George A. Follows

Massive genomic rearrangement acquired in a single catastrophic event during cancer development

Summary Cancer is driven by somatically acquired point mutations and chromosomal rearrangements, conventionally thought to accumulate gradually over time. Using next-generation sequencing, we characterize a phenomenon, which we term chromothripsis, whereby tens to hundreds of genomic rearrangements occur in a one-off cellular crisis. Rearrangements involving one or a few chromosomes crisscross back and forth across involved regions, generating frequent oscillations between two copy number states. These genomic hallmarks are highly improbable if rearrangements accumulate over time and instead imply that nearly all occur during a single cellular catastrophe. The stamp of chromothripsis can be seen in at least 2%–3% of all cancers, across many subtypes, and is present in ∼25% of bone cancers. We find that one, or indeed more than one, cancer-causing lesion can emerge out of the genomic crisis. This phenomenon has important implications for the origins of genomic remodeling and temporal emergence of cancer. PaperClip

Subclonal phylogenetic structures in cancer revealed by ultra-deep sequencing

During the clonal expansion of cancer from an ancestral cell with an initiating oncogenic mutation to symptomatic neoplasm, the occurrence of somatic mutations (both driver and passenger) can be used to track the on-going evolution of the neoplasm. All subclones within a cancer are phylogenetically related, with the prevalence of each subclone determined by its evolutionary fitness and the timing of its origin relative to other subclones. Recently developed massively parallel sequencing platforms promise the ability to detect rare subclones of genetic variants without a priori knowledge of the mutations involved. We used ultra-deep pyrosequencing to investigate intraclonal diversification at the Ig heavy chain locus in 22 patients with B-cell chronic lymphocytic leukemia. Analysis of a non-polymorphic control locus revealed artifactual insertions and deletions resulting from sequencing errors and base substitutions caused by polymerase misincorporation during PCR amplification. We developed an algorithm to differentiate genuine haplotypes of somatic hypermutations from such artifacts. This proved capable of detecting multiple rare subclones with frequencies as low as 1 in 5000 copies and allowed the characterization of phylogenetic interrelationships among subclones within each patient. This study demonstrates the potential for ultra-deep resequencing to recapitulate the dynamics of clonal evolution in cancer cell populations.

Mapping long-range promoter contacts in human cells with high-resolution capture Hi-C

Transcriptional control in large genomes often requires looping interactions between distal DNA elements, such as enhancers and target promoters. Current chromosome conformation capture techniques do not offer sufficiently high resolution to interrogate these regulatory interactions on a genomic scale. Here we use Capture Hi-C (CHi-C), an adapted genome conformation assay, to examine the long-range interactions of almost 22,000 promoters in 2 human blood cell types. We identify over 1.6 million shared and cell type–restricted interactions spanning hundreds of kilobases between promoters and distal loci. Transcriptionally active genes contact enhancer-like elements, whereas transcriptionally inactive genes interact with previously uncharacterized elements marked by repressive features that may act as long-range silencers. Finally, we show that interacting loci are enriched for disease-associated SNPs, suggesting how distal mutations may disrupt the regulation of relevant genes. This study provides new insights and accessible tools to dissect the regulatory interactions that underlie normal and aberrant gene regulation.

Alemtuzumab in Combination With Methylprednisolone Is a Highly Effective Induction Regimen for Patients With Chronic Lymphocytic Leukemia and Deletion of TP53: Final Results of the National Cancer Research Institute CLL206 Trial

PURPOSE In chronic lymphocytic leukemia (CLL), TP53 deletion/mutation is strongly associated with an adverse outcome and resistance to chemotherapy-based treatment. In contrast, TP53 defects are not associated with resistance to the anti-CD52 monoclonal antibody alemtuzumab or methylprednisolone. In an attempt to improve the treatment of TP53-defective CLL, a multicenter phase II study was developed to evaluate alemtuzumab and methylprednisolone in combination. PATIENTS AND METHODS Thirty-nine patients with TP53-deleted CLL (17 untreated and 22 previously treated) received up to 16 weeks of treatment with alemtuzumab 30 mg three times a week and methylprednisolone 1.0 g/m(2) for five consecutive days every 4 weeks. Antimicrobial prophylaxis consisted of cotrimoxazole, itraconazole, and aciclovir (or valganciclovir for asymptomatic cytomegalovirus viremia). The primary end point was response as assigned by an end-point review committee. Secondary end points were safety, progression-free survival (PFS) and overall survival (OS). RESULTS The overall response rate, complete response rate (including with incomplete marrow recovery), median PFS, and median OS were 85%, 36%, 11.8 months, and 23.5 months, respectively, in the entire cohort and 88%, 65%, 18.3 months, and 38.9 months, respectively, in previously untreated patients. Grade 3 to 4 hematologic and glucocorticoid-associated toxicity occurred in 67% and 23% of patients, respectively. Grade 3 to 4 infection occurred in 51% of the overall cohort and in 29% of patients less than 60 years of age. Treatment-related mortality was 5%. CONCLUSION Alemtuzumab plus methypredisolone is the most effective induction regimen hitherto reported in TP53-deleted CLL. The risk of infection is age related and, in younger patients, seems only marginally higher than that associated with rituximab, fludarabine, and cyclophosphamide.

Two distinct auto-regulatory loops operate at the PU.1 locus in B cells and myeloid cells

The transcription factor PU.1 occupies a central role in controlling myeloid and early B-cell development, and its correct lineage-specific expression is critical for the differentiation choice of hematopoietic progenitors. However, little is known of how this tissue-specific pattern is established. We previously identified an upstream regulatory cis element whose targeted deletion in mice decreases PU.1 expression and causes leukemia. We show here that the upstream regulatory cis element alone is insufficient to confer physiologic PU.1 expression in mice but requires the cooperation with other, previously unidentified elements. Using a combination of transgenic studies, global chromatin assays, and detailed molecular analyses we present evidence that PU.1 is regulated by a novel mechanism involving cross talk between different cis elements together with lineage-restricted autoregulation. In this model, PU.1 regulates its expression in B cells and macrophages by differentially associating with cell type-specific transcription factors at one of its cis-regulatory elements to establish differential activity patterns at other elements.

DNA Damage–Induced Bcl-xL Deamidation Is Mediated by NHE-1 Antiport Regulated Intracellular pH

The pro-survival protein Bcl-xL is critical for the resistance of tumour cells to DNA damage. We have previously demonstrated, using a mouse cancer model, that oncogenic tyrosine kinase inhibition of DNA damage–induced Bcl-xL deamidation tightly correlates with T cell transformation in vivo, although the pathway to Bcl-xL deamidation remains unknown and its functional consequences unclear. We show here that rBcl-xL deamidation generates an iso-Asp52/iso-Asp66 species that is unable to sequester pro-apoptotic BH3-only proteins such as Bim and Puma. DNA damage in thymocytes results in increased expression of the NHE-1 Na/H antiport, an event both necessary and sufficient for subsequent intracellular alkalinisation, Bcl-xL deamidation, and apoptosis. In murine thymocytes and tumour cells expressing an oncogenic tyrosine kinase, this DNA damage–induced cascade is blocked. Enforced intracellular alkalinisation mimics the effects of DNA damage in murine tumour cells and human B-lineage chronic lymphocytic leukaemia cells, thereby causing Bcl-xL deamidation and increased apoptosis. Our results define a signalling pathway leading from DNA damage to up-regulation of the NHE-1 antiport, to intracellular alkalanisation to Bcl-xL deamidation, to apoptosis, representing the first example, to our knowledge, of how deamidation of internal asparagine residues can be regulated in a protein in vivo. Our findings also suggest novel approaches to cancer therapy.

Guidelines on the diagnosis, investigation and management of chronic lymphocytic leukaemia

Royal Bournemouth Hospital, Bournemouth, 2 Royal Marsden Hospital, London, 3 Southampton General Hospital, Southampton, Cardiff and Vale NHS Trust, Cardiff Cambridge University Hospitals NHS Foundation Trust, Cambridge UK; St. Jamess Institute of Oncology, Leeds, Christie Hospital NHS Trust, Manchester Royal Marsden Hospital London; Heart of England NHS Foundataion Trust, Birmingham; 10 Royal Liverpool University Hospital, Liverpool; Churchill Hospital, Headington, Oxford, Norfolk and Norwich University Hospital, Norwich

Rituximab Plus Chlorambucil As First-Line Treatment for Chronic Lymphocytic Leukemia: Final Analysis of an Open-Label Phase II Study

PURPOSE Most patients with chronic lymphocytic leukemia (CLL) are elderly and/or have comorbidities that may make them ineligible for fludarabine-based treatment. For this population, chlorambucil monotherapy is an appropriate therapeutic option; however, response rates with chlorambucil are low, and more effective treatments are needed. This trial was designed to assess how the addition of rituximab to chlorambucil (R-chlorambucil) would affect safety and efficacy in patients with CLL. PATIENTS AND METHODS Patients with first-line CLL were treated with rituximab (375 mg/m(2) on day 1, cycle one, and 500 mg/m(2) thereafter) plus chlorambucil (10 mg/m(2)/d all cycles; day 1 through 7) for six 28-day cycles. For patients not achieving complete response (CR), six additional cycles of chlorambucil alone could be administered. The primary end point of the study was safety. RESULTS A total of 100 patients were treated with R-chlorambucil, with a median follow-up of 30 months. Median age of patients was 70 years (range, 43 to 86 years), with patients having a median of seven comorbidities. Hematologic toxicities accounted for most grade 3/4 adverse events reported, with neutropenia and lymphopenia both occurring in 41% of patients and leukopenia in 23%. Overall response rates were 84%, with CR achieved in 10% of patients. Median progression-free survival was 23.5 months; median overall survival was not reached. CONCLUSION These results compare favorably with previously published results for chlorambucil monotherapy, suggesting that the addition of rituximab to chlorambucil may improve efficacy with no unexpected adverse events. R-chlorambucil may improve outcome for patients who are ineligible for fludarabine-based treatments.

Expression of the leukemia oncogene Lmo2 is controlled by an array of tissue-specific elements dispersed over 100 kb and bound by Tal1/Lmo2, Ets, and Gata factors.

The Lmo2 gene encodes a transcriptional cofactor critical for the development of hematopoietic stem cells. Ectopic LMO2 expression causes leukemia in T-cell acute lymphoblastic leukemia (T-ALL) patients and severe combined immunodeficiency patients undergoing retroviral gene therapy. Tightly controlled Lmo2 expression is therefore essential, yet no comprehensive analysis of Lmo2 regulation has been published so far. By comparative genomics, we identified 17 highly conserved noncoding elements, 9 of which revealed specific acetylation marks in chromatin-immunoprecipitation and microarray (ChIP-chip) assays performed across 250 kb of the Lmo2 locus in 11 cell types covering different stages of hematopoietic differentiation. All candidate regulatory regions were tested in transgenic mice. An extended LMO2 proximal promoter fragment displayed strong endothelial activity, while the distal promoter showed weak forebrain activity. Eight of the 15 distal candidate elements functioned as enhancers, which together recapitulated the full expression pattern of Lmo2, directing expression to endothelium, hematopoietic cells, tail, and forebrain. Interestingly, distinct combinations of specific distal regulatory elements were required to extend endothelial activity of the LMO2 promoter to yolk sac or fetal liver hematopoietic cells. Finally, Sfpi1/Pu.1, Fli1, Gata2, Tal1/Scl, and Lmo2 were shown to bind to and transactivate Lmo2 hematopoietic enhancers, thus identifying key upstream regulators and positioning Lmo2 within hematopoietic regulatory networks.

Inhibition of the Bcl-xL Deamidation Pathway in Myeloproliferative Disorders

BACKGROUND The myeloproliferative disorders are clonal disorders with frequent somatic gain-of-function alterations affecting tyrosine kinases. In these diseases, there is an increase in DNA damage and a risk of progression to acute leukemia. The molecular mechanisms in myeloproliferative disorders that prevent apoptosis induced by damaged DNA are obscure. METHODS We searched for abnormalities of the proapoptotic Bcl-x(L) deamidation pathway in primary cells from patients with chronic myeloid leukemia (CML) or polycythemia vera, myeloproliferative disorders associated with the BCR-ABL fusion kinase and the Janus tyrosine kinase 2 (JAK2) V617F mutation, respectively. RESULTS The Bcl-x(L) deamidation pathway was inhibited in myeloid cells, but not T cells, in patients with CML or polycythemia vera. DNA damage did not increase levels of the amiloride-sensitive sodium-hydrogen exchanger isoform 1 (NHE-1), intracellular pH, Bcl-x(L) deamidation, and apoptosis. Inhibition of the pathway was reversed by enforced alkalinization or overexpression of NHE-1, leading to a restoration of apoptosis. In patients with CML, the pathway was blocked in CD34+ progenitor cells and mature myeloid cells. Imatinib or JAK2 inhibitors reversed inhibition of the pathway in cells from patients with CML and polycythemia vera, respectively, but not in cells from a patient with resistance to imatinib because of a mutation in the BCR-ABL kinase domain. CONCLUSIONS BCR-ABL and mutant JAK2 inhibit the Bcl-x(L) deamidation pathway and the apoptotic response to DNA damage in primary cells from patients with CML or polycythemia vera.