Mutational landscape, clonal evolution patterns, and role of RAS mutations in relapsed acute lymphoblastic leukemia.

Although multiagent combination chemotherapy is curative in a significant fraction of childhood acute lymphoblastic leukemia (ALL) patients, 20% of cases relapse and most die because of chemorefractory disease. Here we used whole-exome and whole-genome sequencing to analyze the mutational landscape at relapse in pediatric ALL cases. These analyses identified numerous relapse-associated mutated genes intertwined in chemotherapy resistance-related protein complexes. In this context, RAS-MAPK pathway-activating mutations in the neuroblastoma RAS viral oncogene homolog (NRAS), kirsten rat sarcoma viral oncogene homolog (KRAS), and protein tyrosine phosphatase, nonreceptor type 11 (PTPN11) genes were present in 24 of 55 (44%) cases in our series. Interestingly, some leukemias showed retention or emergence of RAS mutant clones at relapse, whereas in others RAS mutant clones present at diagnosis were replaced by RAS wild-type populations, supporting a role for both positive and negative selection evolutionary pressures in clonal evolution of RAS-mutant leukemia. Consistently, functional dissection of mouse and human wild-type and mutant RAS isogenic leukemia cells demonstrated induction of methotrexate resistance but also improved the response to vincristine in mutant RAS-expressing lymphoblasts. These results highlight the central role of chemotherapy-driven selection as a central mechanism of leukemia clonal evolution in relapsed ALL, and demonstrate a previously unrecognized dual role of RAS mutations as drivers of both sensitivity and resistance to chemotherapy.

Viral diversity and clonal evolution from unphased genomic data.

BACKGROUND: Clonal expansion is a process in which a single organism reproduces asexually, giving rise to a diversifying population. It is pervasive in nature, from within-host pathogen evolution to emergent infectious disease outbreaks. Standard phylogenetic tools rely on full-length genomes of individual pathogens or population consensus sequences (phased genotypes). METHODS: We introduce two measures of diversity to study the evolution of clonal populations using unphased genomic data, which eliminate the need to construct full-length genomes. Our method follows a maximum likelihood approach to estimate evolutionary rates and times to the most recent common ancestor, based on a relaxed molecular clock model; independent of a growth model. Deviations from neutral evolution indicate the presence of selection and bottleneck events. RESULTS: We evaluated our methods in silico and then compared it against existing approaches with the well-characterized 2009 H1N1 influenza pandemic. We then applied our method to high-throughput genomic data from marburgvirus-infected non-human primates and inferred the time of infection and the intra-host evolutionary rate, and identified purifying selection in viral populations. CONCLUSIONS: Our method has the power to make use of minor variants present in less than 1% of the population and capture genomic diversification within days of infection, making it an ideal tool for the study of acute RNA viral infection dynamics.

Euphoric Presentation in Creutzfeldt-Jakob Disease and Its Diagnostic Implications: A Case Report.

Creutzfeldt-Jakob disease (CJD) constitutes an aggressively advancing, terminal neurodegenerative condition classified within the spectrum of transmissible spongiform encephalopathies. The difficulty in establishing a diagnosis before death arises from the condition's rarity and the resulting limited level of suspicion attributed to it. The polymorphic nature of CJD symptoms contributes to the challenge of early diagnostic recognition. Emotional and behavioral changes have been well documented, but the initial presentation of euphoria has not been documented. Here, we present the case of a female patient who was experiencing an unusual state of euphoria followed by intermittently altered mental status. She was ultimately diagnosed with sporadic CJD, discharged home on hospice, and died within six months of discharge.

The unique Morgue ubiquitination protein is conserved in a diverse but restricted set of invertebrates.

Drosophila Morgue is a unique ubiquitination protein that facilitates programmed cell death and associates with DIAP1, a critical cell death inhibitor with E3 ubiquitin ligase activity. Morgue possesses a unique combination of functional domains typically associated with distinct types of ubiquitination enzymes. This includes an F box characteristic of the substrate-binding subunit in Skp, Cullin, and F box (SCF)-type ubiquitin E3 ligase complexes and a variant ubiquitin E2 conjugase domain where the active site cysteine is replaced by a glycine. Morgue also contains a single C4-type zinc finger motif. This architecture suggests potentially novel ubiquitination activities for Morgue. In this study, we address the evolutionary origins of this distinctive protein utilizing a combination of bioinformatics and molecular biology approaches. We find that Morgue exhibits widespread but restricted phylogenetic distribution among metazoans. Morgue proteins were identified in a wide range of Protostome phyla, including Arthropoda, Annelida, Mollusca, Nematoda, and Platyhelminthes. However, with one potential exception, Morgue was not detected in Deuterostomes, including Chordates, Hemichordates, or Echinoderms. Morgue was also not found in Ctenophora, Cnidaria, Placozoa, or Porifera. Characterization of Morgue sequences within specific animal lineages suggests that gene deletion or acquisition has occurred during divergence of nematodes and that at least one arachnid expresses an atypical form of Morgue consisting only of the variant E2 conjugase domain. Analysis of the organization of several morgue genes suggests that exon-shuffling events have contributed to the evolution of the Morgue protein. These results suggest that Morgue mediates conserved and distinctive ubiquitination functions in specific cell death pathways.

Structure and Mechanisms of NT5C2 Mutations Driving Thiopurine Resistance in Relapsed Lymphoblastic Leukemia.

Activating mutations in the cytosolic 5'-nucleotidase II gene NT5C2 drive resistance to 6-mercaptopurine in acute lymphoblastic leukemia. Here we demonstrate that constitutively active NT5C2 mutations K359Q and L375F reconfigure the catalytic center for substrate access and catalysis in the absence of allosteric activator. In contrast, most relapse-associated mutations, which involve the arm segment and residues along the surface of the inter-monomeric cavity, disrupt a built-in switch-off mechanism responsible for turning off NT5C2. In addition, we show that the C-terminal acidic tail lost in the Q523X mutation functions to restrain NT5C2 activation. These results uncover dynamic mechanisms of enzyme regulation targeted by chemotherapy resistance-driving NT5C2 mutations, with important implications for the development of NT5C2 inhibitor therapies.

Recurrent mutations in epigenetic regulators, RHOA and FYN kinase in peripheral T cell lymphomas.

Peripheral T cell lymphomas (PTCLs) are a heterogeneous and poorly understood group of non-Hodgkin lymphomas. Here we combined whole-exome sequencing of 12 tumor-normal DNA pairs, RNA sequencing analysis and targeted deep sequencing to identify new genetic alterations in PTCL transformation. These analyses identified highly recurrent epigenetic factor mutations in TET2, DNMT3A and IDH2 as well as a new highly prevalent RHOA mutation encoding a p.Gly17Val alteration present in 22 of 35 (67%) angioimmunoblastic T cell lymphoma (AITL) samples and in 8 of 44 (18%) PTCL, not otherwise specified (PTCL-NOS) samples. Mechanistically, the RHOA Gly17Val protein interferes with RHOA signaling in biochemical and cellular assays, an effect potentially mediated by the sequestration of activated guanine-exchange factor (GEF) proteins. In addition, we describe new and recurrent, albeit less frequent, genetic defects including mutations in FYN, ATM, B2M and CD58 implicating SRC signaling, impaired DNA damage response and escape from immune surveillance mechanisms in the pathogenesis of PTCL.

Activating mutations in the NT5C2 nucleotidase gene drive chemotherapy resistance in relapsed ALL.

Acute lymphoblastic leukemia (ALL) is an aggressive hematological tumor resulting from the malignant transformation of lymphoid progenitors. Despite intensive chemotherapy, 20% of pediatric patients and over 50% of adult patients with ALL do not achieve a complete remission or relapse after intensified chemotherapy, making disease relapse and resistance to therapy the most substantial challenge in the treatment of this disease. Using whole-exome sequencing, we identify mutations in the cytosolic 5'-nucleotidase II gene (NT5C2), which encodes a 5'-nucleotidase enzyme that is responsible for the inactivation of nucleoside-analog chemotherapy drugs, in 20/103 (19%) relapse T cell ALLs and 1/35 (3%) relapse B-precursor ALLs. NT5C2 mutant proteins show increased nucleotidase activity in vitro and conferred resistance to chemotherapy with 6-mercaptopurine and 6-thioguanine when expressed in ALL lymphoblasts. These results support a prominent role for activating mutations in NT5C2 and increased nucleoside-analog metabolism in disease progression and chemotherapy resistance in ALL.

Reverse engineering of TLX oncogenic transcriptional networks identifies RUNX1 as tumor suppressor in T-ALL.

The TLX1 and TLX3 transcription factor oncogenes have a key role in the pathogenesis of T cell acute lymphoblastic leukemia (T-ALL). Here we used reverse engineering of global transcriptional networks to decipher the oncogenic regulatory circuit controlled by TLX1 and TLX3. This systems biology analysis defined T cell leukemia homeobox 1 (TLX1) and TLX3 as master regulators of an oncogenic transcriptional circuit governing T-ALL. Notably, a network structure analysis of this hierarchical network identified RUNX1 as a key mediator of the T-ALL induced by TLX1 and TLX3 and predicted a tumor-suppressor role for RUNX1 in T cell transformation. Consistent with these results, we identified recurrent somatic loss-of-function mutations in RUNX1 in human T-ALL. Overall, these results place TLX1 and TLX3 at the top of an oncogenic transcriptional network controlling leukemia development, show the power of network analyses to identify key elements in the regulatory circuits governing human cancer and identify RUNX1 as a tumor-suppressor gene in T-ALL.