomegalovirus; replication; RNA Polymerase I; Herpes Simplex type 1 virus; nucleolus Received : July 18, 2017 Accepted : October 13, 2017 Published : October 29, 2017 Abstract Human cytomegalovirus (HCMV) utilizes RNA polymerase II to transcribe viral genes and produce viral mRNAs. It can specifically target the nucleolus to facilitate viral transcription and translation. As RNA polymerase I (Pol I)-mediated transcription is active in the nucleolus, we investigated the role of Pol I, along with relative contributions of the human Pol II and Pol III, to early phases of viral transcription in HCMV infected cells, compared with Herpes Simplex Virus-1 (HSV-1) and Murine cytomegalovirus (MCMV). Inhibition of Pol I with siRNA or the Pol I inhibitors CX-5461 or Actinomycin D (5nM) resulted in significantly decreased IE and pp65 mRNA and protein levels in human fibroblasts at early times post infection. This initially delayed replication was compensated for later during the replication process, at which stage it didn’t significantly affect virus production. Pol I inhibition also reduced HSV-1 ICP0 and gB transcripts, suggesting that some herpesviruses engage Pol I for their early transcription. In contrast, inhibition of Pol I failed to affect MCMV transcription. Collectively, our results contribute to better understanding of the functional interplay between RNA Pol I-mediated nucleolar events and the Herpes viruses, particularly HCMV whose pathogenic impact ranges from congenital malformations and potentially deadly infections among immunosuppressed patients, up to HCMV’s emerging oncomodulatory role in human tumors.

// Jordan M. Reese 1 , Elizabeth S. Bruinsma 2 , David G. Monroe 2 , Vivian Negron 3 , Vera J. Suman 4 , James N. Ingle 5 , Matthew P. Goetz 1,5 and John R. Hawse 2 1 Department of Molecular Pharmacology and Experimental Therapeutics, Mayo Clinic, Rochester, MN, USA 2 Department of Biochemistry and Molecular Biology, Mayo Clinic, Rochester, MN, USA 3 Department of Pathology, Mayo Clinic, Rochester, MN, USA 4 Department of Biomedical Statistics and Informatics, Mayo Clinic, Rochester, MN, USA 5 Department of Oncology, Mayo Clinic, Rochester, MN, USA Correspondence to: John R. Hawse, email: // Keywords : TNBC, ERβ, cell cycle, CDK, estrogen Received : March 21, 2017 Accepted : September 16, 2017 Published : October 11, 2017 Abstract Triple negative breast cancer (TNBC), which comprises approximately 15% of all primary breast cancer diagnoses, lacks estrogen receptor alpha, progesterone receptor and human epidermal growth factor receptor 2 expression. However, we, and others, have demonstrated that approximately 30% of TNBCs express estrogen receptor beta (ERβ), a nuclear hormone receptor and potential drug target. Treatment of ERβ expressing MDA-MB-231 cells with estrogen or the ERβ selective agonist, LY500307, was shown to result in suppression of cell proliferation. This inhibitory effect was due to blockade of cell cycle progression. In vivo , estrogen treatment significantly repressed the growth of ERβ expressing MDA-MB-231 cell line xenografts . Gene expression studies and ingenuity pathway analysis identified a network of ERβ down-regulated genes involved in cell cycle progression including CDK1, cyclin B and cyclin H. siRNA mediated knockdown or drug inhibition of CDK1 and CDK7 in TNBC cells resulted in substantial decreases in proliferation regardless of ERβ expression. These data suggest that the tumor suppressive effects of ERβ in TNBC result from inhibition of cell cycle progression, effects that are in part mediated by suppression of CDK1/7. Furthermore, these data indicate that blockade of CDK1/7 activity in TNBC may be of therapeutic benefit, an area of study that has yet to be explored.

// Mihwa Kim 1 , Liza D. Morales 1,2 , Minwoo Baek 1 , Thomas J. Slaga 3 , John DiGiovanni 4 and Dae Joon Kim 1 1 Department of Biomedical Sciences, School of Medicine, University of Texas Rio Grande Valley, Edinburg, TX, USA 2 South Texas Diabetes and Obesity Institute, School of Medicine, University of Texas Rio Grande Valley, Edinburg, TX, USA 3 Department of Pharmacology, School of Medicine, University of Texas Health Science Center at San Antonio, San Antonio, TX, USA 4 Division of Pharmacology & Toxicology, College of Pharmacy, The University of Texas at Austin, Austin, TX, USA Correspondence to: Dae Joon Kim, email: // Keywords : TC-PTP, nuclear translocation, AKT, 14-3-3σ, keratinocytes Received : June 26, 2017 Accepted : September 15, 2017 Published : October 11, 2017 Abstract Understanding protein subcellular localization is important to determining the functional role of specific proteins. T-cell protein tyrosine phosphatase (TC-PTP) contains bipartite nuclear localization signals (NLSI and NLSII) in its C-terminus. We previously have demonstrated that the nuclear form of TC-PTP (TC45) is mainly localized to the cytoplasm in keratinocytes and it is translocated to the nucleus following UVB irradiation. Here, we report that TC45 is translocated by an AKT/14-3-3σ-mediated mechanism in response to UVB exposure, resulting in increased apoptosis and decreased keratinocyte proliferation. We demonstrate that UVB irradiation increased phosphorylation of AKT and induced nuclear translocation of 14-3-3σ and TC45. However, inhibition of AKT blocked nuclear translocation of TC45 and 14-3-3σ. Site-directed mutagenesis of 14-3-3σ binding sites within TC45 showed that a substitution at Threonine 179 (TC45/T179A) effectively blocked UVB-induced nuclear translocation of ectopic TC45 due to the disruption of the direct binding between TC45 and 14-3-3σ. Overexpression of TC45/T179A in keratinocytes resulted in a decrease of UVB-induced apoptosis which corresponded to an increase in nuclear phosphorylated STAT3, and cell proliferation was higher in TC45/T179A-overexpressing keratinocytes compared to control keratinocytes following UVB irradiation. Furthermore, deletion of TC45 NLSII blocked its UVB-induced nuclear translocation, indicating that both T179 and NLSII are required. Taken together, our findings suggest that AKT and 14-3-3σ cooperatively regulate TC45 nuclear translocation in a critical step of an early protective mechanism against UVB exposure that signals the deactivation of STAT3 in order to promote keratinocyte cell death and inhibit keratinocyte proliferation.

Cyclin dependent kinase 1 (CDK1) is essential for cell viability and plays a vital role in many biological events including cell cycle control, DNA damage repair, and checkpoint activation. Here, we identify an unanticipated role for CDK1 in promoting nascent DNA synthesis during S-phase. We report that a short duration of CDK1 inhibition, which does not perturb cell cycle progression, triggers a replication-associated DNA damage response (DDR). This DDR is associated with a disruption of replication fork progression and leads to genome instability. Moreover, we show that compromised CDK1 activity dramatically increases the efficacy of chemotherapeutic agents that kill cancer cells through perturbing DNA replication, including Olaparib, an FDA approved PARP inhibitor. Our study has revealed an important role for CDK1 in the DNA replication program, and suggests that the therapeutic targeting CDK1 may be a novel approach for combination chemotherapy.

Disruption of RNA splicing causes genome instability, which could contribute to cancer etiology. Furthermore, RNA splicing is an emerging anti-cancer target. Thus, we have evaluated the influence of the spliceosome factor PRPF8 and the splicing inhibitor Pladienolide B (PlaB) on homologous recombination (HR). We find that PRPF8 depletion and PlaB treatment cause a specific defect in homology-directed repair (HDR), and single strand annealing (SSA), which share end resection as a common intermediate, and BRCA1 as a required factor. Furthermore, PRPF8 depletion and PlaB treatment cause reduced end resection detected as chromatin-bound RPA, BRCA1 foci in response to damage, and histone acetylation marks that are associated with BRCA1-mediated HR. We also identified distinctions between PlaB and PRPF8 depletion, in that PlaB also reduces 53BP1 foci, and BRCA1 expression. Furthermore loss of 53BP1, which rescues SSA in BRCA1 depleted cells, and partially rescues SSA in PRPF8 depleted cells, has no effect on SSA in PlaB treated cells. Finally, while PRPF8 depletion has no obvious effect on the integrity of interchromatin granules, PlaB disrupts these structures. These findings indicate that PRPF8 is important for BRCA1-mediated HR, whereas PlaB also has a more general effect on the DNA damage response and nuclear organization.

// Bo Cao 1 , Ziling Fang 1,2 , Peng Liao 1 , Xiang Zhou 1,3 , Jianping Xiong 2 , Shelya Zeng 1 and Hua Lu 1 1 Department of Biochemistry & Molecular Biology, Tulane Cancer Center, Tulane University School of Medicine, New Orleans, LA, USA 2 The First Affiliated Hospital of Nanchang University, Nanchang, People’s Republic of China 3 Fudan University Shanghai Cancer Center and the Institutes of Biomedical Sciences, Fudan University, Shanghai, People’s Republic of China Correspondence to: Hua Lu, email: // Keywords : RPL22/eL22, p53, MDM2, RPL11/uL5, RPL5/uL18 Received : July 25, 2017 Accepted : September 08, 2017 Published : October 06, 2017 Abstract Several ribosomal proteins (RPs) in response to various ribosomal stressors have been shown to play a critical role in p53-dependent regulation of cell cycle arrest, apoptosis and tumor suppression. Here, we report ribosomal protein L22 (RPL22/eL22) as a novel p53 activator highly mutated (mostly deletion mutation) in various types of human cancers, but not essential for ribosomal biogenesis in normal cells. Ectopic expression of RPL22/eL22 suppressed the colony formation of cancer cells in a p53-dependent manner, whereas knockdown of RPL22/eL22 significantly compromised p53 activation by Actinomycin D, rescuing p53-induced G1/G0 cell cycle arrest. Interestingly, human tumors with RPL22/eL22 deletion appeared to sustain wild type p53. Mechanistically, RPL22/eL22 bound to MDM2 acidic domain and inhibited MDM2-mediated p53 ubiquitination and degradation, hence extending the half-life of p53. Ribosome-profiling analysis revealed that induction of ribosomal stress by Actinomycin D leads to the increase of ribosome-free RPL22/eL22 pool. Also, RPL22/eL22 formed a complex with MDM2/RPL5/uL18/RPL11/uL5 and synergized with RPL11/uL5 to activate p53. Furthermore, the N terminus of RPL22/eL22 bound to MDM2, while the C terminus interacted with RPL5/uL18/RPL11/uL5; both of these two fragments activated p53 by inhibiting MDM2. Our study indicates that RPL22/eL22 highly mutated in human cancers plays an anti-cancer role likely through regulation of the MDM2-p53 feedback loop, and also suggests that targeting the RPL22/eL22-MDM2-p53 pathway could be a potential strategy for future development of anti-cancer therapy.

Here we present an innovative computational-based drug discovery strategy, coupled with machine-based learning and functional assessment, for the rational design of novel small molecule inhibitors of the lipogenic enzyme stearoyl-CoA desaturase 1 (SCD1). Our methods resulted in the discovery of several unique molecules, of which our lead compound SSI-4 demonstrates potent anti-tumor activity, with an excellent pharmacokinetic and toxicology profile. We improve upon key characteristics, including chemoinformatics and absorption/distribution/metabolism/excretion (ADME) toxicity, while driving the IC50 to 0.6 nM in some instances. This approach to drug design can be executed in smaller research settings, applied to a wealth of other targets, and paves a path forward for bringing small-batch based drug programs into the Clinic.

// Li Qin 1 , Yixiang Xu 1,2 , Xiaobin Yu 1 , Michael J. Toneff 1 , Dabing Li 1,3 , Lan Liao 1 , Jarrod D. Martinez 1 , Yi Li 1 and Jianming Xu 1,3 1 Department of Molecular and Cellular Biology and Dan L. Duncan Cancer Center, Baylor College of Medicine, Houston, TX, USA 2 Institute of Biosciences and Technology, Texas A&M University Health Science Center, Houston, TX, USA 3 Institute for Cancer Medicine and College of Basic Biomedical Sciences, Southwest Medical University, Sichuan, China Correspondence to: Jianming Xu, email: // Li Qin , email: // Keywords : Kdm3a, knockout mice, mammary gland growth, breast cancer, cyclin D1 Received : July 18, 2017 Accepted : August 31, 2017 Published : September 28, 2017 Abstract Histone modification alters chromatin architecture to regulate gene transcription. KDM3A is a histone demethylase in the JmjC domain-containing protein family. It removes di- and mono- methyl residues from di- or mono-methylated lysine 9 of histone H3 (H3K9me2/me1). Recent studies have shown that Kdm3a plays an important role in self-renewal of embryonic stem cells, spermatogenesis, metabolism, sex determination and tumor angiogenesis. However, its role in mammary gland development and breast carcinogenesis remains unclear. In this study, we found that Kdm3a is expressed in the mouse mammary gland epithelial cells. Knockout of Kdm3a significantly increased H3K9me2/me1 levels in these epithelial cells, which correlated with markedly decreased mammary gland ductal elongation and branching in the intact knockout virgin mice. Furthermore, estrogen replacement in the ovariectomized Kdm3a knockout mice couldn’t rescue the retarded ductal growth. Moreover, transplantation of KO mammary gland pieces to wild type recipient mice showed slower ductal growth compared with that of WT gland pieces. Consistently, knockout of Kdm3a also reduced the proliferation rates and cyclin D1 expression in the mammary gland epithelial cells. In addition, Kdm3a knockout did not significantly change the latency of the polyoma middle T oncogene-induced mammary gland tumorigenesis. Tumor growth, however, was slowed which might be due to the decrease in cyclin D1 expression and tumor cell proliferation. We also found that Kdm3a binds and activates the cyclin D1 promoter. These results demonstrate that Kdm3a plays an important intrinsic role in promoting mammary gland ductal growth and tumor growth probably through enhancing cyclin D1 expression and cell proliferation.

// Keiichi Sakakibara 1 , Takuji Sato 1 , Donald W. Kufe 2 , Daniel D. VonHoff 3 and Takumi Kawabe 1 1 CanBas Co., Ltd., Numazu, Shizuoka, Japan 2 Dana-Farber Cancer Institute, Harvard Medical School, Boston, Massachusetts, USA 3 Translational Genomics Research Institute (TGen), Phoenix, Arizona, USA Correspondence to: Keiichi Sakakibara, email: // Keywords : platinum agent, immunogenic cell death, immune checkpoint inhibitor, Anti-PD-1, CD8+ T cell Received : June 27, 2017 Accepted : August 26, 2017 Published : September 16, 2017 Abstract CBP501, a calmodulin-binding peptide, is an anti-cancer drug candidate and functions as an enhancer of platinum uptake into cancer cells. Here we show that CBP501 promotes immunogenic cell death (ICD) in combination with platinum agents. CBP501 enhanced a clinically relevant low dose of cisplatin (CDDP) in vitro as evidenced by upregulation of ICD markers, including cell surface calreticulin exposure and release of high-mobility group protein box-1. Synergistic induction of ICD by CDDP plus CBP501 as compared to CDDP alone was confirmed in the well-established vaccination assay. Furthermore, cotreatment of CDDP plus CBP501 significantly reduced the tumor growth and upregulated the percentage of tumor infiltrating CD8 + T cell in vivo . Importantly, the antitumor effect of CDDP plus CBP501 was significantly reduced by anti-CD8 antibody treatment. Based on this novel effect of CBP501, we analyzed the combination treatment with immune checkpoint inhibitors in vivo . Mice treated with CBP501 in combination with CDDP and anti-PD-1 or anti-PD-L1 showed an additive antitumor effect. These results support the conclusion that CBP501 enhances CDDP-induced ICD in vitro and in vivo . The findings also support the further clinical development of the CBP501 for enhancing the antitumor activity of immune checkpoint inhibitors in combination with CDDP.

// Payal Jain 1,2,3 , Amanda Silva 1 , Harry J. Han 2 , Shih-Shan Lang 1,2 , Yuankun Zhu 1,3 , Katie Boucher 1,2,3 , Tiffany E. Smith 1,2,3 , Aesha Vakil 4 , Patrick Diviney 5 , Namrata Choudhari 1,2,3 , Pichai Raman 3,6,7 , Christine M. Busch 8 , Tim Delaney 1,2,3 , Xiaodong Yang 9 , Aleksandra K. Olow 10 , Sabine Mueller 9,11,12 , Daphne Haas-Kogan 13 , Elizabeth Fox 8 , Phillip B. Storm 1,2,3 , Adam C. Resnick 1,2,3,6,* and Angela J. Waanders 3,8,14,* 1 Division of Neurosurgery, The Children’s Hospital of Philadelphia, Philadelphia, PA, USA 2 Department of Neurosurgery, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA, USA 3 Center for Data Driven Discovery in Biomedicine (D3b), The Children’s Hospital of Philadelphia, Philadelphia, PA, USA 4 The Fred Hutchinson Cancer Research Center, Seattle, WA, USA 5 Division of Neurology, The Children’s Hospital of Philadelphia, Philadelphia, PA, USA 6 Department of Biomedical and Health Informatics, The Children’s Hospital of Philadelphia, Philadelphia, PA, USA 7 Center for Childhood Cancer Research, Children’s Hospital of Philadelphia, Philadelphia, PA, USA 8 Division of Oncology, Department of Pediatrics, The Children’s Hospital of Philadelphia, Philadelphia, PA, USA 9 Division of Neurology, University of California, San Francisco, CA, USA 10 Amgen, South San Francisco, CA, USA 11 Department of Neurosurgery, University of California, San Francisco, CA, USA 12 Department of Pediatrics, University of California, San Francisco, CA, USA 13 Department of Radiation Oncology, Harvard Medical School, Boston, MA, USA 14 Department of Pediatrics, Perelman School of Medicine at the University of Pennsylvania, Philadelphia, PA, USA * These authors are co-senior authors and have contributed equally to this work Correspondence to: Angela J. Waanders, email: waandersA@email.chop.edu Keywords : pediatric low-grade glioma, BRAF-fusions, trametinib and everolimus, MAPK pathway, PI3K/mTOR pathway Received : March 03, 2017 Accepted : August 23, 2017 Published : September 15, 2017 Abstract Pediatric low-grade gliomas (PLGGs) are frequently associated with activating BRAF gene fusions, such as KIAA1549-BRAF, that aberrantly drive the mitogen activated protein kinase (MAPK) pathway. Although RAF inhibitors (RAFi) have been proven effective in BRAF-V600E mutant tumors, we have previously shown how the KIAA1549-BRAF fusion can be paradoxically activated by RAFi. While newer classes of RAFi, such as PLX8394, have now been shown to inhibit MAPK activation by KIAA1549-BRAF, we sought to identify alternative MAPK pathway targeting strategies using clinically relevant MEK inhibitors (MEKi), along with potential escape mechanisms of acquired resistance to single-agent MAPK pathway therapies. We demonstrate effectiveness of multiple MEKi against diverse BRAF-fusions with novel N-terminal partners, with trametinib being the most potent. However, resistance to MEKi or PLX8394 develops via increased RTK expression causing activation of PI3K/mTOR pathway in BRAF-fusion expressing resistant clones. To circumvent acquired resistance, we show potency of combinatorial targeting with trametinib and everolimus, an mTOR inhibitor (mTORi) against multiple BRAF-fusions. While single-agent mTORi and MEKi PLGG clinical trials are underway, our study provides preclinical rationales for using MEKi and mTORi combinatorial therapy to stave off or prevent emergent drug-resistance in BRAF-fusion driven PLGGs.

// Elke Burgermeister 1,* , Patrick Hode 1,* , Johannes Betge 1 , Tobias Gutting 1 , Andreas Merkel 1 , Wen Wu 1 , Marc Tanzer 2 , Maximilian Mossner 3 , Daniel Nowak 3 , Julia Magdeburg 4 , Felix Ruckert 4 , Carsten Sticht 5 , Katja Breitkopf-Heinlein 1 , Nadine Schulte 1 , Nicolai Hartel 1 , Sebastian Belle 1 , Stefan Post 4 , Timo Gaiser 6 , Barbara Ingold Heppner 7 , Hans-Michael Behrens 8 , Christoph Rocken 8 and Matthias P.A. Ebert 1 1 Department of Medicine II, University Hospital Mannheim, Medical Faculty Mannheim, Heidelberg University, Mannheim, Germany 2 Department of Medicine II, Klinikum rechts der Isar, Technische Universitat Munchen, Munich, Germany 3 Department of Medicine III, University Hospital Mannheim, Medical Faculty Mannheim, Heidelberg University, Mannheim, Germany 4 Department of Surgery, University Hospital Mannheim, Medical Faculty Mannheim, Heidelberg University, Mannheim, Germany 5 Center for Medical Research (ZMF), Medical Faculty Mannheim, Heidelberg University, Mannheim, Germany 6 Institute of Pathology, University Hospital Mannheim, Medical Faculty Mannheim, Heidelberg University, Mannheim, Germany 7 Institute of Pathology, University Hospital Charite, Berlin, Germany 8 Institute of Pathology, Christian-Albrechts University, Kiel, Germany * These authors have contributed equally to this work Correspondence to: Matthias P.A. Ebert, email: // Keywords : TUSC3, colorectal cancer, prognosis, glycosylation, growth factor receptor Received : March 14, 2017 Accepted : August 23, 2017 Published : September 15, 2017 Abstract Colorectal cancer (CRC) is a biologically and clinically heterogeneous disease. Even though many recurrent genomic alterations have been identified that may characterize distinct subgroups, their biological impact and clinical significance as prognostic indicators remain to be defined. The tumor suppressor candidate-3 ( TUSC3/N33 ) locates to a genomic region frequently deleted or silenced in cancers. TUSC3 is a subunit of the oligosaccharyltransferase (OST) complex at the endoplasmic reticulum (ER) which catalyzes bulk N-glycosylation of membrane and secretory proteins. However, the consequences of TUSC3 loss are largely unknown. Thus, the aim of the study was to characterize the functional and clinical relevance of TUSC3 expression in CRC patients’ tissues ( n =306 cases) and cell lines. TUSC3 mRNA expression was silenced by promoter methylation in 85 % of benign adenomas ( n =46 cases) and 35 % of CRCs ( n =74 cases). Epidermal growth factor receptor (EGFR) was selected as one exemplary ER-derived target protein of TUSC3-mediated posttranslational modification. We found that TUSC3 inhibited EGFR-signaling and promoted apoptosis in human CRC cells, whereas TUSC3 siRNA knock-down increased EGFR-signaling. Accordingly, in stage I/II node negative CRC patients ( n =156 cases) loss of TUSC3 protein expression was associated with poor overall survival. In sum, our data suggested that epigenetic silencing of TUSC3 may be useful as a molecular marker for progression of early CRC.

// Georgios N. Pongas 1,2,3 , Christina M. Annunziata 2,3 and Louis M. Staudt 1 1 Lymphoid Malignancies Branch, National Cancer Institute, National Institutes of Health, Bethesda, MD, USA 2 Medical Oncology Service, National Cancer Institute, National Institutes of Health, Bethesda, MD, USA 3 Women’s Malignancies Branch, National Cancer Institute, National Institutes of Health, Bethesda, MD, USA Correspondence to: Louis M. Staudt, email: // Keywords : DLBCL, PI3K, Idelalisib, ABC DLBCL, BYL719 Received : August 07, 2017 Accepted : August 15, 2017 Published : September 13, 2017 Abstract Cell line models of the activated B cell-like (ABC) subtype of diffuse large B cell (DLBCL) depend on both NF-κB and phosphatidylinositol 3-kinase (PI3K) signaling pathways for survival, especially those with gain-of-function B cell receptor (BCR) mutations. Here we show that these cells depend specifically on the PI3Kδ isoform, but that PI3K pathway interruption by PI3Kδ inhibitors is short-lived due to feedback activation of the PI3Kα isoform. PI3Kδ and PI3Kα inhibition cooperated in killing ABC DLBCL lines, and genetic knockdown of PI3Kα sensitized cells to PI3Kδ inhibition and prolonged the interruption of PI3K signaling. PI3Kδ inhibition evoked feedback activation of proximal BCR signaling, which increased the association of PI3Kα with BCAP and CD19 and increased overall PI3K activity. These results support the clinical evaluation of dual PI3Kδ and PI3Kα inhibition in patients with ABC DLBCL.

// Evgeny Teplyakov 1,3 , Qiongfang Wu 1 , Jian Liu 1 , Elena M. Pugacheva 2 , Dmitry Loukinov 2 , Abdelhalim Boukaba 1 , Victor Lobanenkov 2 and Alexander Strunnikov 1,3 1 Molecular Epigenetics Laboratory, Guangzhou Institutes of Biomedicine and Health, Guangzhou, China 2 NIH, NIAID, Laboratory of Immunogenetics, Rockville, MD, USA 3 The University of the Chinese Academy of Sciences, Beijing, China Correspondence to: Alexander Strunnikov, email: // Keywords : transcriptome, chromatin, CML, CTA, sncRNA Received : June 16, 2017 Accepted : August 23, 2017 Published : September 02, 2017 Abstract The BORIS/CTCFL gene, is a testis-specific CTCF paralog frequently erroneously activated in cancer, although its exact role in cancer remains unclear. BORIS is both a transcription factor and an architectural chromatin protein. BORIS’ normal role is to establish a germline-like gene expression and remodel the epigenetic landscape in testis; it similarly remodels chromatin when activated in human cancer. Critically, at least one cancer cell line, K562, is dependent on BORIS for its self-renewal and survival. Here, we downregulate BORIS expression in the K562 cancer cell line to investigate downstream pathways regulated by BORIS. RNA-seq analyses of both mRNA and small ncRNAs, including miRNA and piRNA, in the knock-down cells revealed a set of differentially expressed genes and pathways, including both testis-specific and general proliferation factors, as well as proteins involved in transcription regulation and cell physiology. The differentially expressed genes included important transcriptional regulators such as SOX6 and LIN28A . Data indicate that both direct binding of BORIS to promoter regions and locus-control activity via long-distance chromatin domain regulation are involved. The sum of findings suggests that BORIS activation in leukemia does not just recapitulate the germline, but creates a unique regulatory network.

The permanent defects in bone growth observed in preclinical studies of hedgehog (Hh) pathway inhibitors were not substantiated in early phase clinical studies of vismodegib in children. Consequently, vismodegib advanced into pediatric trials for malignancies suspected of being driven by aberrant activation of the Hh pathway. In one multicenter phase II trial, vismodegib was added to the therapy regimen for newly diagnosed Hh pathway activated medulloblastoma. Herein, we report on 3 children (2 on trial and one off trial) treated with vismodegib who developed widespread growth plate fusions that persist long after cessation of therapy. Currently, all 3 patients exhibit profound short stature and disproportionate growth, and 2 subsequently developed precocious puberty. Notably, the growth plate fusions only developed after a prolonged exposure to the drug (> 140 days). These findings resulted in a major trial amendment to restrict the agent to skeletally mature patients as well as a product label warning and update. Moreover, these findings alter the risk-benefit ratio of Hh inhibitors and underscore the importance of careful study of targeted agents in children.

There is substantial evidence that early growth response-1 (Egr1) gene, a zinc-finger transcription factor, behaves as a tumor suppressor in leukemia. This includes reports from this laboratory that constitutive Egr1 overrides leukemia conferred by deregulated c-Myc or E2F-1 in the M1 myeloid leukemic cell line by promoting differentiation. To investigate the effect of Egr1 on the initiation and progression of Chronic Myelogenous Leukemia (CML), lethally irradiated syngeneic wild type mice were reconstituted with bone marrow (BM) from either wild type or Egr1 null mice transduced with a 210-kD BCR-ABL-expressing MSCV-retrovirus (bone marrow transplantation {BMT}). Loss of Egr1 was observed to accelerate the development of BCR-ABL driven leukemia in recipient mice, resulting in the development of a more aggressive disease, a significantly shortened median survival time, and increased BCR-ABL expressing leukemic stem/progenitor cells (GFP+Lin-cKit+Sca+). Egr1 deficient progenitors expressing BCR-ABL exhibited decreased apoptosis, and increased cell viability and proliferation relative to WT counterparts. Secondary BMT of BCR-ABL BM revealed that loss of Egr1 resulted in enrichment of LSCs, consistent with shorter survival time and more aggressive disease of these mice compared to WT counterparts. Furthermore, serial re-plating colony assays indicated that loss of Egr1 increased self-renewal ability of BCR-ABL expressing BM. These novel findings on the tumor suppressor role of Egr1 in CML provide the impetus to study the effect of altering Egr1 expression in AML, where the overall five year survival rate remains low. The effect of loss of Egr1 in CML could reflect its established functions in normal hematopoiesis, maintaining quiescence of HSCs and driving terminal differentiation to the monocyte/macrophage lineage. Gain of function studies should validate these conclusions and provide further rationale for increased Egr1 as a therapeutic target in AML.

// Robert J. Cardnell 1 , Lerong Li 2 , Triparna Sen 1 , Rasha Bara 1 , Pan Tong 2 , Junya Fujimoto 3 , Abbie S. Ireland 4 , Matthew R. Guthrie 4 , Sheila Bheddah 5 , Upasana Banerjee 1 , Nene N. Kalu 1 , You-Hong Fan 1 , Scott J. Dylla 5 , Faye M. Johnson 1,6 , Ignacio I. Wistuba 3 , Trudy G. Oliver 4 , John V. Heymach 1 , Bonnie S. Glisson 1 , Jing Wang 2,4,* and Lauren A. Byers 1,6,* 1 Department of Thoracic/Head and Neck Medical Oncology, The University of Texas MD Anderson Cancer Center, Houston, TX, USA 2 Department of Bioinformatics and Computational Biology, The University of Texas MD Anderson Cancer Center, Houston, TX, USA 3 Department of Molecular Translational Pathology, The University of Texas MD Anderson Cancer Center, Houston, TX, USA 4 Department of Oncological Sciences, University of Utah, Huntsman Cancer Institute, Salt Lake City, UT, USA 5 AbbVie StemCentrx, LLC, San Franciso, CA, USA 6 The University of Texas Graduate School of Biomedical Sciences, Houston, TX, USA * Co-senior authors Correspondence to: Lauren A. Byers, email: // Keywords : Alisertib, DLL3, rovalpituzumab tesirine, SCLC, TTF1 Received : July 04, 2017 Accepted : August 14, 2017 Published : September 01, 2017 Abstract Small cell lung cancer (SCLC) is a recalcitrant cancer for which no new treatments have been approved in over 30 years. While molecular subtyping now guides treatment selection for patients with non-small cell lung cancer and other cancers, SCLC is still treated as a single disease entity. Using model-based clustering, we found two major proteomic subtypes of SCLC characterized by either high thyroid transcription factor-1 (TTF1)/low cMYC protein expression or high cMYC/low TTF1. Applying “drug target constellation” (DTECT) mapping, we further show that protein levels of TTF1 and cMYC predict response to targeted therapies including aurora kinase, Bcl2, and HSP90 inhibitors. Levels of TTF1 and DLL3 were also highly correlated in preclinical models and patient tumors. TTF1 (used in the diagnosis lung cancer) could therefore be used as a surrogate of DLL3 expression to identify patients who may respond to the DLL3 antibody-drug conjugate rovalpituzumab tesirine. These findings suggest that TTF1, cMYC or other protein markers identified here could be used to identify subgroups of SCLC patients who may respond preferentially to several emerging targeted therapies.

// Alison K. Esser 1,* , Daniel A. Rauch 1,* , Jingyu Xiang 1 , John C. Harding 1 , Nicole A. Kohart 2 , Michael H. Ross 1 , Xinming Su 1 , Kevin Wu 1 , Devra Huey 2 , Yalin Xu 1 , Kiran Vij 1 , Patrick L. Green 2 , Thomas J. Rosol 2 , Stefan Niewiesk 2 , Lee Ratner 1 and Katherine N. Weilbaecher 1 1 Department of Medicine, Division of Oncology, Washington University School of Medicine, St. Louis, MO, USA 2 Department of Veterinary Biosciences, School of Veterinary Medicine, The Ohio State University, Columbus, OH, USA * Co-authors Correspondence to: Katherine N. Weilbaecher, email: // Keywords : HTLV-1, ATL, leukemia, HBZ, bone Received : June 10, 2017 Accepted : August 03, 2017 Published : August 27, 2017 Abstract Adult T-cell leukemia/lymphoma (ATL) is an aggressive T cell malignancy that occurs in HTLV-1 infected patients. Most ATL patients develop osteolytic lesions and hypercalcemia of malignancy, causing severe skeletal related complications and reduced overall survival. The HTLV-1 virus encodes 2 viral oncogenes, Tax and HBZ. Tax, a transcriptional activator, is critical to ATL development, and has been implicated in pathologic osteolysis. HBZ, HTLV-1 basic leucine zipper transcription factor, promotes tumor cell proliferation and disrupts Wnt pathway modulators; however, its role in ATL induced osteolytic bone loss is unknown. To determine if HBZ is sufficient for the development of bone loss, we established a transgenic Granzyme B HBZ (Gzmb-HBZ) mouse model. Lymphoproliferative disease including tumors, enlarged spleens and/or abnormal white cell counts developed in two-thirds of Gzmb-HBZ mice at 18 months. HBZ positive cells were detected in tumors, spleen and bone marrow. Importantly, pathologic bone loss and hypercalcemia were present at 18 months. Bone-acting factors were present in serum and RANKL, PTHrP and DKK1, key mediators of hypercalcemia and bone loss, were upregulated in Gzmb-HBZ T cells. These data demonstrate that Gzmb-HBZ mice model ATL bone disease and express factors that are current therapeutic targets for metastatic and bone resident tumors.

// Matthew L. Fisher 1 , Nicholas Ciavattone 1 , Daniel Grun 1 , Gautam Adhikary 1 and Richard L. Eckert 1,2,3,4 1 Department of Biochemistry and Molecular Biology, University of Maryland School of Medicine, Baltimore, Maryland, USA 2 Department of Dermatology, University of Maryland School of Medicine, Baltimore, Maryland, USA 3 Department of Reproductive Biology, University of Maryland School of Medicine, Baltimore, Maryland, USA 4 The Marlene and Stewart Greenebaum Comprehensive Cancer Center, University of Maryland School of Medicine, Baltimore, Maryland, USA Correspondence to: Richard L. Eckert, email: // Keywords : YAP, TAZ, hippo signaling, ∆Np63α;, sulforaphane Received : July 06, 2017 Accepted : July 31, 2017 Published : August 27, 2017 Abstract Epidermal squamous cell carcinoma (SCC) is among the most common cancers. SCC can be treated by surgical excision, but recurrence of therapy-resistant disease is a major problem. We recently showed that YAP1, the Hippo signaling transcription adaptor protein, and ∆Np63α, a key epidermal stem cell survival protein, form a complex to drive epidermal cancer stem cell survival. In the present study, we demonstrate that YAP1 and ∆Np63α are important sulforaphane cancer prevention targets. We show that sulforaphane treatment increases YAP1 phosphorylation and proteolytic degradation. The loss of YAP1 is associated with a reduction in ∆Np63α level and a reduction in ECS cell survival, spheroid formation, invasion and migration. Loss of YAP1 and ∆Np63α is mediated by the proteasome and can be inhibited by lactacystin treatment. YAP1 or ∆Np63α knockdown replicates the responses to sulforaphane, and restoration of YAP1 or ∆Np63α antagonizes sulforaphane action. Sulforaphane suppresses ECS cell tumor formation and this is associated with reduced levels of YAP1 and ∆Np63α. These studies suggest that YAP1 and ∆Np63α may be important sulforaphane cancer preventive targets in epidermal squamous cell carcinoma.

// Yasmin A. Lyons 1 , Sunila Pradeep 1 , Sherry Y. Wu 1 , Monika Haemmerle 1 , Jean M. Hansen 1 , Michael J. Wagner 1 , Alejandro Villar-Prados 1 , Archana S. Nagaraja 1 , Robert L. Dood 1 , Rebecca A. Previs 1 , Wei Hu 1 , Yang Zhao 4 , Duncan H. Mak, 7 Zhilan Xiao 5 , Brenda D. Melendez 5 , Gregory A. Lizee 5 , Imelda Mercado-Uribe 6 , Keith A. Baggerly 4 , Patrick Hwu 5 , Jinsong Liu 6 , Willem W. Overwijk 5 , Robert L. Coleman 1 and Anil K. Sood 1,2,3 1 Departments of Gynecologic Oncology and Reproductive Medicine, The University of Texas MD Anderson Cancer Center, Houston, TX, USA 2 Center for RNAi and Non-Coding RNA, The University of Texas MD Anderson Cancer Center, Houston, TX, USA 3 Department of Cancer Biology, The University of Texas MD Anderson Cancer Center, Houston, TX, USA 4 Department of Bioinformatics and Computational Biology, Division of Quantitative Sciences, The University of Texas MD Anderson Cancer Center, Houston, TX, USA 5 Department of Melanoma Medical Oncology-Research, Division of Cancer Medicine, The University of Texas MD Anderson Cancer Center, Houston, TX, USA 6 Department of Pathology, Division of Pathology and Laboratory Medicine, Section of Gynecologic Pathology, The University of Texas MD Anderson Cancer Center, Houston, TX, USA 7 Department of Leukemia, The University of Texas MD Anderson Cancer Center, Houston, TX, USA Correspondence to: Anil K. Sood, email: // Keywords : adaptive resistance, anti-VEGF therapy, tumor associated macrophages, tumor microenvironment, CSF1R inhibition Received : April 18, 2017 Accepted : July 20, 2017 Published : August 24, 2017 Abstract Anti-angiogenesis therapy has shown clinical benefit in patients with high-grade serous ovarian cancer (HGSC), but adaptive resistance rapidly emerges. Thus, approaches to overcome such resistance are needed. We developed the setting of adaptive resistance to anti-VEGF therapy, and performed a series of in vivo experiments in both immune competent and nude mouse models. Given the pro-angiogenic properties of tumor-associated macrophages (TAMs) and the dominant role of CSF1R in macrophage function, we added CSF1R inhibitors following emergence of adaptive resistance to anti-VEGF antibody. Mice treated with a CSF1R inhibitor (AC708) after anti-VEGF antibody resistance had little to no measurable tumor burden upon completion of the experiment while those that did not receive a CSF1R inhibitor still had abundant tumor. To mimic clinically used regimens, mice were also treated with anti-VEGF antibody and paclitaxel until resistance emerged, and then a CSF1R inhibitor was added. The addition of a CSF1R inhibitor restored response to anti-angiogenesis therapy, resulting in 83% lower tumor burden compared to treatment with anti-VEGF antibody and paclitaxel alone. Collectively, our data demonstrate that the addition of a CSF1R inhibitor to anti-VEGF therapy and taxane chemotherapy results in robust anti-tumor effects.

// Shyam Kumar Gudey 1,* , Reshma Sundar 1,* , Carl-Henrik Heldin 2 , Anders Bergh 1 and Marene Landstrom 1 1 Department of Medical Biosciences, Umea University, Umea, Sweden 2 Ludwig Institute for Cancer Research, Science for Life Laboratory, Uppsala University, Uppsala, Sweden * These authors have contributed equally to this work Correspondence to: Marene Landstrom, email: // Keywords : signal transduction, tumor biology, Snail1, sumoylation, prostate cancer Received : March 03, 2017 Accepted : May 19, 2017 Published : August 09, 2017 Abstract Transforming growth factor β (TGFβ) is a key regulator of epithelial-to-mesenchymal transition (EMT) during embryogenesis and in tumors. The effect of TGFβ, on EΜΤ, is conveyed by induction of the pro-invasive transcription factor Snail1. In this study, we report that TGFβ stimulates Snail1 sumoylation in aggressive prostate, breast and lung cancer cells. Sumoylation of Snail1 lysine residue 234 confers its transcriptional activity, inducing the expression of classical EMT genes, as well as TGFβ receptor I (TβRI) and the transcriptional repressor Hes1. Mutation of Snail1 lysine residue 234 to arginine (K234R) abolished sumoylation of Snail1, as well as its migratory and invasive properties in human prostate cancer cells. An increased immunohistochemical expression of Snail1, Sumo1, TβRI, Hes1, and c-Jun was observed in aggressive prostate cancer tissues, consistent with their functional roles in tumorigenesis.

// Tsun Yee Tsang 1,2 , Wei Wei 1,2 , Hiroaki Itamochi 3 , Rosemary Tambouret 1 and Michael J. Birrer 1,2 1 Department of Medicine, Massachusetts General Hospital, Boston, Massachusetts, USA 2 Harvard Medical School, Boston, Massachusetts, USA 3 Department of Obstetrics and Gynecology, Tottori University School of Medicine, Yonago, Tottori, Japan Correspondence to: Michael J. Birrer, email: // Keywords : clear cell ovarian cancer, PRKCI, genomics, microarray, therapeutic target Received : April 07, 2017 Accepted : May 29, 2017 Published : August 04, 2017 Abstract Clear cell ovarian cancer (CCOC) is an epithelial ovarian cancer histotype with unique pathologic, biologic and clinical features. Despite its worse prognosis than serous ovarian cancer (SOC), the genomic landscape of CCOC is less well defined. Integrated genomic analysis of CCOC allows the identification of potential therapeutic targets to improve the treatment of this tumor. Using comparative genomic hybridization and gene expression profiling, we have screened 12 CCOC cell lines and 40 tumors to identify 45 amplified and overexpressed genes. Pathways analysis of these genes identified 19 genes with cancer-related functions. Of these, PRKCI is one of the most frequently amplified and overexpressed genes and its expression induced cancer cell proliferation and migration/invasion in vitro as well as tumor growth in vivo . Targeting PRKCI by small molecule inhibitor, sodium aurothiomalate (ATM), significantly reduced the in vivo tumor growth and may be a new therapeutic strategy to improve the treatment of CCOC.

// Hilaire C. Lam 1 , Heng-Jia Liu 1 , Christian V. Baglini 1 , Harilaos Filippakis 1 , Nicola Alesi 1 , Julie Nijmeh 1 , Heng Du 1 , Alicia Llorente Lope 1 , Katherine A. Cottrill 2 , Adam Handen 2 , John M. Asara 3 , David J. Kwiatkowski 1 , Issam Ben-Sahra 4 , William M. Oldham 1 , Stephen Y. Chan 2 and Elizabeth P. Henske 1 1 Department of Medicine, Pulmonary and Critical Care Medicine, Brigham and Women’s Hospital and Harvard Medical School, Boston, MA, USA 2 Department of Medicine, Division of Cardiology, Center for Pulmonary Vascular Biology and Medicine, Pittsburgh Heart, Lung, Blood, and Vascular Medicine Institute, University of Pittsburgh School of Medicine and University of Pittsburgh Medical Center, Pittsburgh, PA, USA 3 Department of Medicine, Division of Signal Transduction, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA, USA 4 Department of Biochemistry and Molecular Genetics, Northwestern University, Chicago, IL, USA Correspondence to: Elizabeth P. Henske, email: // Keywords : tuberous sclerosis complex, mTORC1, rapamycin, miR-21, mitochondria Received : June 20, 2017 Accepted : June 25, 2017 Published : August 04, 2017 Abstract mTORC1 hyperactivation drives the multi-organ hamartomatous disease tuberous sclerosis complex (TSC). Rapamycin inhibits mTORC1, inducing partial tumor responses; however, the tumors regrow following treatment cessation. We discovered that the oncogenic miRNA, miR-21, is increased in Tsc2-deficient cells and, surprisingly, further increased by rapamycin. To determine the impact of miR-21 in TSC, we inhibited miR-21 in vitro . miR-21 inhibition significantly repressed the tumorigenic potential of Tsc2-deficient cells and increased apoptosis sensitivity. Tsc2-deficient cells’ clonogenic and anchorage independent growth were reduced by ~50% ( p

// Yoshihiro Ito 1 , Peter K. Vogt 1 and Jonathan R. Hart 1 1 Department of Molecular Medicine, The Scripps Research Institute, La Jolla, San Diego, CA, USA Correspondence to: Peter K. Vogt, email: // Keywords : SH2 domain, SH3 domain, RhoGAP domain, iSH2 domain, domain exchange Received : November 11, 2016 Accepted : July 12, 2017 Published : August 03, 2017 Abstract Our understanding of isoform-specific activities of phosphatidylinositol 3-kinase (PI3K) is still rudimentary, and yet, deep knowledge of these non-redundant functions in the PI3K family is essential for effective and safe control of PI3K in disease. The two major isoforms of the regulatory subunits of PI3K are p85α and p85β, encoded by the genes PIK3R1 and PIK3R2, respectively. These isoforms show distinct functional differences that affect and control cellular PI3K activity and signaling [ 1 - 4 ]. In this study, we have further explored the differences between p85α and p85β by genetic truncations and substitutions. We have discovered unexpected activities of the mutant proteins that reflect regulatory functions of distinct p85 domains. These results can be summarized as follows: Deletion of the SH3 domain increases oncogenic and PI3K signaling activity. Deletion of the combined SH3-RhoGAP domains abolishes these activities. In p85β, deletion of the cSH2 domain reduces oncogenic and signaling activities. In p85α, such a deletion has an activating effect. The deletions of the combined cSH2 and iSH2 domains and also the deletion of the cSH2, iSH2 and nSH2 domains yield results that go in the same direction, generally activating in p85α and reducing activity in p85β. The contrasting functions of the cSH2 domains are verified by domain exchanges with the cSH2 domain of p85β exerting an activating effect and the cSH2 domain of p85α an inactivating effect, even in the heterologous isoform. In the cell systems studied, protein stability was not correlated with oncogenic and signaling activity. These observations significantly expand our knowledge of the isoform-specific activities of p85α and p85β and of the functional significance of specific domains for regulating the catalytic subunits of class IA PI3K.

Neutropenia represents one of the major dose-limiting toxicities of many current cancer therapies. To circumvent the off-target effects of cytotoxic chemotherapeutics, kinase inhibitors are increasingly being used as an adjunct therapy to target leukemia. In this study, we conducted a screen of leukemic cell lines in parallel with primary neutrophils to identify kinase inhibitors with the capacity to induce apoptosis of myeloid and lymphoid cell lines whilst sparing primary mouse and human neutrophils. We have utilized a high-throughput live cell imaging platform to demonstrate that cytotoxic drugs have limited effects on neutrophil viability but are toxic to hematopoietic progenitor cells, with the exception of the topoisomerase I inhibitor SN-38. The parallel screening of kinase inhibitors revealed that mouse and human neutrophil viability is dependent on cyclin-dependent kinase (CDK) activity but surprisingly only partially dependent on PI3 kinase and JAK/STAT signaling, revealing dominant pathways contributing to neutrophil viability. Mcl-1 haploinsufficiency sensitized neutrophils to CDK inhibition, demonstrating that Mcl-1 is a direct target for CDK inhibitors. This study reveals a therapeutic window for the kinase inhibitors BEZ235, BMS-3, AZD7762, and (R)-BI-2536 to induce apoptosis of leukemia cell lines whilst maintaining immunocompetence and hemostasis.

// Saqer F. Alsaqer 1,* , Mustafa M. Tashkandi 1,* , Vinay K. Kartha 2,3 , Ya-Ting Yang 1 , Yazeed Alkheriji 1 , Andrew Salama 4 , Xaralabos Varelas 5 , Maria Kukuruzinska 1 , Stefano Monti 2,3 and Manish V. Bais 1 1 Department of Molecular and Cell Biology, Boston University Henry M. Goldman School of Dental Medicine, Boston, MA, USA 2 Bioinformatics Program, Boston University, Boston, MA, USA 3 Section of Computational Biomedicine, Boston University School of Medicine, Boston, MA, USA 4 Department of Oral and Maxillofacial Surgery, Boston University Henry M. Goldman School of Dental Medicine, Boston, MA, USA 5 Department of Biochemistry, Boston University School of Medicine, Boston, MA, USA * These authors have contributed equally to this work Correspondence to: Manish V. Bais, email: // Keywords : LSD1, CTGF, OSCC, PDX, orthotopic mouse model Received : December 28, 2016 Accepted : July 14, 2017 Published : July 27, 2017 Abstract Lysine-specific demethylase 1 (LSD1) is a nuclear histone demethylase and a member of the amine oxidase (AO) family. LSD1 is a flavin-containing AO that specifically catalyzes the demethylation of mono- and di-methylated histone H3 lysine 4 through an FAD-dependent oxidative reaction. LSD1 is inappropriately upregulated in lung, liver, brain and esophageal cancers, where it promotes cancer initiation, progression, and metastasis. However, unlike other lysine-specific demethylases, the role and specific targets of LSD1 in oral squamous cell carcinoma (OSCC) pathogenesis remain unknown. We show that LSD1 protein expression was increased in malignant OSCC tissues in a clinical tissue microarray, and its expression correlated with progressive tumor stages. In an orthotopic oral cancer mouse model, LSD1 overexpression in aggressive HSC-3 cells promoted metastasis whereas knockdown of LSD1 inhibited tumor spread, suggesting that LSD1 is a key regulator of OSCC metastasis. Pharmacological inhibition of LSD1 using a specific small molecule inhibitor, GSK-LSD1, down-regulated EGF signaling pathway. Further, GSK-LSD1 attenuates CTGF/CCN2, MMP13, LOXL4 and vimentin expression but increased E-cadherin expression in pre-existing, patient-derived tonsillar OSCC xenografts. Similarly, GSK-LSD1 inhibited proliferation and CTGF expression in mesenchymal cells, including myoepithelial cells and osteosarcoma cells. In addition, gene set enrichment analysis revealed that GSK-LSD1 increased p53 expression and apoptosis while inhibiting c-myc, β-catenin and YAP-induced oncogenic transcriptional networks. These data reveal that aberrant LSD1 activation regulates key OSCC microenvironment and EMT promoting factors, including CTGF, LOXL4 and MMP13.

// Lin Chen 1,2 , Shauna N. Vasilatos 1,3 , Ye Qin 1,3 , Tiffany A. Katz 5 , Chunyu Cao 1,3,8 , Hao Wu 1,6 , Nilgun Tasdemir 1,3 , Kevin M. Levine 1,4 , Steffi Oesterreich 1,3 , Nancy E. Davidson 7 and Yi Huang 1,3 1 Women’s Cancer Research Center, UPMC Hillman Cancer Center, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA 2 School of Medicine, Tsinghua University, Beijing, P.R. China 3 Department of Pharmacology & Chemical Biology, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA 4 Department of Pathology, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA 5 Center for Precision Environmental Health, Baylor College of Medicine, Houston, TX, USA 6 Department of Oncology, The First Affiliated Hospital of Nanjing Medical University, Nanjing, Jiangsu, P.R. China 7 Fred Hutchinson Cancer Research Center and Department of Medicine, University of Washington, Seattle, WA, USA 8 China Three Gorges University, Yichang, Hubei, P. R. China Correspondence to: Yi Huang, email: // Keywords : LSD2/KDM1B, breast cancer, cell growth, migration, invasion Received : April 19, 2017 Accepted : July 03, 2017 Published : July 19, 2017 Abstract Flavin-dependent histone demethylases govern histone H3K4 methylation and act as important chromatin modulators that are extensively involved in regulation of DNA replication, gene transcription, DNA repair, and heterochromatin gene silencing. While the activities of lysine-specific demethylase 1 (LSD1/KDM1A) in facilitating breast cancer progression have been well characterized, the roles of its homolog LSD2 (KDM1B) in breast oncogenesis are relatively less understood. In this study, we showed that LSD2 protein level was significantly elevated in malignant breast cell lines compared with normal breast epithelial cell line. TCGA- Oncomine database showed that LSD2 expression is significantly higher in basal-like breast tumors compared to other breast cancer subtypes or normal breast tissue. Overexpression of LSD2 in MDA-MB-231 cells significantly altered the expression of key important epigenetic modifiers such as LSD1, HDAC1/2, and DNMT3B; promoted cellular proliferation; and augmented colony formation in soft agar; while attenuating motility and invasion. Conversely, siRNA-mediated depletion of endogenous LSD2 hindered growth of multiple breast cancer cell lines while shRNA-mediated LSD2 depletion augmented motility and invasion. Moreover, LSD2 overexpression in MDA-MB-231 cells facilitated mammosphere formation, enriched the subpopulation of CD49f + /EpCAM - and ALDH high , and induced the expression of pluripotent stem cell markers, NANOG and SOX2. In xenograft studies using immune-compromised mice, LSD2-overexpressing MDA-MB-231 cells displayed accelerated tumor growth but significantly fewer lung metastases than controls. Taken together, our findings provide novel insights into the critical and multifaceted roles of LSD2 in the regulation of breast cancer progression and cancer stem cell enrichment.