<p>Lung cancer is the third most prevalent cancer in the world; however it is the leading cause of cancer related deaths worldwide. Non-small cell lung cancer (NSCLC) accounts for ~85% of the lung cancer cases. The current strategies to treat NSCLC patients with frequent causal genetic mutations is through targeted therapeutics. Approximately 10-35% of NSCLC patient tumors have activated mutations in the Epidermal Growth Factor Receptor (EGFR) resulting in uncontrolled cellular proliferation. The standard-of care for such patients is EGFR-Tyrosine Kinase Inhibitors (EGFR-TKIs), a class of targeted therapeutics that specifically inhibit EGFR activity. One such EGFR-TKI used in this study is erlotinib. Following erlotinib treatment, tumors rapidly regress at first; however, over 50% of patients develop erlotinib resistance within a year post treatment. Development of resistance remains to be the major challenge in treatment of NSCLC using EGFR-TKIs such as erlotinib. </p> <p>In approximately 60% of cases, acquired erlotinib resistance in patients is attributed to a secondary mutation in EGFR, whereas in about 20% of cases, activation of alternative signaling pathways is the reported mechanism. For the remaining 15-20% of <a>cases</a> the mechanism of resistance remains unknown. Therefore, it can be speculated that the common methods used to identify genetic mutations in tumors post erlotinib treatment, such as histologic analysis and genetic screening may fail to identify alterations in epigenetic mediators of erlotinib resistance, also including microRNAs (miRNAs). MiRNAs are short non-coding RNAs that post-transcriptionally negatively regulate their target transcripts. Hence, in this study two comprehensive screens were simultaneously conducted in erlotinib sensitive cells: 1) a genome-wide knock-out screen, conducted with the hypothesis that loss of function of certain genes drive erlotinib resistance, 2) a miRNA overexpression screen, conducted with the hypothesis that certain miRNAs drive the development of erlotinib resistance when overexpressed. The overreaching goal of the study was to identify novel drivers of erlotinib resistance such as microRNAs or other epigenetic factors in NSCLC.</p><p>The findings of this study led to the identification of a tumor suppressive protein and an epigenetic regulator, SUV420H2 (KMT5C) that has never been reported to be involved in erlotinib resistance. On the other hand, the miRNA overexpression screen identified five miRNAs that contribute to erlotinib resistance that were extensively analyzed using multiple bioinformatic tools. It was predicted that the miRNAs mediate erlotinib resistance via multiple pathways, owing to the ability of each miRNA to target multiple transcripts via partial complementarity. Importantly, a correlation between the two screens was identified clearly supporting the use of two simultaneous screens as a reliable technique to determine highly significant miRNA-target interactions. Overall, the findings from this study suggest that epigenetic factors, such as histone modifiers and miRNAs function as critical mediators of erlotinib resistance, possibly belonging to the 15-20% of NSCLC cases with unidentified mechanisms involved in erlotinib resistance.</p><p></p>

Since the discovery of cell-free fetal DNA in peripheral blood of pregnant women, cell-free nucleic acids in maternal plasma are explored in relation to non-invasive prenatal diagnosis of various fetal conditions and pregnancy complications. Non-invasive prenatal diagnosis of monogenic diseases represented by TSC1-linked tuberous sclerosis could be achieved by detection of paternally-inherited mutant allele in the pool of maternal alleles in plasma. Reliability of detection of mutant allele could be improved by simultaneous mutation haplotype analysis or detection of universal fetal marker. None of the 3 methods (allele- specific real-time PCR, SNaPshot minisequencing and quantitative fluorescent PCR) evaluated using artificial mixtures and maternal plasma samples reliably and accurately detected low-frequency allele distinguished by point mutation, SNP or microsatellite in TSC1 gene or in its close proximity. We developed a strategy for prediction of proportion of informative couples for panel of SNPs of interest that can be applied to any monogenic disease. Exploiting differential methylation of promoters of genes RASSF1A, HLCS and OLIG2 in maternal and fetal genome, we failed to establish functional fetal marker. MicroRNAs of placental origin released into plasma could serve as biomarkers of...

Drosophila Dicer-2 generates small interfering RNAs (siRNAs) from long double-stranded RNA (dsRNA), whereas Dicer-1 produces microRNAs from premicroRNA. My thesis focuses on the functional characteristics of two Drosophila Dicers that makes them specific for their biological substrates. We found that RNA binding protein partners of Dicers and two small molecules, ATP and phosphate are key in regulating Drosophila Dicers’ specificity. Without any additional factor, recombinant Dicer-2 cleaves pre-miRNA, but its product is shorter than the authentic miRNA. However, the protein R2D2 and inorganic phosphate block pre-miRNA processing by Dicer-2. In contrast, Dicer-1 is inherently capable of processing the substrates of Dicer, long dsRNAs. Yet, partner protein of Dicer-1, Loqs-PB and ATP increase the efficiency of miRNA production from pre-miRNAs by Dicer-1, therefore enhance substrate specificity of Dicer-1. Our data highlight the role of ATP and regulatory dsRNA-binding partner proteins to achieve substrate specificity in Drosophila RNA silencing. Our study also sheds light onto the function of the helicase domain in Drosophila Dicers. Although Dicer-1 doesn’t hydrolyze ATP, ATP enhances miRNA production by increasing Dicer-1’s substrate specificity through lowering its KM. On the other hand, Dicer-2 is a dsRNA-stimulated ATPase that hydrolyzes ATP to ADP, and ATP hydrolysis is required for Dicer-2 to process long dsRNA. Wild-type Dicer-2, but not a mutant defective in ATP hydrolysis, is processive; generating siRNAs faster than it can dissociate from a long dsRNA substrate. We propose that the Dicer-2 helicase domain uses ATP to generate many siRNAs from a single molecule of dsRNA before dissociating from its substrate. Piwi-dependent small RNAs, namely piRNAs, are a third class of small RNAs that are distinct from miRNAs and siRNAs. Their primary function is to repress transposons in the animal germline. piRNAs are Dicer-independent, and require Piwi family proteins for their biogenesis and function. Recently in addition to their presence in animal germlines, the presence and function of piRNA-like RNAs in the somatic tissues have been suggested (Yan et al. 2011; Morazzani et al. 2012; Rajasethupathy et al. 2012). We have investigated whether the piRNA-like reads in our many Drosophila head libraries come from the germline as a contaminant or are soma-specific. Most of the piRNA reads in our published head libraries show high similarity to germline piRNAs. However, piRNA-like reads from manually dissected heads are distinct from germline piRNAs, proving the presence of somatic piRNA-like small RNAs. We are currently asking the question whether these distinct piRNA-like reads in the heads are dependent on the Piwi family proteins, like the germline piRNAs.

Leukemia is a hematopoietic cancer that is characterized by the abnormal differentiation and proliferation of hematopoietic cells. It is ranked 7th by death rate among cancer types in USA, even though it is not one of the top 10 cancers by incidence (USCS, 2010). This indicates an urgent need for more effective treatment strategies. In order to design the new ways of prevention and treatment of leukemia, it is important to understand the molecular mechanisms involved in development of the disease. In this study, we investigated mechanisms involved in the development of acute myeloid leukemia (AML) that is associated with CBF fusion genes. The RUNX1 and CBFB genes that encode subunits of a transcriptional regulator complex CBF, are mutated in a subset (20 – 25%) of AML cases. As a result of these mutations, fusion genes called CBFB-MYH11 and RUNX1-ETO arise. The chimeric proteins encoded by the fusion genes provide block in proliferation for myeloid progenitors, but are not sufficient for AML development. Genetic studies have indicated that activation of cytokine receptor signaling is a major oncogenic pathway that cooperates in leukemia development. The main goal of my work was to determine a role of two factors that regulate cytokine signaling activity, the microRNA cluster miR-17-92 and the thrombopoietin receptor MPL, in their potential cooperation with the CBF fusions in AML development. We determined that the miR-17-92 miRNA cluster cooperates with Cbfb-MYH11 in AML development in a mouse model of human CBFB-MYH11 AML. We found that the miR-17-92 cluster downregulates Pten and activates the PI3K/Akt pathway in the leukemic blasts. We also demonstrated that miR-17-92 provides an anti-apoptotic effect in the leukemic cells, but does not seem to affect proliferation. The anti-apoptotic effect was mainly due to activity of miR-17 and miR-20a, but not miR-19a and miR-19b. Our second study demonstrated that wild type Mpl cooperated with RUNX1-ETO fusion in development of AML in mice. Mpl induced PI3K/Akt, Ras/Raf/Erk and Jak2/Stat5 signaling pathways in the AML cells. We showed that PIK3/Akt pathway plays a role in AML development both in vitro and in vivo by increasing survival of leukemic cells. The levels of MPL transcript in the AML samples correlated with their response to thrombopoietin (THPO). Moreover, we demonstrated that MPL provides pro-proliferative effect for the leukemic cells, and that the effect can be abrogated with inhibitors of PI3K/AKT and MEK/ERK pathways. Taken together, these data confirm important roles for the PI3K/AKT and RAS/RAF/MEK pathways in the pathogenesis of AML, identifies two novel genes that can serve as secondary mutations in CBF fusions-associated AML, and in general expands our knowledge of mechanisms of leukemogenesis.

Small interfering RNAs (siRNAs), microRNAs (miRNAs), and piRNAs (piRNA) are conserved classes of small single-stranded ~21-30 nucleotide (nt) RNA guides that repress eukaryotic gene expression using distinct RNA Induced Silencing Complexes (RISCs). At its core, RISC is composed of a single-stranded small RNA guide bound to a member of the Argonaute protein family, which together bind and repress complementary target RNA. miRNAs target protein coding mRNAs—a function essential for normal development and broadly involved in pathways of human disease; small interfering RNAs (siRNA) defend against viruses, but can also be engineered to direct experimental or therapeutic gene silencing; piwi associated RNAs (piRNAs) protect germline genomes from expansion of parasitic nucleic acids such as transposons. Using the fruit fly, Drosophila melanogaster, as a model organism we seek to understand how small silencing RNAs are made and how they function. In Drosophila, miRNAs and siRNAs are proposed to have parallel, but separate biogenesis and effector machinery. miRNA duplexes are excised from imperfectly paired hairpin precursors by Dicer1 and loaded into Ago1; siRNA duplexes are hewn from perfectly paired long dsRNA by Dicer2 and loaded into Ago2. Contrary to this model we found one miRNA, miR-277, is made by Dicer1, but partitions between Ago1 and Ago2 RISCs. These two RISCs are functionally distinct—Ago2 could silence a perfectly paired target, but not a centrally bulged target; Ago1 could silence a bulged target, but not a perfect target. This was surprising since both Ago1 and Ago2 have endonucleolytic cleavage activity necessary for perfect target cleavage in vitro. Our detailed kinetic studies suggested why—Ago2 is a robust multiple turnover enzyme, but Ago1 is not. Along with a complementary in vitro study our data supports a duplex sorting mechanism in which Diced duplexes are released, and rebind to Ago1 or Ago2 loading machinery, regardless of which Dicer produced them. This allows structural information embedded in small RNA duplexes to direct small RNA loading into Ago1 and/or Ago2, resulting in distinct regulatory outputs. Small RNA sorting also has chemical consequences for the small RNA guide. Although siRNAs were presumed to have the signature 2′, 3′ hydroxyl ends left by Dicer, we found that small RNAs loaded into Ago2 or Piwi proteins, but not Ago1, are modified at their 3´ ends by the RNA 2´-O-methyltransferase DmHen1. In plants Hen1 modifies the 3´ ends all small RNAs duplexs, protecting and stabilizing them. Implying a similar function in flies, piRNAs are smaller, less abundant, and their function is perturbed in hen1 mutants. But unlike plants, small RNAs are modified as single-strands in RISC rather than as duplexes. This nicely explains why the dsRNA binding domain in plant Hen1 was discarded in animals, and why both dsRNA derived siRNAs and ssRNA derived piRNAs are modified. The recent discovery that both piRNAs and siRNAs target transposons links terminal modification and transposon silencing, suggesting that it is specialized for this purpose.

In the preceding chapters, I have discussed my doctoral research on studying the siRNA loading pathway in Drosophila using both biochemical and genetic approaches. We established a gel shift system to identify the intermediate complexes formed during siRNA loading. We detected at least three complexes, named complex B, RISC loading complex (RLC) and RISC. Using kinetic modeling, we determined that the siRNA enters complex B and RLC early during assembly when it remains double-stranded, and then matures in RISC to generate Argonaute bearing only the single-stranded guide. We further characterized the three complexes. We showed that complex B comprises Dcr-1 and Loqs, while both RLC and RISC contain Dcr-2 and R2D2. Our study suggests that the Dcr-2/R2D2 heterodimer plays a central role in RISC assembly. We observed that Dcr-1/Loqs, which function together to process pre-miRNA into mature miRNA, were also involved in siRNA loading. This was surprising, because it has been proposed that the RNAi pathway and miRNA pathway are separate and parallel, with each using a unique set of proteins to produce small RNAs, to assemble functional RNA-guided enzyme complexes, and to regulate target mRNAs. We further examined the molecular function of Dcr-1/Loqs in RNAi pathway. Our data suggest that, in vivo and in vitro, the Dcr-1/Loqs complex binds to siRNA. In vitro, the binding of the Dcr-1/Loqs complex to siRNA is the earliest detectable step in siRNA-triggered Ago2-RISC assembly. Futhermore, the binding of Dcr-1/Loqs to siRNA appears to facilitate dsRNA dicing by Dcr-2/R2D2, because the dicing activity is much lower in loqslysate than in wild type. Long inverted repeat (IR) triggered white silencing in fly eyes is an example of endogenous RNAi. Consistent with our finding that Dcr-1/Loqs function to load siRNA, less white siRNA accumulates in loqs mutant eyes compared to wild type. As a result, loqs mutants are partially defective in IR trigged whitesilencing. Our data suggest considerable functional and genetic overlap between the miRNA and siRNA pathways, with the two sharing key components previously thought to be confined to just one of the two pathways. Based on our study on siRNA loading pathway, we also elucidated the molecular function of Armitage (Armi) protein in RNAi. We showed that armi is required for RNAi. Lysates from armi mutant ovaries are defective for RNAi in vitro. Native gel analysis of protein-siRNA complexes suggests that armi mutants support early steps in the RNAi pathway, i.e., the formation of complex B and RLC, but are defective in the production of the RISC.

Short noncoding RNAs play roles in regulating nearly every biological process, in nearly every organism, yet the exact function and importance of these molecules remains a subject of some debate. In order to gain a better understanding of the contexts in which these regulators have evolved, I have undertaken a variety of approaches to study the evolutionary history of the components that make up these pathways, in the form of two main research efforts. In the first chapter, I have used a combination of population genetics and molecular evolution techniques to show that proteins involved in the piRNA pathway are rapidly evolving, and that different components of the pathway seem to be evolving rapidly on different timescales. These rapidly evolving piRNA pathway proteins can be loosely separated into two groups. The first group appears to evolve quickly at the species level, perhaps in response to transposons that invade across species lines, while the second group appears to evolve quickly at the level of individual populations, perhaps in response to transposons that are paternally present yet novel to the maternal genome. In the second chapter of my research, I have used molecular evolution techniques and carefully devised controls to show that the binding sites of well-conserved miRNAs are among the most slowly changing short motifs in the genome, consistent with a conserved function for these short RNAs in regulatory pathways that are ancient and extremely slow to change. I have additionally discovered a major flaw in an existing approach to motif turnover calculations, which may lead to systematic biases in the published literature toward the false inference of increased regulatory complexity over time. I have implemented a revised approach to motif turnover that addresses this flaw.

Pancreatic ductal adenocarcinoma (PDAC) is among the most lethal malignancies in the United States, with an average five-year survival rate of just 6.7%. One unifying aspect of PDAC is mutational activation of the KRAS oncogene, which occurs in over 90% of PDAC. Therefore, inhibiting KRAS function is likely an effective therapeutic strategy for this disease, and current research in our lab and others is focused on identifying downstream effectors of KRAS signaling that may be therapeutic targets. miRNAs are powerful regulators of gene expression that can behave as oncogenes or tumor suppressors. Dysregulation of miRNA expression is commonly observed in human tumors, including PDAC. The mir-17~92 cluster of miRNAs is an established oncogene in a variety of tumor contexts, and members of the mir-17~92 cluster are upregulated in PDAC, but their role has not been explored in vivo. This dissertation encompasses two studies exploring the role of miRNAs in pancreatic tumorigenesis. In Chapter II, I demonstrate that deletion of the mir-17~92 cluster impairs PDAC precursor lesion formation and maintenance, and correlates with reduced ERK signaling in these lesions. mir-17~92 deficient tumors and cell lines are also less invasive, which I attribute to the loss of the miR-19 family of miRNAs. In Chapter III, I find that Dicer heterozygosity inhibits PDAC metastasis, and that this phenotype is attributable to an increased sensitivity to anoikis. Ongoing experiments will determine whether shifts in particular miRNA signatures between cell lines can be attributed to this phenotype. Together these findings illustrate the importance of miRNA biogenesis, and the mir-17~92 cluster in particular, in supporting PDAC development and progression.

Small RNAs are single-stranded, 18–36 nucleotide RNAs that can be categorized as miRNA, siRNA, and piRNA. miRNA are expressed ubiquitously in tissues and at particular developmental stages. They fine-tune gene expression by regulating the stability and translation of mRNAs. piRNAs are mainly expressed in the animal gonads and their major function is repressing transposable elements to ensure the faithful transfer of genetic information from generation to generation. My thesis research focused on the biogenesis of miRNAs and piRNAs using both experimental and computational strategies. The biogenesis of miRNAs involves sequential processing of their precursors by the RNase III enzymes Drosha and Dicer to generate miRNA/miRNA* duplexes, which are subsequently loaded into Argonaute proteins to form the RNA-induced silencing complex (RISC). We discovered that, after assembled into Ago1, more than a quarter of Drosophila miRNAs undergo 3′ end trimming by the 3′-to-5′ exoribonuclease Nibbler. Such trimming occurs after removal of the miRNA* strand from pre-RISC and may be the final step in RISC assembly, ultimately enhancing target messenger RNA repression. Moreover, by developing a specialized Burrow-Wheeler Transform based short reads aligner, we discovered that in the absence of Nibbler a subgroup of miRNAs undergoes increased tailing—non-templated nucleotide addition to their 3′ ends, which are usually associated with miRNA degradation. Therefore, the 3′ trimming by Nibbler might increase miRNA stability by protecting them from degradation. In Drosophila germ line, piRNAs associate with three PIWI-clade Argonaute proteins, Piwi, Aub, and Ago3. piRNAs bound by Aub and Ago3 are generated by reciprocal cleavages of sense and antisense transposon transcripts (a.k.a., the “Ping-Pong” cycle), which amplifies piRNA abundance and degrades transposon transcripts in the cytoplasm. On the other hand, Piwi and its associated piRNA repress the transcription of transposons in the nucleus. We discovered that Aub- and Ago3-mediated transposon RNA cleavage not only generates piRNAs bound to each other, but also produces substrates for the endonuclease Zucchini, which processively cleaves those substrates in a periodicity of ~26 nt and generates piRNAs that predominantly load into Piwi. Without Aub or Ago3, the abundance of Piwi-bound piRNAs drops and transcriptional silencing is compromised. Our discovery revises the current model of piRNA biogenesis.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by loss of motor neurons, resulting in progressive muscle weakness, atrophy, paralysis and death within five years of diagnosis. About ten percent of cases are inherited, of which twenty percent are due to mutations in the superoxide dismutase 1 (SOD1) gene. Since the only FDA approved ALS drug prolongs survival by just a few months, new therapies for this disease are needed. Experiments in transgenic ALS mouse models have shown that decreasing levels of mutant SOD1 protein alters and in some cases entirely prevents disease progression. We explored this potential therapeutic approach by using a single stranded AAV9 vector encoding an artificial microRNA against human SOD1 injected bilaterally into the cerebral lateral ventricles of neonatal SOD1G93A mice. This therapy extended median survival from 135 to 206 days (a 50% increase) and delayed hind limb paralysis. Animals remained ambulatory until endpoint, as defined by a sharp drop in body weight. Treated animals had a reduction of mutant human SOD1 mRNA levels in upper and lower motor neurons. As compared to untreated SOD1G93A mice, the AAV9 treated mice also had significant improvements in multiple parameters including the number of motor neurons, diameter of ventral root axons, and degree of neuroinflammation in the spinal cord. These studies clearly show that an AAV9-delivered artificial microRNA is a translatable therapeutic approach for ALS.