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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.

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.

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.

We recently documented that gain-of-function (GOF) mutant p53 (mtp53) R273H in triple negative breast cancer (TNBC) cells interacts with replicating DNA and PARP1. The missense R273H GOF mtp53 has a mutated central DNA binding domain that renders it unable to bind specifically to DNA, but maintains the capacity to interact tightly with chromatin. Both the C-terminal domain (CTD) and oligomerization domain (OD) of GOF mtp53 proteins are intact and it is unclear whether these regions of mtp53 are responsible for chromatin-based DNA replication activities. We generated MDA-MB-468 cells with CRISPR-Cas9 edited versions of the CTD and OD regions of mtp53 R273H. These included a frame-shift mtp53 R273Hfs387, which depleted mtp53 protein expression; mtp53 R273HΔ381-388, which had a small deletion within the CTD; and mtp53 R273HΔ347-393, which had both the OD and CTD regions truncated. The mtp53 R273HΔ347-393 existed exclusively as monomers and disrupted the chromatin interaction of mtp53 R273H. The CRISPR variants proliferated more slowly than the parental cells and mt53 R273Hfs387 showed the most extreme phenotype. We uncovered that after thymidine-induced G1/S synchronization, but not hydroxyurea or aphidicholin, R273Hfs387 cells displayed impairment of S-phase progression while both R273HΔ347-393 and R273HΔ381-388 displayed only moderate impairment. Moreover, reduced chromatin interaction of MCM2 and PCNA in mtp53 depleted R273Hfs387 cells post thymidine-synchronization revealed delayed kinetics of replisome assembly underscoring the slow S-phase progression. Taken together our findings show that the CTD and OD domains of mtp53 R273H play critical roles in mutant p53 GOF that pertain to processes associated with DNA replication.

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.

Ulcerative colitis has a significant impact on the quality of life for the patients, and can substantially increase the risk of colon cancer in patients suffering long-term. Conventional treatments provide only modest relief paired with a high risk of side effects, while complementary and alternative medicines can offer safe and effective options. Over the past decade, we have shown that both American ginseng and its hexane fraction (HAG) have anti-oxidant and anti-inflammatory properties that can suppress mouse colitis and prevent colitis-associated colon cancer. With the goal of isolating a single active compound, we further fractionated HAG, and found the most abundant molecule in this fraction was the polyacetylene, panaxynol (PA). After isolating and characterizing PA, we tested the efficacy of PA in the treatment and prevention of colitis in mice and studied the mechanism of action. We demonstrate here that PA effectively treats colitis in a Dextran Sulfate Sodium mouse model by targeting macrophages for DNA damage and apoptosis. This study provides additional mechanistic evidence that American ginseng can be used for conventional treatment of colitis and other diseases associated with macrophage dysfunction.

Purpose Metformin combined with the mTOR inhibitor rapamycin showed potential synergistic anti-tumor activity in preclinical studies in pancreatic ductal adenocarcinoma (PDA). This phase 1b study (NCT02048384) was conducted to evaluate the feasibility and activity of metformin +/- rapamycin in the maintenance setting for unselected patients with metastatic PDA (mPDA) treated with chemotherapy. Materials and methods Eligible patients with stable or responding mPDA after ≥ 6 months on chemotherapy were randomized 1:1 to metformin alone (Arm A) or with rapamycin (Arm B), stratified by prior treatment with FOLFIRINOX. Fluorodeoxyglucose (FDG) PET scans and peripheral blood mononuclear cells were obtained for exploratory analyses. Results 22 subjects (11 per arm) received treatment per protocol. Median PFS/OS were 3.5 and 13.2 months respectively, with 2 year OS rate of 37%; there were no differences between arms. No responses were observed by RECIST; however, decreases in FDG avidity and/or CA19-9 were observed in several long-term survivors. Treatment related adverse events of Grade ≥ 3 occurred in 0% vs 27% of patients in Arm A vs B and were asymptomatic hematologic or electrolyte abnormalities that were not clinically significant. Improved survival was associated with low baseline neutrophil: lymphocyte ratio, baseline lack of assessable disease by PET, and greater expansion of dendritic cells following treatment. Conclusions Metformin +/- rapamycin maintenance for mPDA was well-tolerated and several patients achieved stable disease associated with exceptionally long survival. Further prospective studies are needed to clarify the role of these agents in the maintenance setting and to enhance patient selection for such approaches.

Aberrant activation of mitogenic signaling pathways in cancer promotes growth and proliferation of cells by activating mTOR and S6 phosphorylation, and D-cyclin kinases and Rb phosphorylation, respectively. Correspondingly, inhibition of phosphorylation of both Rb and S6 is required for robust anti-tumor efficacy of drugs that inhibit cell signaling. The best-established mechanism of mTOR activation in cancer is via PI3K/Akt signaling, but mTOR activity can also be stimulated by CDK4 and PIM kinases. In this study, we show that the CDK4/6 inhibitor abemaciclib inhibits PIM kinase and S6 phosphorylation in cancer cells and concurrent inhibition of PIM, CDK4, and CDK6 suppresses both S6 and Rb phosphorylation. TSC2 or PIK3CA mutations obviate the requirement for PIM kinase and circumvent the inhibition of S6 phosphorylation by abemaciclib. Combination with a PI3K inhibitor restored suppression of S6 phosphorylation and synergized to curtail cell growth. By combining abemaciclib with a PI3K inhibitor, three pathways (Akt, PIM, and CDK4) to mTOR activation are neutralized, suggesting a potential combination strategy for the treatment of PIK3CA-mutant ER+ breast cancer.

TP53 is a critical tumor suppressor. In approximately 50% of human cancers the TP53 gene is either lost or mutated. The expression level of TP53 in the cells is tightly controlled by a fine-tune machinery, mainly through the Mdm2-mediated ubiquitination pathway. On the other side, the ubiquitinated TP53 could be reversed and stabilized by USP7 and USP10, to keep the amount of TP53 in check. MicroRNAs can negatively regulate TP53 expression levels through direct targeting or positively regulate TP53 function through the repression of negative regulators of TP53. Here we report that microRNA-138 controls TP53 expression by directly targeting USP10. Furthermore, TP53 represses microRNA-138 expression, forming a negative feedback regulatory loop. This finding adds another layer of complexity to the TP53 network.

// 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.

// Hsu Wei-Shan 1,* , Vas C. Amit 1,2,* and Duncan J. Clarke 1 1 Department of Genetics, Cell Biology and Development, University of Minnesota Medical School, Minneapolis, MN, USA 2 Present address: Cargill Inc., Wayzata, MN, USA * Co-first authors Correspondence to: Duncan J. Clarke, email: clark140@umn.edu Vas C. Amit, email: acjvas@gmail.com Keywords : condensin; Smc4; Smc2; Mad2; anaphase promoting complex Received: July 27, 2018 Accepted: December 04, 2018 Published: January 08, 2019 Abstract The condensin complex is a conserved ATPase which promotes the compaction of chromatin during mitosis in eukaryotic cells. Condensin complexes have in addition been reported to contribute to interphase processes including sister chromatid cohesion. It is not understood how condensins specifically become competent to facilitate chromosome condensation in preparation for chromosome segregation in anaphase. Here we describe evidence that core condensin subunits are regulated at the level of protein stability in budding yeast. In particular, Smc2 and Smc4 abundance is cell cycle regulated, peaking at mitosis and falling to low levels in interphase. Smc4 degradation at the end of mitosis is dependent on the Anaphase Promoting Complex/Cyclosome and is mediated by the proteasome. Overproduction of Smc4 results in delayed decondensation, but has a limited ability to promote premature condensation in interphase. Unexpectedly, the Mad2 spindle checkpoint protein is required for mitotic Smc4 degradation. These studies have revealed the novel finding that condensin protein levels are cell cycle regulated and have identified the factors necessary for Smc4 proteolysis.

Aberrant activation of CDK4/6 kinase is the most common somatic event in non-small cell lung cancer (NSCLC). Palbociclib is a highly specific CDK4/6 inhibitor shown to inhibit cell cycle progression and promote cellular senescence. We conducted a phase 2 clinical trial of palbociclib in 19 previously-treated patients with advanced NSCLC. Only patients with p16-null staining by immunohistochemistry and documented tumor progression were eligible. The primary endpoint was tumor response rate. Palbociclib therapy alone was well-tolerated. Of 16 evaluable patients who received > 1 month of therapy, there were no objective responses. However, 8 patients (50%) with previously progressive NSCLC had stable disease (SD) lasting a range of 4-10.5 months. Median overall survival (OS) for all cases was 5.1 months, and median overall survival for the subset of patients with SD was 16.6 months. We also performed preclinical testing of palbociclib in combination with 13 different targeted or cytotoxic chemotherapeutic agents using a cell viability assay. Only the combination of palbociclib and mTOR inhibitors resulted in synergistic growth inhibition, particularly in tumors carrying RAS mutations. Our findings warrant further clinical investigation of the combination of palbociclib and mTOR inhibitors, especially in patients carrying activated RAS mutations.

ONC201 was initially identified as an inducer of cell death through the tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) pathway. The compound is currently being tested in patients with hematological malignancies and solid tumors, including those of the breast. We investigated strategies to convert the response of breast cancers to ONC201 from anti-proliferative to apoptotic. ONC201 treatment upregulates TRAIL and primes TRAIL-resistant non-triple negative breast cancer (TNBC) cells to undergo cell death through the extrinsic pathway. Remarkably, the addition of exogenous recombinant human TRAIL (rhTRAIL) converts the response of TRAIL-resistant non-TNBC cells to ONC201 from anti-proliferative to apoptotic in a death receptor 5 (DR5)-dependent manner in vitro. Importantly, normal fibroblasts do not undergo apoptosis following rhTRAIL plus ONC201. In vivo, MDA-MB-361 tumor growth rate is significantly reduced following treatment with a combination of ONC201 and rhTRAIL as compared to control tumors. Natural killer (NK) cells which use TRAIL to kill DR5-expressing cancer cells, exhibit greater cytotoxicity against ONC201-treated breast cancer cells compared to controls. rhTRAIL also converts the response of cells from other tumor types to ONC201 from anti-proliferative to apoptotic. A monoclonal DR5-agonistic antibody converts the response of non-TNBC cells to ONC201 from anti-proliferative to apoptotic. Our findings describe a novel therapeutic strategy that potently converts the response of a cancer cell to ONC201 from anti-proliferative to apoptotic. This approach may be clinically relevant and has potential to induce tumor regression of patient tumors with relative resistance to ONC201 monotherapy.

E2F1-3a overexpression due to amplification or to mutation or loss of the retinoblastoma gene, induces genes involved in DNA synthesis and leads to abnormal cellular proliferation, tumor growth, and invasion. Therefore, inhibiting the overexpression of one or more of these activating E2Fs is a recognized target in cancer therapeutics. In previous studies we identified by phage display, a novel 7-mer peptide (PEP) that bound tightly to an immobilized consensus E2F1 promoter sequence, and when conjugated to penetratin to increase its uptake into cells, was cytotoxic to several malignant cell lines and human prostate and small cell lung cancer xenografts. Based on molecular simulation studies that showed that the D-Arg penetratin peptide (D-Arg PEP) secondary structure is more stable than the L-Arg PEP, the L-Arg in the peptide was substituted with D-Arg. In vitro studies confirmed that it was more stable than the L- form and was more cytotoxic as compared to the L-Arg PEP when tested against the human castrate resistant cell line, DU145 and the human lung cancer H196 cell line. When encapsulated in PEGylated liposomes, the D-Arg-PEP potently inhibited growth of the DU145 xenograft in mice. Our findings validate D- Arg PEP, an inhibitor of E2F1and 3a transcription, as an improved second generation drug candidate for targeted molecular therapy of cancers with elevated levels of activated E2F(s).

CDK4 is emerging as a target in KRAS-mutant non-small cell lung cancer (NSCLC). We demonstrate that KRAS-mutant NSCLC cell lines are initially sensitive to the CDK4/6 inhibitor palbociclib, but readily acquire resistance associated with increased expression of CDK6, D-type cyclins and cyclin E. Resistant cells also demonstrated increased ERK1/2 activity and sensitivity to MEK and ERK inhibitors. Moreover, MEK inhibition reduced the expression and activity of cell cycle proteins mediating palbociclib resistance. In resistant cells, ERK activated mTOR, driven in part by upstream FGFR1 signaling resulting from the extracellular secretion of FGF ligands. A genetically-engineered mouse model of KRAS-mutant NSCLC initially sensitive to palbociclib similarly developed acquired resistance with increased expression of cell cycle mediators, ERK1/2 and FGFR1. In this model, resistance was delayed with combined palbociclib and MEK inhibitor treatment. These findings implicate an FGFR1-MAP kinase-mTOR pathway resulting in increased expression of D-cyclins and CDK6 that confers palbociclib resistance and indicate that CDK4/6 inhibition acts to promote MAP kinase dependence.

The outgrowth of primary melanoma, the deadliest skin cancer, and generation of metastasis is supported by the tumor microenvironment (TME) which includes non-cancerous cells. Since the TME plays an important role in melanoma pathogenesis, its targeting is a promising therapeutic approach. Thus, it is important to identify proteins in the melanoma TME that may serve as therapeutic targets. Here we show that the nicotinamide adenine dinucleotide glycohydrolase CD38 is a suitable target for this purpose. Loss of CD38 in the TME as well as inhibition of its enzymatic activity restrained outgrowth of primary melanoma generated by two transplantable models of melanoma, B16F10 and Ret-mCherry-sorted (RMS) melanoma cells. Pathological analysis indicated that loss of CD38 increased cell death and reduced the amount of cancer-associated fibroblasts (CAFs) and blood vessels. Importantly, in addition to inhibiting outgrowth of primary melanoma tumors, loss of CD38 also inhibited spontaneous occurrence of RMS pulmonary and brain metastasis. The underlying mechanism may involve, at least in the brain, inhibition of metastasis expansion, since loss of CD38 inhibited the outgrowth of B16F10 and RMS brain tumors that were generated by direct intracranial implantation. Collectively, our results suggest that targeting CD38 in the melanoma TME provides a new therapeutic approach for melanoma treatment.

Glioblastoma (GBM) is the most common primary brain tumor and is invariably fatal. Heat shock proteins (HSPs) provide protein signatures/biomarkers for GBM that afford potential as targets for developing anti-GBM drugs. In GBM, elevated expression of hypoxia inducible factors under the influence of Ets family proteins significantly promotes the expression of HSPs. RNAseq analysis identified HSPB1 as a prominent upregulated HSP in GBM and in radiation resistant/temozolomide resistant (radio/TMZR) GBM. Here, we established friend leukemia integration 1 (Fli-1), a member of Ets family to be playing a transcriptional regulatory role on the HSPB1 gene. Fli-1 binds to nucleotide residues GGAA at binding sites 3, 6 and 7 in the 5-kb upstream region of HSPB1. Fli-1 has been linked to oncogenic transformation with upregulation in radio/TMZR GBM. Overexpression of Fli-1 in GBM promotes resistance, whereas Fli-1 knockdown in radio/TMZR GBM cells suppresses resistance. We identify the underlying molecular mechanisms of Fli-1-mediated regulation of HSPB1 that drive extracellular matrix remodeling and epithelial to mesenchymal transition in radio/TMZR GBM cells. This study uncovers Fli-1 as a potential therapeutic target for combating radiation and temozolomide resistance in GBM.

// 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.

The overexpression and hyperactivity of p21-activated serine/threonine kinases (PAKs) is known to facilitate tumorigenesis; however, the contribution of cancer-associated PAK mutations to tumor initiation and progression remains unclear. Here, we identify p21-activated serine/threonine kinase 5 (PAK5) as the most frequently altered PAK family member in human melanoma. More than 60% of melanoma-associated PAK5 gene alterations are missense mutations, and distribution of these variants throughout the protein coding sequence make it difficult to distinguish oncogenic drivers from passengers. To address this issue, we stably introduced the five most common melanoma-associated PAK5 missense mutations into human immortalized primary melanocytes (hMELTs). While expression of these mutants did not promote single-cell migration or induce temozolomide resistance, a subset of variants drove aberrant melanocyte proliferation. These mitogenic mutants, PAK5 S364L and D421N, clustered within an unstructured, serine-rich domain of the protein and inappropriately activated ERK and PKA through kinase-independent and -dependent mechanisms, respectively. Together, our findings establish the ability of mutant PAK5 to enhance PKA and MAPK signaling in melanocytes and localize the engagement of mitogenic pathways to a serine-rich region of PAK5.

// Amanda B. Pilling 1,2 , Jihye Kim 1 , Adriana Estrada-Bernal 1 , Qiong Zhou 1 , Anh T. Le 1 , Katherine R. Singleton 1 , Lynn E. Heasley 1 , Aik Choon Tan 1 , James DeGregori 1 and Robert C. Doebele 1 1 University of Colorado Cancer Center, Aurora, CO, USA 2 Henry Ford Cancer Institute, Detroit, MI, USA Correspondence to: Robert C. Doebele, email: // Keywords : ALK; ROS1; NSCLC; MYC; synthetic lethality Received : July 28, 2017 Accepted : January 09, 2018 Published : January 16, 2018 Abstract A subset of lung cancers is dependent on the anaplastic lymphoma kinase ( ALK ) oncogene for survival, a mechanism that is exploited by the use of the ALK inhibitor crizotinib. Despite exceptional initial tumor responses to ALK inhibition by crizotinib, durable clinical response is limited and the emergence of drug resistance occurs. Furthermore, intrinsic resistance is frequently observed, where patients fail to respond initially to ALK-inhibitor therapy. These events demonstrate the underlying complexity of a molecularly-defined oncogene-driven cancer and highlights the need to identify compensating survival pathways. Using a loss-of-function whole genome short-hairpin (shRNA) screen, we identified MYCBP as a determinant of response to crizotinib, implicating the MYC signaling axis in resistance to crizotinib-treated ALK+ NSCLC. Further analysis reveals that ALK regulates transcriptional expression of MYC and activates c-MYC transactivation of c-MYC target genes. Inhibition of MYC by RNAi or small molecules sensitizes ALK+ cells to crizotinib. Taken together, our findings demonstrate a dual oncogene mechanism, where ALK positively regulates the MYC signaling axis, providing an additional oncogene target whose inhibition may prevent or overcome resistance.

// Ning Kon 1,* , Donglai Wang 1,* , Tongyuan Li 1 , Le Jiang 1 , Li Qiang 2,3,** and Wei Gu 1,3 1 Institute for Cancer Genetics, College of Physicians and Surgeons of Columbia University, New York, New York, USA 2 Naomi Berrie Diabetes Center, Department of Medicine, College of Physicians and Surgeons of Columbia University, New York, New York, USA 3 Department of Pathology and Cell Biology, College of Physicians and Surgeons of Columbia University, New York, New York, USA * These two authors contributed equally to this work ** This author is listed as a co-corresponding author Correspondence to: Wei Gu, email: // Keywords : p53; Mdmx; Mdm4; metabolism; adipose tissues Received : September 26, 2017 Accepted : December 08, 2017 Published : January 02, 2018 Abstract Although cell-cycle arrest, senescence and apoptosis remain as major canonical activities of p53 in tumor suppression, the emerging role of p53 in metabolism has been a topic of great interest. Nevertheless, it is not completely understood how p53-mediated metabolic activities are regulated in vivo and whether this part of the activities has an independent role beyond tumor suppression. Mdmx (also called Mdm4), like Mdm2, acts as a major suppressor of p53 but the embryonic lethality of mdmx-null mice creates difficulties to evaluate its physiological significance in metabolism. Here, we report that the embryonic lethality caused by the deficiency of mdmx , in contrast to the case for mdm2 , is fully rescued in the background of p53 3KR/3KR , an acetylation-defective mutant unable to induce cell-cycle arrest, senescence and apoptosis. p53 3KR/3KR /mdmx -/- mice are healthy but skinny without obvious developmental defects. p53 3KR/3KR /mdmx -/- mice are resistant to fat accumulation in adipose tissues upon high fat diet. Notably, the levels of p53 protein are only slightly increased and can be further induced upon DNA damage in p53 3KR/3KR /mdmx -/- mice, suggesting that Mdmx is only partially required for p53 degradation in vivo . Further analyses indicate that the anti-obesity phenotypes in p53 3KR/3KR /mdmx -/- mice are caused by activation of lipid oxidation and thermogenic programs in adipose tissues. These results demonstrate the specific effects of the p53/Mdmx axis in lipid metabolism and adipose tissue remodeling and reveal a surprising role of Mdmx inhibition in anti-obesity effects beyond, commonly expected, tumor suppression. Thus, our study has significant implications regarding Mdmx inhibitors in the treatment of obesity related diseases.

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.

The NM23/NME gene was identified as a metastasis suppressor. It's re-expression inhibited cancer cell motility and suppressed metastasis, without effecting primary tumor size in multiple model systems. The mechanisms of NME suppression of motility and metastasis are incompletely known. Of particular interest, has been NME histidine 118 phosphorylation, involved in nucleoside diphosphate kinase (NDPK) and histidine protein kinase (HPK) activities. Using recently developed monoclonal antibodies to phosphohistidine, we have addressed the correlation of NME phosphohistidine with motility suppression, and distinguished the NDPK and HPK contributions. While general levels of NME correlated with its 1-phosphohistidine form in two cell line model systems, two exceptions were noted: Tumor cells actively migrating in scratch assays, even if expressing high levels of NME1, were low in its 1-phosphohistidine form. Site-directed mutagenesis of NME1 histidine 118 and proline 96 was examined by transfection experiments and partial purification of recombinant proteins. NME1P96S overexpressing tumor cells exhibited high motility and migration phenotypes despite high 1-phosphohistidine content and NDPK activity; HPK activity using succinate thiokinase as a substrate was poor. The data suggest the importance of NME 1-phosphohistidine levels in potential mechanistic pathways of metastasis suppression and point toward the HPK activity of NME1 downstream of autophosphorylation.

Microtubule targeting agents (MTAs) are some of the most effective anticancer drugs used to treat a wide variety of adult and pediatric cancers. Building evidence suggests that these drugs inhibit interphase signaling events and that this contributes to their anticancer actions. The effects of diverse MTAs were evaluated following a 2 hour incubation with clinically relevant concentrations to test the hypothesis that these drugs rapidly and differentially disrupt epithelial-to-mesenchymal transition (EMT)-related signaling. The MTAs rapidly promoted the cortical localization of internal pools of E-cadherin in HCC1937 breast cancer cells, with the most robust effects observed with the microtubule destabilizers eribulin and vinorelbine. Cortical E-cadherin localization was also promoted by the Src kinase inhibitor dasatinib or by siRNA-mediated depletion of the p130Cas scaffold. Mechanistic studies demonstrate that eribulin disrupts the interaction between p130Cas and Src, leading to decreased cortical Src phosphorylation that precedes the accumulation of cortical E-cadherin. These results suggest that microtubules can be actively co-opted by cancer cells to inhibit cortical E-cadherin localization, a hallmark of EMT, and provide a direct link between the initial disruption of the microtubule network and reversal of EMT phenotypes demonstrated by eribulin in long-term studies.

Besides somatic mutations or drug efflux, epigenetic reprogramming can lead to acquired drug resistance. We recently have identified early stress-induced multi-drug tolerant cancer cells termed induced drug-tolerant cells (IDTCs). Here, IDTCs were generated using different types of cancer cell lines; melanoma, lung, breast and colon cancer. A common loss of the H3K4me3 and H3K27me3 and gain of H3K9me3 mark was observed as a significant response to drug exposure or nutrient starvation in IDTCs. These epigenetic changes were reversible upon drug holidays. Microarray, qRT-PCR and protein expression data confirmed the up-regulation of histone methyltransferases (SETDB1 and SETDB2) which contribute to the accumulation of H3K9me3 concomitantly in the different cancer types. Genome-wide studies suggest that transcriptional repression of genes is due to concordant loss of H3K4me3 and regional increment of H3K9me3. Conversely, genome-wide CpG site-specific DNA methylation showed no common changes at the IDTC state. This suggests that distinct histone methylation patterns rather than DNA methylation are driving the transition from parental to IDTCs. In addition, silencing of SETDB1/2 reversed multi drug tolerance. Alterations of histone marks in early multi-drug tolerance with an increment in H3K9me3 and loss of H3K4me3/H3K27me3 is neither exclusive for any particular stress response nor cancer type specific but rather a generic response.

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.

PAX8 is a lineage-restricted transcription factor that is expressed in epithelial ovarian cancer (EOC) precursor tissues, and in the major EOC histotypes. Frequent overexpression of PAX8 in primary EOCs suggests this factor functions as an oncogene during tumorigenesis, however, the biological role of PAX8 in EOC development is poorly understood. We found that stable knockdown of PAX8 in EOC models significantly reduced cell proliferation and anchorage dependent growth in vitro, and attenuated tumorigenicity in vivo. Chromatin immunoprecipitation followed by next generation sequencing (ChIP-seq) and transcriptional profiling were used to create genome-wide maps of PAX8 binding and putative target genes. PAX8 binding sites were significantly enriched in promoter regions (p < 0.05) and superenhancers (p < 0.05). MEME-ChIP analysis revealed that PAX8 binding sites overlapping superenhancers or enhancers, but not promoters, were enriched for JUND/B and ARNT/AHR motifs. Integrating PAX8 ChIP-seq and gene expression data identified PAX8 target genes through their associations within shared topological association domains. Across two EOC models we identified 62 direct regulatory targets based on PAX8 binding in promoters and 1,330 putative enhancer regulatory targets. SEPW1, which is involved in oxidation-reduction, was identified as a PAX8 target gene in both cell line models. While the PAX8 cistrome exhibits a high degree of cell-type specificity, analyses of PAX8 target genes and putative cofactors identified common molecular targets and partners as candidate therapeutic targets for EOC.

Significant limitations exist in our ability to predict breast cancer risk at the individual level. Circulating microRNAs (C-miRNAs) have emerged as measurable biomarkers (liquid biopsies) for cancer detection. We evaluated the ability of C-miRNAs to identify women most likely to develop breast cancer by profiling miRNA from serum obtained long before diagnosis. 24 breast cancer cases and controls (matched for risk and age) were identified from women enrolled in the High-Risk Breast Program at the UVM Cancer Center. Isolated RNA from serum was profiled for over 2500 human miRNAs. The miRNA expression data were input into a stepwise linear regression model to discover a multivariable miRNA signature that predicts long-term risk of breast cancer. 25 candidate miRNAs were identified that individually classified cases and controls based on statistical methodologies. A refined 6-miRNA risk-signature was discovered following regression modeling that distinguishes cases and controls (AUC0.896, CI 0.804-0.988) in this cohort. A functional relationship between miRNAs that cluster together when cases are contrasted against controls was suggested and confirmed by pathway analyses. The discovered 6 miRNA risk-signature can discriminate high-risk women who ultimately develop breast cancer from those who remain cancer-free, improving current risk assessment models. Future studies will focus on functional analysis of the miRNAs in this signature and testing in larger cohorts. We propose that the combined signature is highly significant for predicting cancer risk, and worthy of further screening in larger, independent clinical cohorts.

// Sarah L. Dombernowsky 1,* , Jeanette Schwarz 1,* , Jacob Samsoe-Petersen 1 , Reidar Albrechtsen 1 , Kim B. Jensen 1,2 , Gary Thomas 3 and Marie Kveiborg 1 1 Biotech Research and Innovation Centre, University of Copenhagen, Copenhagen, Denmark 2 Novo Nordisk Foundation Center for Stem Cell Biology, University of Copenhagen, Copenhagen, Denmark 3 Department of Microbiology and Molecular Genetics, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA * These authors have contributed equally to this work Correspondence to: Marie Kveiborg, email: // Keywords : DSS-induced colitis; colon cancer; ApcMin model; PACS-2; ADAM17 Received : May 07, 2017 Accepted : October 28, 2017 Published : November 26, 2017 Abstract PACS-2 is a multifunctional sorting protein that mediates cell homeostasis. We recently identified PACS-2 in a functional genome-wide siRNA screen for novel regulators of the metalloproteinase ADAM17, the main sheddase for ligands of the ErbB receptor family. Of note, we showed that Pacs2 -/- mice have significantly reduced EGFR activity and proliferative index in the intestinal epithelium. As EGFR signaling is highly mitogenic for intestinal epithelial stem cells, and plays essential roles in intestinal epithelial regeneration and tumor development, we have now examined the role of PACS-2 in these processes. Specifically, we analyzed the role of Pacs 2-deficiency in a DSS-induced colitis model as well as in the genetic Apc Min colon cancer model. We now report that loss of PACS-2 delays tissue regeneration after colonic injury with little effect on key inflammatory parameters. We did however not observe any apparent effects on tumor formation driven by excessive proliferative signaling downstream from APC-deficiency. Our findings reveal that the role of PACS-2 in regulating ADAM17-mediated shedding is not an obligate requirement for the epithelium to respond to the strong inflammatory or tumorigenic inducers in the models assessed here.

// 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.

// Antonio Cigliano 1,* , Maria G. Pilo 2,* , Lei Li 3,* , Gavinella Latte 2 , Marta Szydlowska 1 , Maria M. Simile 2 , Panagiotis Paliogiannis 2 , Li Che 4 , Giovanni M. Pes 2 , Giuseppe Palmieri 5 , Maria C. Sini 5 , Antonio Cossu 6 , Alberto Porcu 2 , Gianpaolo Vidili 2 , Maria A. Seddaiu 2 , Rosa M. Pascale 2 , Silvia Ribback 1 , Frank Dombrowski 1 , Xin Chen 4 and Diego F. Calvisi 2 1 Institut fur Pathologie, Universitatsmedizin Greifswald, Greifswald, Germany 2 Department of Clinical and Experimental Medicine, University of Sassari, Sassari, Italy 3 School of Pharmacy, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei, China 4 Department of Bioengineering and Therapeutic Sciences and Liver Center, University of California, San Francisco, CA, USA 5 Institute of Biomolecular Chemistry, National Research Council, Sassari, Italy 6 Unit of Pathology, Azienda Ospedaliero Universitaria Sassari, Sassari, Italy * These Authors contributed equally to the work Correspondence to: Diego F. Calvisi, email: // Xin Chen, email: // Keywords : hepatocellular carcinoma, liver cancer, HSF1, c-Myc, signalling pathways Received : August 31, 2017 Accepted : September 21, 2017 Published : October 03, 2017 Abstract Deregulated activity of the c-Myc protooncogene is a frequent molecular event underlying mouse and human hepatocarcinogenesis. Nonetheless, the mechanisms sustaining c-Myc oncogenic activity in liver cancer remain scarcely delineated. Recently, we showed that the mammalian target of rapamycin complex 1 (mTORC1) cascade is induced and necessary for c-Myc dependent liver tumor development and progression. Since the heat shock factor 1 (HSF1) transcription factor is a major positive regulator of mTORC1 in the cell, we investigated the functional interaction between HSF1 and c-Myc using in vitro and in vivo approaches. We found that ablation of HSF1 restrains the growth of c-Myc-derived mouse hepatocellular carcinoma (HCC) cell lines, where it induces downregulation of c-Myc levels. Conversely, silencing of c-Myc gene in human and mouse HCC cells led to downregulation of HSF1 expression. Most importantly, overexpression of a dominant negative form of HSF1 (HSF1dn) in the mouse liver via hydrodynamic gene delivery resulted in the complete inhibition of mouse hepatocarcinogenesis driven by overexpression of c-Myc. Altogether, the present results indicate that a functional HSF1 is necessary for c-Myc-driven hepatocarcinogenesis. Consequently, targeting HSF1 might represent a novel and effective therapeutic strategy for the treatment of HCC subsets with activated c-Myc signaling.

// 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.

// Angelica M. Lagunas 1 , Jianchun Wu 1 and David L. Crowe 1 1 University of Illinois Cancer Center, Chicago, IL, USA Correspondence to: David L. Crowe, email: // Keywords : genomic instability, telomerase, kinase signaling, differentiation Received : October 07, 2016 Accepted : August 25, 2017 Published : September 16, 2017 Abstract Chromosome ends are protected by telomeres that prevent DNA damage response and degradation. When telomeres become critically short, the DNA damage response is activated at chromosome ends which induces cellular senescence or apoptosis. Telomeres are protected by the double stranded DNA binding protein TRF2 and maintained by telomerase or a recombination based mechanism known as alternative lengthening of telomeres (ALT). Telomerase is expressed in the basal layer of the epidermis, and stem cells in epidermis have longer telomeres than proliferating populations. Stem cell expansion has been associated with epithelial-mesenchymal transition (EMT) in cancer. EMT is a critical process in cancer progression in which cells acquire spindle morphology, migrate from the primary tumor, and spread to distant anatomic sites. Our previous study demonstrated that loss of TRF2 expression observed in human squamous cell carcinomas expanded metastatic cancer stem cells during mouse skin carcinogenesis. To determine if telomerase inhibition could block the TRF2-null mediated expansion of metastatic clones, we characterized skin carcinogenesis in a conditional TRF2/Terc double null mutant mouse. Loss of TRF2 and Terc expression resulted in telomere DNA damage, severely depleted CD34 + and Lgr6+ cancer stem cells, and induced terminal differentiation of metastatic cancer cells. However a novel cancer stem cell population evolved in primary tumors exhibiting genomic instability, ALT, and EMT. Surprisingly we discovered that metastatic clones evolved prior to histopathologic onset of primary tumors. These results have important implications for understanding the evolution and treatment of metastatic cancer.

// 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.