Michelle Nahas

A four-way junction accelerates hairpin ribozyme folding via a discrete intermediate

The natural form of the hairpin ribozyme comprises two major structural elements: a four-way RNA junction and two internal loops carried by adjacent arms of the junction. The ribozyme folds into its active conformation by an intimate association between the loops, and the efficiency of this process is greatly enhanced by the presence of the junction. We have used single-molecule spectroscopy to show that the natural form fluctuates among three distinct states: the folded state and two additional, rapidly interconverting states (proximal and distal) that are inherited from the junction. The proximal state juxtaposes the two loop elements, thereby increasing the probability of their interaction and thus accelerating folding by nearly three orders of magnitude and allowing the ribozyme to fold rapidly in physiological conditions. Therefore, the hairpin ribozyme exploits the dynamics of the junction to facilitate the formation of the active site from its other elements. Dynamic interplay between structural elements, as we demonstrate for the hairpin ribozyme, may be a general theme for other functional RNA molecules.

Observation of internal cleavage and ligation reactions of a ribozyme

We have used single-molecule spectroscopy to untangle conformational dynamics and internal chemistry in the hairpin ribozyme. The active site of the ribozyme is stably formed by docking two internal loops, but upon cleavage undocking is accelerated by two orders of magnitude. The markedly different kinetic properties allow us to differentiate cleaved and ligated forms, and thereby observe multiple cycles of internal cleavage and ligation of a ribozyme in a uniquely direct way. The position of the internal equilibrium is biased toward ligation, but the cleaved ribozyme undergoes several undocking events before ligation, during which products may dissociate. Formation of the stably docked active site, rapid undocking after cleavage, and a strong bias toward ligation should combine to generate a stable circular template for the synthesis of the viral (+) strand and thus ensure a productive replication cycle.

Clinical next‐generation sequencing successfully applied to fine‐needle aspirations of pulmonary and pancreatic neoplasms

Next‐generation sequencing was performed on pulmonary and pancreatic fine‐needle aspirations (FNAs) and on paired FNAs and resected primary tumors from the same patient.

Integrated genomic DNA/RNA profiling of hematologic malignancies in the clinical setting

The spectrum of somatic alterations in hematologic malignancies includes substitutions, insertions/deletions (indels), copy number alterations (CNAs), and a wide range of gene fusions; no current clinically available single assay captures the different types of alterations. We developed a novel next-generation sequencing-based assay to identify all classes of genomic alterations using archived formalin-fixed paraffin-embedded blood and bone marrow samples with high accuracy in a clinically relevant time frame, which is performed in our Clinical Laboratory Improvement Amendments-certified College of American Pathologists-accredited laboratory. Targeted capture of DNA/RNA and next-generation sequencing reliably identifies substitutions, indels, CNAs, and gene fusions, with similar accuracy to lower-throughput assays that focus on specific genes and types of genomic alterations. Profiling of 3696 samples identified recurrent somatic alterations that impact diagnosis, prognosis, and therapy selection. This comprehensive genomic profiling approach has proved effective in detecting all types of genomic alterations, including fusion transcripts, which increases the ability to identify clinically relevant genomic alterations with therapeutic relevance.

A High Frequency of Activating Extracellular Domain ERBB2 (HER2) mutation in Micropapillary Urothelial Carcinoma

Purpose: Micropapillary urothelial carcinoma (MPUC) is a rare and aggressive form of bladder cancer. We conducted genomic analyses [next-generation sequencing (NGS)] of MPUC and non-micropapillary urothelial bladder carcinomas (non-MPUC) to characterize the genomic landscape and identify targeted treatment options. Experimental Design: DNA was extracted from 40 μm of formalin-fixed paraffin-embedded sections from 15 MPUC and 64 non-MPUC tumors. Sequencing (NGS) was performed on hybridization-captured, adaptor ligation–based libraries to high coverage for 3,230 exons of 182 cancer-related genes plus 37 introns from 14 genes frequently rearranged in cancer. The results were evaluated for all classes of genomic alteration. Results: Mutations in the extracellular domain of ERBB2 were identified in 6 of 15 (40%) of MPUC: S310F (four cases), S310Y (one case), and R157W (one case). All six cases of MPUC with ERBB2 mutation were negative for ERBB2 amplification and Erbb2 overexpression. In contrast, 6 of 64 (9.4%) non-MPUC harbored an ERBB2 alteration, including base substitution (three cases), amplification (two cases), and gene fusion (one case), which is higher than the 2 of 159 (1.3%) protein-changing ERBB2 mutations reported for urinary tract cancer in COSMIC. The enrichment of ERBB2 alterations in MPUC compared with non-MPUC is significant both between this series (P < 0.0084) and for all types of urinary tract cancer in COSMIC (P < 0.001). Conclusions: NGS of MPUC revealed a high incidence of mutation in the extracellular domain of ERBB2, a gene for which there are five approved targeted therapies. NGS can identify genomic alteration, which inform treatment options for the majority of MPUC patients. Clin Cancer Res; 20(1); 68–75. ©2013 AACR.

Genomic and functional analysis of leukemic transformation of myeloproliferative neoplasms

Significance Myeloproliferative neoplasms (MPN) are chronic hematopoietic disorders characterized by clonal proliferation of mature myeloid elements. A subset of MPNs transforms to acute myeloid leukemia (AML). The mechanisms and pathways that contribute to transformation from MPN to AML have not been well delineated. We have characterized the somatic mutational spectrum of post-MPN AML and demonstrate that somatic tumor protein 53 (TP53) mutations are common in JAK2V617F-mutant, post-MPN AML but not in chronic-phase MPN. We demonstrate that expression of JAK2V617F combined with Tp53 loss in a murine model leads to fully penetrant AML in vivo. We have characterized this model and used it to test therapeutic strategies. These data reveal novel insights into the pathogenesis of, and potential therapeutic strategies for, leukemic transformation. Patients with myeloproliferative neoplasms (MPNs) are at significant, cumulative risk of leukemic transformation to acute myeloid leukemia (AML), which is associated with adverse clinical outcome and resistance to standard AML therapies. We performed genomic profiling of post-MPN AML samples; these studies demonstrate somatic tumor protein 53 (TP53) mutations are common in JAK2V617F-mutant, post-MPN AML but not in chronic-phase MPN and lead to clonal dominance of JAK2V617F/TP53-mutant leukemic cells. Consistent with these data, expression of JAK2V617F combined with Tp53 loss led to fully penetrant AML in vivo. JAK2V617F-mutant, Tp53-deficient AML was characterized by an expanded megakaryocyte erythroid progenitor population that was able to propagate the disease in secondary recipients. In vitro studies revealed that post-MPN AML cells were sensitive to decitabine, the JAK1/2 inhibitor ruxolitinib, or the heat shock protein 90 inhibitor 8-(6-iodobenzo[d][1.3]dioxol-5-ylthio)-9-(3-(isopropylamino)propyl)-9H-purine-6-amine (PU-H71). Treatment with ruxolitinib or PU-H71 improved survival of mice engrafted with JAK2V617F-mutant, Tp53-deficient AML, demonstrating therapeutic efficacy for these targeted therapies and providing a rationale for testing these therapies in post-MPN AML.

Multivector fluorescence analysis of the xpt guanine riboswitch aptamer domain and the conformational role of guanine.

Purine riboswitches are RNA regulatory elements that control purine metabolism in response to intracellular concentrations of the purine ligands. Conformational changes of the guanine riboswitch aptamer domain induced by guanine binding lead to transcriptional regulation of genes involved in guanine biosynthesis. The guanine riboswitch aptamer domain has three RNA helices designated P1, P2, and P3. An overall model for the Mg2+- and guanine-dependent relative orientations and dynamics of P1, P2, and P3 has not been reported, and the conformational role of guanine under physiologically relevant conditions has not been fully elucidated. In this study, an ensemble and single-molecule fluorescence resonance energy transfer (FRET) study was performed on three orthogonally labeled variants of the xpt guanine riboswitch aptamer domain. The combined FRET data support a model in which the unfolded state of the aptamer domain has a highly dynamic P2 helix that switches rapidly between two orientations relative to nondynamic P1 and P3. At ≪1 mM Mg2+ (in the presence of a saturating level of guanine) or ≥1 mM Mg2+ (in the absence of guanine), the riboswitch starts to adopt a folded conformation in which loop−loop interactions lock P2 and P3 into place. At >5 mM Mg2+, further compaction occurs in which P1 more closely approaches P3. Our data help to explain the biological role of guanine as stabilizing the globally folded aptamer domain conformation at physiologically relevant Mg2+ concentrations (≤1 mM), whereas in the absence of guanine, much higher Mg2+ concentrations are required to induce this folding event.

Folding and catalysis of the hairpin ribozyme

The hairpin ribozyme undergoes a site-specific transesterification cleavage of the phosphodiester backbone. The natural form of the ribozyme is a four-way helical junction, where two arms contain unpaired loops. This folds by pairwise coaxial stacking of helical arms, and a rotation into an antiparallel conformation in which there is close association between the loops. This probably generates the local conformation required to facilitate the trajectory into an in-line SN2 transition state. Folding is induced by the cooperative binding of at least two divalent metal ions, which are probably distributed between the junction and the loop-loop interface. The junction forms the structural scaffold on which the geometry of the ribozyme is built, and structural perturbation of the junction leads to impaired catalytic activity.

Single-molecule studies of DNA and RNA four-way junctions

Branched helical junctions are common in nucleic acids. In DNA, the four-way junction (Holliday junction) is an essential intermediate in homologous recombination and is a highly dynamic structure, capable of stacking conformer transitions and branch migration. Our single-molecule fluorescence studies provide unique insight into the energy landscape of Holliday junctions by visualizing these processes directly. In the hairpin ribozyme, an RNA four-way junction is an important structural element that enhances active-site formation by several orders of magnitude. Our single-molecule studies suggest a plausible mechanism for how the junction achieves this remarkable feat; the structural dynamics of the four-way junction bring about frequent contacts between the loops that are needed to form the active site. The most definitive evidence for this is the observation of three-state folding in single-hairpin ribozymes, the intermediate state of which is populated due to the intrinsic properties of the junction.

Patient-derived xenotransplants can recapitulate the genetic driver landscape of acute leukemias.

Genomic studies have identified recurrent somatic mutations in acute leukemias. However, current murine models do not sufficiently encompass the genomic complexity of human leukemias. To develop preclinical models, we transplanted 160 samples from patients with acute leukemia (acute myeloid leukemia, mixed lineage leukemia, B-cell acute lymphoblastic leukemia, T-cell ALL) into immunodeficient mice. Of these, 119 engrafted with expected immunophenotype. Targeted sequencing of 374 genes and 265 frequently rearranged RNAs detected recurrent and novel genetic lesions in 48 paired primary tumor (PT) and patient-derived xenotransplant (PDX) samples. Overall, the frequencies of 274 somatic variant alleles correlated between PT and PDX samples, although the data were highly variable for variant alleles present at 0–10%. Seventeen percent of variant alleles were detected in either PT or PDX samples only. Based on variant allele frequency changes, 24 PT-PDX pairs were classified as concordant while the other 24 pairs showed various degree of clonal discordance. There was no correlation of clonal concordance with clinical parameters of diseases. Significantly more bone marrow samples than peripheral blood samples engrafted discordantly. These data demonstrate the utility of developing PDX banks for modeling human leukemia, and emphasize the importance of genomic profiling of PDX and patient samples to ensure concordance before performing mechanistic or therapeutic studies.