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ETP-ALL (Early T cell Progenitor Acute Lymphoblastic Leukemia) represents a high-risk subtype of T cell acute lymphocytic leukemia (T-ALL). Therapeutically, ETP-ALL patients frequently relapse after conventional chemotherapy highlighting the need for alternative therapeutic approaches. Using our ZEB2Tg ETP-ALL mouse model we previously documented the potential utility of the catalytic LSD1 inhibitor (GSK2879552) for treating mouse/human ETP-ALL. However, this approach proved to be inefficient, especially in killing human LOUCY cell ETP-ALL xenografts in vivo. Here we have revealed the novel involvement of ZEB2/LSD1 complexes in repressing the intrinsic apoptosis pathway by inhibiting the expression of several pro-apoptotic proteins such as BIM (BCL2L11) as a major driver for ETP-ALL survival. Treatment with LSD1i (particularly with the steric inhibitor SP2509) restored the expression of ZEB2/LSD1 pro-apoptotic BIM (BCL2L11) target. In combination with a JAK/STAT pathway inhibitor (JAKi, Ruxolitinib) or with a direct inhibitor of the anti-apoptotic BCL2 protein (BCL2i, ABT-199) resistance of human and mouse ETP-ALL to LSD1i was reversed. This new combination approach efficiently inhibited the growth of human and mouse ETP-ALL cells in vivo by enhancing their differentiation and triggering an apoptotic response. These results set the stage for novel combination therapies to be used in clinical trials to treat ETP-ALL patients.

Chemical structure of beta-catenin antagonist, BC2059.

Despite a clear link between ataxia-telangiectasia mutated (ATM)–dependent phosphorylation of p53 and cell cycle checkpoint control, the intracellular biology and subcellular localization of p53 phosphoforms during the initial sensing of DNA damage is poorly understood. Using G0-G1 confluent primary human diploid fibroblast cultures, we show that endogenous p53, phosphorylated at Ser15 (p53Ser15), accumulates as discrete, dose-dependent and chromatin-bound foci within 30 minutes following induction of DNA breaks or DNA base damage. This biologically distinct subpool of p53Ser15 is ATM dependent and resistant to 26S-proteasomal degradation. p53Ser15 colocalizes and coimmunoprecipitates with γ-H2AX with kinetics similar to that of biochemical DNA double-strand break (DNA-dsb) rejoining. Subnuclear microbeam irradiation studies confirm p53Ser15 is recruited to sites of DNA damage containing γ-H2AX, ATMSer1981, and DNA-PKcsThr2609 in vivo. Furthermore, studies using isogenic human and murine cells, which express Ser15 or Ser18 phosphomutant proteins, respectively, show defective nuclear foci formation, decreased induction of p21WAF, decreased γ-H2AX association, and altered DNA-dsb kinetics following DNA damage. Our results suggest a unique biology for this p53 phosphoform in the initial steps of DNA damage signaling and implicates ATM-p53 chromatin-based interactions as mediators of cell cycle checkpoint control and DNA repair to prevent carcinogenesis.

Supplementary Figures 1-3 from Evidence for the Direct Binding of Phosphorylated p53 to Sites of DNA Breaks In vivo

Supplementary Figure Legends from Evidence for the Direct Binding of Phosphorylated p53 to Sites of DNA Breaks In vivo

Cancer cell growth is dependent upon the sustainability of proliferative signaling and resisting cell death. Macroautophagy/autophagy promotes cancer cell growth by providing nutrients to cells and preventing cell death. This is in contrast to autophagy promoting cell death under some conditions. The mechanism regulating autophagy-mediated cancer cell growth remains unclear. Herein, we demonstrate that TSSC4 (tumor suppressing subtransferable candidate 4) is a novel tumor suppressor that suppresses cancer cell growth and tumor growth and prevents cell death induction during excessive growth by inhibiting autophagy. The oncogenic proteins ERBB2 (erb-b2 receptor tyrosine kinase 2) and the activation EGFR mutant (EGFRvIII, epidermal growth factor receptor variant III) promote cell growth and TSSC4 expression in breast cancer and glioblastoma multiforme (GBM) cells, respectively. In EGFRvIII-expressing GBM cells, TSSC4 knockout shifted the function of autophagy from a pro-cell survival role to a pro-cell death role during prolonged cell growth. Furthermore, the interaction of TSSC4 with MAP1LC3/LC3 (microtubule associated protein 1 light chain 3) via its conserved LC3-interacting region (LIR) contributes to its inhibition of autophagy. Finally, TSSC4 suppresses tumorsphere formation and tumor growth by inhibiting autophagy and maintaining cell survival in tumorspheres. Taken together, sustainable cancer cell growth can be achieved by autophagy inhibition via TSSC4 expression. Abbreviations: 3-MA: 3-methyladenine; ACTB: actin beta; CQ: chloroquine; EGFRvIII: epidermal growth factor receptor variant III; ERBB2: erb-b2 receptor tyrosine kinase 2; GBM: glioblastoma multiforme; LIR: LC3-interacting region; MAP1LC3/LC3: microtubule Associated protein 1 light chain 3; TSSC4: tumor suppressing subtransferable candidate 4

Nationally, racial and ethnic disparities in childhood asthma plateaued from 2005 to 2013. We assessed trends in childhood asthma in Miami, Florida using Youth Risk Behavior Surveillance System (YRBSS) data and emergency department (ED) utilization and hospitalization rates by zip code population characteristics. Asthma prevalence in Miami did not vary significantly by race/ethnicity in YRBSS respondents in 2005 (16.2–17.2%, all groups), but rose in African–Americans and Hispanics and declined in Whites by 2013 to 27.9, 20.9 and 12.6%, respectively (P = 0.02). Median asthma ED visit rates rose from 106.8 (2006–2008) to 138.2 (2011–2013; P = 0.004) per 10,000 children. High-poverty and majority African–American zip codes were 6.3 and 7.3 times more likely to have asthma ED visit rates > 200 than others (P

Engineered nanoparticles with multiple complementary imaging modalities are of great benefit to the rapid treatment and diagnosis of disease in various organs. Herein, we report the formulation of cubosomes and hexosomes that carry multiple amphiphilic imaging contrast agents in their self-assembled lipid bilayers. This is the first report of the use of both near infrared fluorescent (NIRF) imaging and gadolinium lipid based magnetic resonance (MR) imaging modalities in cubosomes and hexosomes. High-throughput screening was used to rapidly optimize formulations with desirable nano-architectures and low in vitro cytotoxicity. The dual-modal imaging nanoparticles in vivo biodistribution and organ specific contrast enhancement were then studied. The NIRF in vivo imaging results indicated accumulation of both cubosomes and hexosomes in the liver and spleen of mice up to 20 h post-injection. Remarkably, the biodistribution of the nanoparticle formulations was affected by the mesophase (i.e. cubic or hexagonal), a finding of significant importance for the future use of these compounds, with hexosomes showing higher accumulation in the spleen than the liver compared to cubosomes. Furthermore, in vivo MRI data of animals injected with either type of lyotropic liquid crystal nanoparticle displayed enhanced contrast in the liver and spleen.

New molecular cancer treatment strategies aim to reconstitute wild-type p53 (WTp53) function in mutant p53 (MTp53)–expressing tumors as a means of resensitizing cells to chemotherapy or radiotherapy. The success of this approach may depend on whether MTp53 proteins are acting in a dominant-negative or independent gain-of-function mode. Herein, we describe an isogenic, temperature-sensitive p53 model (p53A138V) in p53-null human H1299 lung cancer cells in which WTp53 can be selectively coexpressed with a temperature-sensitive MTp53 allele (A138V) during initial DNA damage and subsequent DNA repair. Cells expressing MTp53 alone or coexpressing induced WTp53 and MTp53 were tested for p53 transcription, G1 and G2 cell cycle checkpoints, apoptosis, and long-term clonogenic survival following DNA damage. Transient transfection of WTp53 into H1299 cells, or shift-down of H1299-p53A138V stable transfectants to 32°C to induce WTp53, led to increased p21WAF1 expression and G1 and G2 arrests following DNA damage but did not increase BAX expression or apoptosis. In contrast, both transient and stable expression of the p53A138V mutant in p53-null H1299 cells (e.g. testing gain-of-function) at 37°C blocked p21WAF1 induction following DNA damage. Cell death was secondary to mitotic catastrophe and/or tumor cell senescence. Overexpression of WTp53 did not resensitize resistant MTp53-expressing cells to ionizing radiation, cisplatinum, or mitomycin C. Our results suggest that human MTp53 proteins can lead to resistant phenotypes independent of WTp53-mediated transcription and checkpoint control. This should be considered when using p53 as a prognostic factor and therapeutic target. [Mol Cancer Ther 2008;7(4):980–92]

In response to DNA breaks, human cells delay their progression through the G1, S, and G2 phases of the cell cycle. This response requires the coordinated effort of the ATM-CHK2-p53 and ATR-CHK1 DNA damage-sensing pathways and DNA repair (eg, DNA-PK and RAD51 complexes). The turnover of many of these DNA damage-associated proteins is controlled by the 26S proteasome. In this article, we review molecular strategies that target each of these pathways using silencing RNA (siRNA), antisense, or small-molecule inhibition. Although these agents can radiosensitize tumor cells, little data are available regarding potential effects on normal tissues to determine the potential therapeutic ratio of these strategies after fractionated radiotherapy. Clinical trials using such agents will require novel correlative science endpoints to track DNA repair and cell-cycle arrest and will need careful assessment of normal tissue toxicity and stability.

Background and purpose Intratumoral hypoxia has been correlated with poor clinical outcome in prostate cancer. Prostate cancer cells can be genetically unstable and have altered DNA repair. We, therefore, hypothesized that the expression of DNA double-strand break (DNA-dsb) repair genes in normal and malignant prostate cultures can be altered under hypoxic conditions. Methods and materials The expression of homologous recombination (HR) and non-homologous recombination (NHEJ) genes following gas hypoxia (0.2%) or exposure to HIF1α-inducing agent, CoCl 2 (100μM), was determined for normal diploid fibroblasts (GM05757) and the pre-malignant and malignant prostate cell lines, BPH-1, 22RV-1, DU145 and PC3. RNA and protein levels were determined using RT-PCR and Western blotting. Additionally, p53 genotype and function, the level of hypoxia-induced apoptosis, and cell cycle distribution, were determined to correlate to changes in DNA-dsb gene expression. Results Induction of hypoxia was confirmed using HIF1α and VEGF expression in gas- and CoCl 2 -treated cultures. Hypoxia (48–72h of 0.2% O 2 ) decreased RNA expression of a number of HR-related genes (e.g. Rad51 , Rad52 , Rad54 , BRCA1 , BRCA2 ) in both normal and malignant cultures. Similar decreases in RNA pertaining to the NHEJ-related genes (e.g. Ku70 , DNA-PKcs , DNA Ligase IV , Xrcc4 ) were observed. In selected cases, hypoxia-mediated decreases in RNA expression led to decreased DNA-dsb protein expression. CoCl 2 -treated cultures did not show decreased DNA-dsb protein expression. The ability of hypoxia to down-regulate Rad51 and other HR-associated genes under hypoxia was not correlated to c-Abl or c-Myc gene expression, p53 genotype or function, propensity for hypoxia-mediated apoptosis, or specific changes in cell cycle distribution. Conclusions Hypoxia can down-regulate expression of DNA-dsb repair genes in both normal and cancer cells. If associated with a functional decrease in DNA-dsb repair, this observation could provide a potential basis for the observed genetic instability within tumor cells exposed to hypoxia.

The p53 tumor suppressor protein is a key mediator of an ATM-dependent DNA damage response cascade following cellular exposure to ionizing radiation. The p53-family members, p63 and p73, are highly similar to p53, yet are differentially activated by IR, UV and cis-platinum via ATM and c-abl/ATR signaling pathways. Loss of function of p53 can occur by mutation or degradation; giving rise to alterations in G(1) and G(2) cell cycle checkpoint control, cell death, DNA repair and genetic stability. The end result of these alterations can be the generation of radioresistant mutant tumor cells. Indeed, in isogenic systems, loss of p53 or p73 function has been associated with decreased chemosensitivity and radiosensitivity, in vitro. However, clinical data supporting a role for p53 genotype as an independent predictive factor for radiotherapy outcome continues to be controversial due to variable endpoints in clinical trial design and in methodology in detecting p53 function. Nonetheless, in carefully controlled radiotherapy studies where mutations in p53 have been detected using DNA sequencing or functional assays, the presence of mutant p53 can be associated with decreased local control following radiotherapy. This suggests that novel molecular treatment strategies specifically designed to re-institute normal p53 function within resistant tumors can be used as combined modality protocols to improve local control and maintain a therapeutic ratio. A future challenge lies in the pre-therapy determination of a 'molecular therapeutic ratio' for individual patients which could allow for specific prognostication based on p53 functional status and subsequent individualized therapy.

Asthma is the most common chronic condition in childhood and disproportionately impacts the poorer and ethnic minorities. The objectives of the study were to estimate the prevalence of asthma in Miami-Dade County (MDC) schoolchildren to aid case-finding and linkage to care.We used the CDC Youth Risk Behavior Surveillance System (YRBSS) definition of possible asthma ("Ever told by a doctor or nurse that they had asthma and still had asthma") and analyzed data from four sources. These included the: (1)-MDC YRBSS 2009, (2)-MDC Health Connect Asthma Survey of school nurses (2008), (3)-parents' survey in the five HealthConnect survey elementary schools with highest asthma prevalence, and (4)-focus group with parents of asthmatic children.(1)-MDC YRBSS data showed that 21.3% of high- and 21.4% of middle-school students had been diagnosed with possible asthma. Prevalence was the highest in African-American middle-school girls (26.9%). (2)-HealthConnect survey responders reported that 4.9% of the students in 131 MDC schools had possible asthma. Asthma prevalence was higher in elementary schools (median = 7.1%) and in low-income MDC zip codes. (3)-Of the parent survey responders, 24.9% indicated that their child had possible asthma, and 19.2% reported that their children had no usual source of care. (4)-Focus group participants reported frequent loss of Medicaid coverage for their children, landlords' indifference to the role of poorly maintained housing in asthma, and unmet needs regarding knowledge of health system navigation.Asthma may be common in MDC schoolchildren, particularly in poor communities. Formidable structural factors limit the caregivers' abilities to manage childhood asthma.

Despite a clear link between ataxia-telangiectasia mutated (ATM)–dependent phosphorylation of p53 and cell cycle checkpoint control, the intracellular biology and subcellular localization of p53 phosphoforms during the initial sensing of DNA damage is poorly understood. Using G0-G1 confluent primary human diploid fibroblast cultures, we show that endogenous p53, phosphorylated at Ser15 (p53Ser15), accumulates as discrete, dose-dependent and chromatin-bound foci within 30 minutes following induction of DNA breaks or DNA base damage. This biologically distinct subpool of p53Ser15 is ATM dependent and resistant to 26S-proteasomal degradation. p53Ser15 colocalizes and coimmunoprecipitates with γ-H2AX with kinetics similar to that of biochemical DNA double-strand break (DNA-dsb) rejoining. Subnuclear microbeam irradiation studies confirm p53Ser15 is recruited to sites of DNA damage containing γ-H2AX, ATMSer1981, and DNA-PKcsThr2609 in vivo. Furthermore, studies using isogenic human and murine cells, which express Ser15 or Ser18 phosphomutant proteins, respectively, show defective nuclear foci formation, decreased induction of p21WAF, decreased γ-H2AX association, and altered DNA-dsb kinetics following DNA damage. Our results suggest a unique biology for this p53 phosphoform in the initial steps of DNA damage signaling and implicates ATM-p53 chromatin-based interactions as mediators of cell cycle checkpoint control and DNA repair to prevent carcinogenesis.