Niklas Feldhahn

A New Human Somatic Stem Cell from Placental Cord Blood with Intrinsic Pluripotent Differentiation Potential

Here a new, intrinsically pluripotent, CD45-negative population from human cord blood, termed unrestricted somatic stem cells (USSCs) is described. This rare population grows adherently and can be expanded to 1015 cells without losing pluripotency. In vitro USSCs showed homogeneous differentiation into osteoblasts, chondroblasts, adipocytes, and hematopoietic and neural cells including astrocytes and neurons that express neurofilament, sodium channel protein, and various neurotransmitter phenotypes. Stereotactic implantation of USSCs into intact adult rat brain revealed that human Tau-positive cells persisted for up to 3 mo and showed migratory activity and a typical neuron-like morphology. In vivo differentiation of USSCs along mesodermal and endodermal pathways was demonstrated in animal models. Bony reconstitution was observed after transplantation of USSC-loaded calcium phosphate cylinders in nude rat femurs. Chondrogenesis occurred after transplanting cell-loaded gelfoam sponges into nude mice. Transplantation of USSCs in a noninjury model, the preimmune fetal sheep, resulted in up to 5% human hematopoietic engraftment. More than 20% albumin-producing human parenchymal hepatic cells with absence of cell fusion and substantial numbers of human cardiomyocytes in both atria and ventricles of the sheep heart were detected many months after USSC transplantation. No tumor formation was observed in any of these animals.

53BP1 inhibits homologous recombination in Brca1-deficient cells by blocking resection of DNA breaks.

Defective DNA repair by homologous recombination (HR) is thought to be a major contributor to tumorigenesis in individuals carrying Brca1 mutations. Here, we show that DNA breaks in Brca1-deficient cells are aberrantly joined into complex chromosome rearrangements by a process dependent on the nonhomologous end-joining (NHEJ) factors 53BP1 and DNA ligase 4. Loss of 53BP1 alleviates hypersensitivity of Brca1 mutant cells to PARP inhibition and restores error-free repair by HR. Mechanistically, 53BP1 deletion promotes ATM-dependent processing of broken DNA ends to produce recombinogenic single-stranded DNA competent for HR. In contrast, Lig4 deficiency does not rescue the HR defect in Brca1 mutant cells but prevents the joining of chromatid breaks into chromosome rearrangements. Our results illustrate that HR and NHEJ compete to process DNA breaks that arise during DNA replication and that shifting the balance between these pathways can be exploited to selectively protect or kill cells harboring Brca1 mutations.

Broad diversity of neutralizing antibodies isolated from memory B cells in HIV-infected individuals

Antibodies to conserved epitopes on the human immunodeficiency virus (HIV) surface protein gp140 can protect against infection in non-human primates, and some infected individuals show high titres of broadly neutralizing immunoglobulin (Ig)G antibodies in their serum. However, little is known about the specificity and activity of these antibodies. To characterize the memory antibody responses to HIV, we cloned 502 antibodies from HIV envelope-binding memory B cells from six HIV-infected patients with broadly neutralizing antibodies and low to intermediate viral loads. We show that in these patients, the B-cell memory response to gp140 is composed of up to 50 independent clones expressing high affinity neutralizing antibodies to the gp120 variable loops, the CD4-binding site, the co-receptor-binding site, and to a new neutralizing epitope that is in the same region of gp120 as the CD4-binding site. Thus, the IgG memory B-cell compartment in the selected group of patients with broad serum neutralizing activity to HIV is comprised of multiple clonal responses with neutralizing activity directed against several epitopes on gp120.

Rif1 Prevents Resection of DNA Breaks and Promotes Immunoglobulin Class Switching

Fixing Broken DNA Some physiological processes, such as immunoglobulin class switching and telomere attrition, result in double-stranded DNA breaks. The DNA damage repair protein, 53BP1, prevents nucleolytic processing of these breaks, but the proteins it partners with to do this are unknown (see the Perspective by Lukas and Lukas). Di Virgilio et al. (p. 711, published online 10 January), using mass spectroscopy–based methods, and Zimmermann et al. (p. 700, published online 10 January), using a telomere-based assay, identify Rif1 as a 53BP1 phosphorylation- and DNA damage–dependent interaction partner. Mice with a B cell–specific deletion in Rif1 showed impaired immunoglobulin class switching. Rif1-deficient cells exhibited extensive 5′-3′ resection at DNA ends, with enhanced genetic instability. Thus, Rif1 partners with 53BP1 to promote the proper repair of double-stranded DNA breaks. In mammalian cells, Rap1-interacting factor 1 protects DNA ends against resection. [Also see Perspective by Lukas and Lukas] DNA double-strand breaks (DSBs) represent a threat to the genome because they can lead to the loss of genetic information and chromosome rearrangements. The DNA repair protein p53 binding protein 1 (53BP1) protects the genome by limiting nucleolytic processing of DSBs by a mechanism that requires its phosphorylation, but whether 53BP1 does so directly is not known. Here, we identify Rap1-interacting factor 1 (Rif1) as an ATM (ataxia-telangiectasia mutated) phosphorylation-dependent interactor of 53BP1 and show that absence of Rif1 results in 5′-3′ DNA-end resection in mice. Consistent with enhanced DNA resection, Rif1 deficiency impairs DNA repair in the G1 and S phases of the cell cycle, interferes with class switch recombination in B lymphocytes, and leads to accumulation of chromosome DSBs.

53BP1 regulates DNA resection and the choice between classical and alternative end joining during class switch recombination

Class switch recombination (CSR) diversifies antibodies by joining highly repetitive DNA elements, which are separated by 60–200 kbp. CSR is initiated by activation-induced cytidine deaminase, an enzyme that produces multiple DNA double-strand breaks (DSBs) in switch regions. Switch regions are joined by a mechanism that requires an intact DNA damage response and classical or alternative nonhomologous end joining (A-NHEJ). Among the DNA damage response factors, 53BP1 has the most profound effect on CSR. We explore the role of 53BP1 in intrachromosomal DNA repair using I-SceI to introduce paired DSBs in the IgH locus. We find that the absence of 53BP1 results in an ataxia telangiectasia mutated–dependent increase in DNA end resection and that resected DNA is preferentially repaired by microhomology-mediated A-NHEJ. We propose that 53BP1 favors long-range CSR in part by protecting DNA ends against resection, which prevents A-NHEJ–dependent short-range rejoining of intra–switch region DSBs.

Regulation of DNA End Joining, Resection, and Immunoglobulin Class Switch Recombination by 53BP1

53BP1 is a DNA damage protein that forms phosphorylated H2AX (γ-H2AX) dependent foci in a 1 Mb region surrounding DNA double-strand breaks (DSBs). In addition, 53BP1 promotes genomic stability by regulating the metabolism of DNA ends. We have compared the joining rates of paired DSBs separated by 1.2 kb to 27 Mb on chromosome 12 in the presence or absence of 53BP1. 53BP1 facilitates joining of intrachromosomal DSBs but only at distances corresponding to γ-H2AX spreading. In contrast, DNA end protection by 53BP1 is distance independent. Furthermore, analysis of 53BP1 mutants shows that chromatin association, oligomerization, and N-terminal ATM phosphorylation are all required for DNA end protection and joining as measured by immunoglobulin class switch recombination. These data elucidate the molecular events that are required for 53BP1 to maintain genomic stability and point to a model wherein 53BP1 and H2AX cooperate to repress resection of DSBs.

A method for identification of HIV gp140 binding memory B cells in human blood

Antibodies to HIV are potentially important reagents for basic and clinical studies. Historically, these reagents have been produced by random cloning of heavy and light chains in phage display libraries [Burton, D.R., Barbas, C.F. III, Persson, M.A.A., Koenig, S., Chanock, R.M., and Lerner, R.A., (1991), A large array of human monoclonal antibodies to type 1 immunodeficiency virus from combinatorial libraries of asymptomatic seropositive individuals. Proc. Natl. Acad. Sci. U. S. A. 88, 10134-10137.] and electrofusion techniques [Buchacher, A., Predl, R., Tauer, C., Purtscher, M., Gruber, G., Heider, R., Steindl, F., Trkola, A., Jungbauer, A., and Katinger, H., (1992), Human monoclonal antibodies against gp41 and gp120 as potential agent for passive immunization. Vaccines 92, 191-195]. Here we describe a method to identify and potentially enrich human memory B cells from HIV infected patients that show serum titers of neutralizing antibodies. When biotinylated gp140 is used to stain peripheral blood mononuclear cells it identifies a distinct population of gp140 binding B cells by flow cytometry.

The B cell mutator AID promotes B lymphoid blast crisis and drug resistance in chronic myeloid leukemia.

Chronic myeloid leukemia (CML) is induced by BCR-ABL1 and can be effectively treated for many years with Imatinib until leukemia cells acquire drug resistance through BCR-ABL1 mutations and progress into fatal B lymphoid blast crisis (LBC). Despite its clinical significance, the mechanism of progression into LBC is unknown. Here, we show that LBC but not CML cells express the B cell-specific mutator enzyme AID. We demonstrate that AID expression in CML cells promotes overall genetic instability by hypermutation of tumor suppressor and DNA repair genes. Importantly, our data uncover a causative role of AID activity in the acquisition of BCR-ABL1 mutations leading to Imatinib resistance, thus providing a rationale for the rapid development of drug resistance and blast crisis progression.

Activation-induced cytidine deaminase acts as a mutator in BCR-ABL1–transformed acute lymphoblastic leukemia cells

The Philadelphia chromosome (Ph) encoding the oncogenic BCR-ABL1 kinase defines a subset of acute lymphoblastic leukemia (ALL) with a particularly unfavorable prognosis. ALL cells are derived from B cell precursors in most cases and typically carry rearranged immunoglobulin heavy chain (IGH) variable (V) region genes devoid of somatic mutations. Somatic hypermutation is restricted to mature germinal center B cells and depends on activation-induced cytidine deaminase (AID). Studying AID expression in 108 cases of ALL, we detected AID mRNA in 24 of 28 Ph+ ALLs as compared with 6 of 80 Ph− ALLs. Forced expression of BCR-ABL1 in Ph− ALL cells and inhibition of the BCR-ABL1 kinase showed that aberrant expression of AID depends on BCR-ABL1 kinase activity. Consistent with aberrant AID expression in Ph+ ALL, IGH V region genes and BCL6 were mutated in many Ph+ but unmutated in most Ph− cases. In addition, AID introduced DNA single-strand breaks within the tumor suppressor gene CDKN2B in Ph+ ALL cells, which was sensitive to BCR-ABL1 kinase inhibition and silencing of AID expression by RNA interference. These findings identify AID as a BCR-ABL1–induced mutator in Ph+ ALL cells, which may be relevant with respect to the particularly unfavorable prognosis of this leukemia subset.

Mimicry of a constitutively active pre–B cell receptor in acute lymphoblastic leukemia cells

Pre–B cells undergo apoptosis unless they are rescued by pre–B cell receptor–dependent survival signals. We previously showed that the BCR-ABL1 kinase that is expressed in pre–B lymphoblastic leukemia bypasses selection for pre–B cell receptor–dependent survival signals. Investigating possible interference of BCR-ABL1 with pre–B cell receptor signaling, we found that neither SYK nor SLP65 can be phosphorylated in response to pre–B cell receptor engagement. Instead, Brutons tyrosine kinase (BTK) is constitutively phosphorylated by BCR-ABL1. Activated BTK is essential for survival signals that otherwise would arise from the pre–B cell receptor, including activation of PLCγ1, autonomous Ca2+ signaling, STAT5-phosphorylation, and up-regulation of BCLX L. Inhibition of BTK activity specifically induces apoptosis in BCR-ABL1 + leukemia cells to a similar extent as inhibition of BCR-ABL1 kinase activity itself. However, BCR-ABL1 cannot directly bind to full-length BTK. Instead, BCR-ABL1 induces the expression of a truncated splice variant of BTK that acts as a linker between the two kinases. As opposed to full-length BTK, truncated BTK lacks kinase activity yet can bind to BCR-ABL1 through its SRC-homology domain 3. Acting as a linker, truncated BTK enables BCR-ABL1–dependent activation of full-length BTK, which initiates downstream survival signals and mimics a constitutively active pre–B cell receptor.