Phosphatases and microRNAs: Investigations in the Context of DNA Damage

Integrity of the cellular genome is constantly threatened by various sources of DNA damage that cause a variety of lesions including double strand breaks. Cells respond to such insults by upregulating the DNA damage response, a carefully orchestrated set of molecular events that leads to transcriptional changes, cell cycle arrest and/or apoptosis. In this dissertation we provide evidence supporting a role for phosphatases and microRNAs (miRNAs) in the cellular response to DNA damage. 53BP1 (tumor suppressor p53 binding protein 1) is a critical mediator of DNA repair signaling and its activity is regulated by several post-translational modifications. Here we show that 53BP1 is phosphorylated during mitosis on two residues, T1609 and S1618, located in the ubiquitination-dependent recruitment motif. These residues are dephosphorylated in late mitosis/early G1 by the PP4C/R3β phosphatase complex, which is required for 53BP1 accumulation at DNA breaks. We discovered that R3β preferentially interacts with 53BP1 in stalled mitosis and determined that this interaction is dependent on the phosphorylation of R3β residue S840 by the cyclin-dependent kinase family member CDK5. We also found that 53BP1 is deliberately excluded from chromatin during mitosis to prevent genomic instability. Ectopic reactivation of 53BP1 in mitosis causes increased micronuclei and lagging chromosome formation, which can be partially reversed by inhibiting the non-homologous end-joining pathway. In a more clinical scenario, accidental radiation exposure can cause DNA damage that manifests itself in complex malignancies including bone marrow failure and cancer. In order to accurately predict radiation exposure for better management of radiation accidents, we identified serum microRNA (miRNA) signatures capable of indicating the long-term impact of total body irradiation in animals. Using different doses of radiation we systematically studied the impact of TBI on the hematopoietic system and then identified three miRNA signatures that effectively distinguished between animals exposed to control, sublethal, and lethal radiation. Furthermore, we used radioprotective and radiomitigating agents to show that serum miRNAs can predict not only the dose of radiation but also its impact on animal health. Finally, to investigate the relevance of these miRNAs in humans, we validated our findings in a humanized mouse model.Medical Science

Circulating miR-29a and miR-150 correlate with delivered dose during thoracic radiation therapy for non-small cell lung cancer

Background: Risk of normal tissue toxicity limits the amount of thoracic radiation therapy (RT) that can be routinely prescribed to treat non-small cell lung cancer (NSCLC). An early biomarker of response to thoracic RT may provide a way to predict eventual toxicities—such as radiation pneumonitis—during treatment, thereby enabling dose adjustment before the symptomatic onset of late effects. MicroRNAs (miRNAs) were studied as potential serological biomarkers for thoracic RT. As a first step, we sought to identify miRNAs that correlate with delivered dose and standard dosimetric factors. Methods: We performed miRNA profiling of plasma samples obtained from five patients with Stage IIIA NSCLC at five dose-points each during radical thoracic RT. Candidate miRNAs were then assessed in samples from a separate cohort of 21 NSCLC patients receiving radical thoracic RT. To identify a cellular source of circulating miRNAs, we quantified in vitro miRNA expression intracellularly and within secreted exosomes in five NSCLC and stromal cell lines. Results: miRNA profiling of the discovery cohort identified ten circulating miRNAs that correlated with delivered RT dose as well as other dosimetric parameters such as lung V20. In the validation cohort, miR-29a-3p and miR-150-5p were reproducibly shown to decrease with increasing radiation dose. Expression of miR-29a-3p and miR-150-5p in secreted exosomes decreased with radiation. This was concomitant with an increase in intracellular levels, suggesting that exosomal export of these miRNAs may be downregulated in both NSCLC and stromal cells in response to radiation. Conclusions: miR-29a-3p and miR-150-5p were identified as circulating biomarkers that correlated with delivered RT dose. miR-150 has been reported to decrease in the circulation of mammals exposed to radiation while miR-29a has been associated with fibrosis in the human heart, lungs, and kidneys. One may therefore hypothesize that outlier levels of circulating miR-29a-3p and miR-150-5p may eventually help predict unexpected responses to radiation therapy, such as toxicity. Electronic supplementary material The online version of this article (doi:10.1186/s13014-016-0636-4) contains supplementary material, which is available to authorized users

Inhibiting stromal cell heparan sulfate synthesis improves stem cell mobilization and enables engraftment without cytotoxic conditioning

The glycosyltransferase gene, Ext1, is essential for heparan sulfate production. Induced deletion of Ext1 selectively in Mx1-expressing bone marrow (BM) stromal cells, a known population of skeletal stem/progenitor cells, in adult mice resulted in marked changes in hematopoietic stemand progenitor cell (HSPC) localization.HSPCegressed fromBMto spleen after Ext1 deletion. This was associated with altered signaling in the stromal cells and with reduced vascular cell adhesion molecule 1 production by them. Further, pharmacologic inhibition of heparan sulfate mobilized qualitatively more potent and quantitatively more HSPC from the BM than granulocyte colony-stimulating factor alone, including in a setting of granulocyte colony-stimulating factor resistance. The reduced presence of endogenous HSPC after Ext1 deletion was associated with engraftment of transfused HSPC without any toxic conditioning of the host. Therefore, inhibiting heparan sulfate production may provide a means for avoiding the toxicities of radiation or chemotherapy in HSPC transplantation for nonmalignant conditions. (Blood. 2014;124(19):2937-2947).Stem Cell and Regenerative Biolog

AKT/FOXO signaling enforces reversible differentiation blockade in myeloid leukemias

AKT activation is associated with many malignancies, where AKT acts, in part, by inhibiting FOXO tumor suppressors. We show a converse role for AKT/FOXOs in acute myeloid leukemia (AML). Rather than decreased FOXO activity, we observed that FOXOs are active in similar to 40% of AML patient samples regardless of genetic subtype. We also observe this activity in human MLL-AF9 leukemia allele-induced AML in mice, where either activation of Akt or compound deletion of FoxO1/3/4 reduced leukemic cell growth, with the latter markedly diminishing leukemia-initiating cell (LIC) function in vivo and improving animal survival. FOXO inhibition resulted in myeloid maturation and subsequent AML cell death. FOXO activation inversely correlated with JNK/c-JUN signaling, and leukemic cells resistant to FOXO inhibition responded to JNK inhibition. These data reveal a molecular role for AKT/FOXO and JNK/c-JUN in maintaining a differentiation blockade that can be targeted to inhibit leukemias with a range of genetic lesions