Milena Pantic

A highly selective telomerase inhibitor limiting human cancer cell proliferation

Telomerase, the ribonucleoprotein enzyme maintaining the telomeres of eukaryotic chromosomes, is active in most human cancers and in germline cells but, with few exceptions, not in normal human somatic tissues. Telomere maintenance is essential to the replicative potential of malignant cells and the inhibition of telomerase can lead to telomere shortening and cessation of unrestrained proliferation. We describe novel chemical compounds which selectively inhibit telomerase in vitro and in vivo. Treatment of cancer cells with these inhibitors leads to progressive telomere shortening, with no acute cytotoxicity, but a proliferation arrest after a characteristic lag period with hallmarks of senescence, including morphological, mitotic and chromosomal aberrations and altered patterns of gene expression. Telomerase inhibition and telomere shortening also result in a marked reduction of the tumorigenic potential of drug‐treated tumour cells in a mouse xenograft model. This model was also used to demonstrate in vivo efficacy with no adverse side effects and uncomplicated oral administration of the inhibitor. These findings indicate that potent and selective, non‐nucleosidic telomerase inhibitors can be designed as novel cancer treatment modalities.

Telomere maintenance in human B lymphocytes

Summary. Telomere shortening has been causally linked to replicative senescence in human cells. To characterize telomere‐length heterogeneity in peripheral blood cells of normal individuals, we analysed the mean length of telomeric repeat sequences in subpopulations of peripheral blood leucocytes, using fluorescence in situ hybridization and flow cytometry (flow‐FISH). Although the telomere length of most haematopoietic subsets was within the same range, the mean telomere length was found to be 15% higher in B compared with T lymphocytes in adult peripheral blood. Whereas telomere loss with ageing corresponded to 33 base pairs (bp) per year in T cells, telomere shortening was slower in B cells, corresponding to 15 bp per year. Separation of adult B‐lymphocyte subpopulations based on CD27 expression revealed that telomere length was almost 2 kb longer in CD19+CD27+ (memory) compared with CD19+CD27– (naive) cells. Furthermore, peripheral blood B cells were activated in vitro. Whereas B‐cell activation with Staphylococcus aureus Cowan strain (SAC) did not increase telomere length, a striking telomere elongation was observed when cells were stimulated with SAC and interleukin 2 to induce plasma cell differentiation. Our observations support the concept that telomere dynamics in B cells are distinct from other haematopoietic cell lineages and that telomere elongation may play an essential role in the generation of long‐term B memory cells.

BRAF inhibitor-associated ERK activation drives development of chronic lymphocytic leukemia.

Patients with BRAFV600E/K-driven melanoma respond to the BRAF inhibitor vemurafenib due to subsequent deactivation of the proliferative RAS/RAF/MEK/ERK pathway. In BRAF WT cells and those with mutations that activate or result in high levels of the BRAF activator RAS, BRAF inhibition can lead to ERK activation, resulting in tumorigenic transformation. We describe a patient with malignant melanoma who developed chronic lymphocytic leukemia (CLL) in the absence of RAS mutations during vemurafenib treatment. BRAF inhibition promoted patient CLL proliferation in culture and in murine xenografts and activated MEK/ERK in primary CLL cells from additional patients. BRAF inhibitor-driven ERK activity and CLL proliferation required B cell antigen receptor (BCR) activation, as inhibition of the BCR-proximal spleen tyrosine kinase (SYK) reversed ERK hyperactivation and proliferation of CLL cells from multiple patients, while inhibition of the BCR-distal Bruton tyrosine kinase had no effect. Additionally, the RAS-GTP/RAS ratio in primary CLL cells exposed to vemurafenib was reduced upon SYK inhibition. BRAF inhibition increased mortality and CLL expansion in mice harboring CLL xenografts; however, SYK or MEK inhibition prevented CLL proliferation and increased animal survival. Together, these results suggest that BRAF inhibitors promote B cell malignancies in the absence of obvious mutations in RAS or other receptor tyrosine kinases and provide a rationale for combined BRAF/MEK or BRAF/SYK inhibition.

Genome-wide Profiling in AML Patients Relapsing after Allogeneic Hematopoietic Cell Transplantation

Molecular pathogenesis of relapse after allogeneic hematopoietic cell transplantation is poorly understood. Data regarding relapse mechanisms after transplantation is scarcely available. We investigated genomic aberrations (GAs) in 21 patients undergoing related and unrelated HLA-matched transplantation in leukemic blasts before transplant and at relapse after transplantation. We found a higher number of GAs after transplantation, suggesting increased genomic instability during relapse. Two of 21 patients showed a large homozygous region spanning the whole HLA-locus on chromosome 6p in the relapse sample. In both patients sequence-based HLA typing of the blasts revealed a loss of the patient-specific allele at the mismatched locus leading to homozygosity for the HLA haplotype shared by the patient and the donor. In addition, GAs were found in critical regions such as 12p13, 13q12.2, and 17p13. Our results suggest that escape from immunologic surveillance may be a relevant mechanism of relapse after transplantation in patients with GAs on chromosome 6p. A combination of continuous immunologic pressure mediated by donor T cells and clonal evolution of myeloid leukemia may result in acquired GAs after transplantation.

Proteomic profiling in distinct cellular compartments of tumor cells reveals p53-dependent upregulation of S100A6 upon induction of telomere dysfunction

Telomere dysfunction is evoking a DNA damage response which leads to replicative senescence or apoptosis. Tumor cells feature telomerase, a ribonucleoprotein complex counteracting telomere shortening and proliferation limitation as a prerequisite of immortal cell growth. Recently, we demonstrated the effects of telomerase inhibition on the proteome of tumor cell clones in whole cell lysates by SELDI‐TOF‐MS profiling and MS/MS protein identification (Zimmermann et al., Proteomics 2009, 9, 521–534). We continued proteomic analyses of such clones after telomerase‐inhibition using fractionation of cellular compartments. Among the differentially expressed peaks found in different fractions, a cytoplasmic 10.1 kDa protein upregulated in telomerase‐inhibited clones (p<0.0001) was identified by nanoflow‐HPLC‐MS/MS as S100A6. S100A6 upregulation was confirmed by immunoblotting in telomerase‐inhibited HCT‐116, A‐549, and NCI‐H460 clones. S100A6 and other proteins involved in telomere dysfunction were further analyzed in derivative p53−/− and p21−/− HCT‐116 cell lines indicating an overall reduced number of significant changes in these lines compared to wild type HCT‐116 cells. In addition, post‐translational modification of S100A6 was demonstrated with a potential role in mediating the cellular response to telomere dysfunction. In conclusion, proteomic profiling in distinct cellular compartments led to the identification of a novel p53‐dependent biomarker of telomere dysfunction, S100A6.

Proteomic profiling of tumor cells after induction of telomere dysfunction

Cell division in the absence of telomerase causes progressive telomere shortening which ultimately leads to telomere dysfunction and initiation of genome instability. In order to identify factors related to loss of telomere function, the effects of telomerase inhibition on the proteome of five tumor cell lines were followed by SELDI‐TOF‐MS. Five differentially expressed protein peaks (p<0.01) were found in a total of 60 clones of five cell lines representing four tissues (lung, breast, prostate, and colon) in which telomerase was inhibited by retroviral overexpression of a dominant negative (DN) mutant of human telomerase reverse transcriptase (hTERT). Among these, a 11.3 kDa peak diminished in DN‐hTERT clones was identified as histone H4 by nanoflow‐HPLC‐MS/MS. Immunoblot analysis not only confirmed the decline of histone H4, but also of other core histone proteins including histone H3. Furthermore, upregulation of several cytokeratins was found to be associated with telomere attrition. In conclusion, loss of telomere function is associated with alterations in the proteome which may represent novel biomarkers for the detection of replicative senescence.

B-cell signaling in persistent polyclonal B lymphocytosis (PPBL).

Persistent polyclonal B lymphocytosis (PPBL) is a benign hematological disorder characterized by a selective expansion of circulating polyclonal marginal zone (MZ)‐like B cells. Previous reports demonstrated that cases of PPBL showed poor activation, proliferation and survival of B cells in vitro, yet the underlying defect remains unknown. Here we report for the first time an attenuated activation of the canonical NF‐κB (nuclear factor of kappa light polypeptide gene enhancer in B cells) and mitogen‐activated protein kinase/extracellular signal‐regulated kinase pathway after CD40 stimulation. This defect was selective, as alternative NF‐κB signaling after CD40 stimulation and both B‐cell receptor‐ and Toll‐like receptor 9‐mediated activation remained unaffected. Reduced canonical NF‐κB activation resulted in decreased IκBα and CD40 expression in resting cells. In PPBL patients, expression of Bcl‐xL in MZ‐like B cells did not increase upon activation, consistent with the high apoptosis rates of PPBL‐derived B cells that were observed in vitro. The B‐cell phenotype of mice with selective knockouts of early components of the CD40 signaling pathway resembles PPBL, but sequencing corresponding genes in sorted MZ‐like B cells of PPBL patients did not reveal relevant genetic alterations. Nevertheless, the frequently observed mutations in early signaling components of the NF‐κB pathway in MZ lymphomas underline the relevance of our findings for the pathogenesis of PPBL.

Recurrence of persistent polyclonal B lymphocytosis (PPBL) after rituximab treatment

Dear Editor, Persistent polyclonal B lymphocytosis (PPBL) is a rare entity with a polyclonal increase of marginal zone-like B lymphocytes in the peripheral blood [1, 2], most prevalent in smoking middle-aged women [3]. Patients display elevated immunoglobulin M (IgM) and sometimes decreased IgG levels in the serum [4–6]. Splenomegaly and/or polyclonal lymphocytic infiltration of the bone marrow may be present [7, 8]. The disease has been associated with HLA-DR7 positivity and chromosomal abnormalities [6], but its etiology remains elusive. Herein, we report its recurrence after rituximab treatment, revealing interesting aspects of its putative pathophysiology. A 44-year-old Caucasian womanwith a history of Sudeck’s dystrophy and hysterectomy presented with epigastric pain. The patient was an active smoker. Awork-up revealed splenomegaly (3.6×16.5 cm) in the absence of enlarged lymph nodes. A differential blood count showed normal leukocyte count (9440/μl) with lymphocytosis (4863/μl, normal 1000– 2800/μl), and blood smear showed atypical, agranular large lymphocytes with bilobated nuclei (Fig. 1a). IgM was elevated (3.18 g/l, normal 0.4–2.3 g/l, Fig. 1b) without monoclonal bands in immunofixation, and IgG was reduced (4.09 g/l, normal 7–16 g/l). A bone marrow biopsy showed normal hematopoiesis and mild lymphocytosis without blasts or light chain restriction. Relative lymphocytosis in the peripheral blood was traced back to a B lymphocytosis (3457/μl, normal 100–500/μl, Fig. 1c) with no detectablemonoclonality in flow cytometry and IgH clonality analysis (Fig. 1d). The patient was HLA-DR7 negative. Albeit a watch-and-wait strategy is generally indicated in this situation, the patient was treated four times with 375 mg/ m rituximab resulting in the normalization of her spleen size (3.7×10.8 cm) and IgM level (2.1 g/l), as well as an eradication of her peripheral blood B lymphocytes (0/μl). Eighteen months after rituximab treatment, her B cell counts were back to normal (307/μl) and a mild polyclonal IgM elevation had returned (2.47 g/l). B cell numbers continued to rise over the next months (Fig. 1c), lymphocytes with bilobated nuclei reappeared, and B cell subpopulation analysis 2 years after the rituximab treatment showed an expansion of marginal zone-like B lymphocytes in the peripheral blood (52.1 % of B cells, normal <30.8 %) indicating recurrence of PPBL. However, her spleen size was still normal. FISH analysis revealed one or two 3q gains in 3.5 % of B cells, respectively (Fig. 1e, cutoffs for non-B cells 0.5–1.0 %). The recurrence of the PPBL phenotype after eradication of the recirculating B cell pool is remarkable and gives rise to various speculations on the etiology of PPBL. Given the high cellular burden in PPBL, an insufficient eradication and re-expansion of affected B cells in peripheral tissues is a possible explanation; however, the eradication from peripheral blood was complete. Alternatively, as CD20 expression is C. Wehr : L. Houet : S. Gutenberger :K. Warnatz (*) Center for Chronic Immunodeficiency (CCI), University Medical Center Freiburg, University of Freiburg, Breisacherstrasse 117, 79106 Freiburg, Germany e-mail: klaus.warnatz@uniklinik-freiburg.de

Diagnostik und moderne Behandlungsstrategien

ZusammenfassungIn den letzten 20 Jahren hat sich die Lebenserwartung von Patienten mit multiplem Myelom aufgrund neuer Diagnosemöglichkeiten und moderner Therapieoptionen stetig verbessert. Für die Wahl der optimalen Behandlungsstrategie müssen sowohl krankheitsspezifische Faktoren als auch das individuelle Risikoprofil des Patienten, sein Alter und seine Komorbiditäten berücksichtigt werden.