Catrin Roolf

Inbred mouse strains reveal biomarkers that are pro-longevity, antilongevity or role switching

Traditionally, biomarkers of aging are classified as either pro‐longevity or antilongevity. Using longitudinal data sets from the large‐scale inbred mouse strain study at the Jackson Laboratory Nathan Shock Center, we describe a protocol to identify two kinds of biomarkers: those with prognostic implication for lifespan and those with longitudinal evidence. Our protocol also identifies biomarkers for which, at first sight, there is conflicting evidence. Conflict resolution is possible by postulating a role switch. In these cases, high biomarker values are, for example, antilongevity in early life and pro‐longevity in later life. Role‐switching biomarkers correspond to features that must, for example, be minimized early, but maximized later, for optimal longevity. The clear‐cut pro‐longevity biomarkers we found reflect anti‐inflammatory, anti‐immunosenescent or anti‐anaemic mechanisms, whereas clear‐cut antilongevity biomarkers reflect inflammatory mechanisms. Many highly significant blood biomarkers relate to immune system features, indicating a shift from adaptive to innate processes, whereas most role‐switching biomarkers relate to blood serum features and whole‐body phenotypes. Our biomarker classification approach is applicable to any combination of longitudinal studies with life expectancy data, and it provides insights beyond a simplified scheme of biomarkers for long or short lifespan.

Phosphoproteome Analysis Reveals Differential Mode of Action of Sorafenib in Wildtype and Mutated FLT3 Acute Myeloid Leukemia (AML) Cells

Constitutively activating internal tandem duplication (ITD) alterations of the receptor tyrosine kinase FLT3 (Fms-like tyrosine kinase 3) are common in acute myeloid leukemia (AML) and classifies FLT3 as an attractive therapeutic target. So far, applications of FLT3 small molecule inhibitors have been investigated primarily in FLT3-ITD+ patients. Only recently, a prolonged event-free survival has been observed in AML patients who were treated with the multikinase inhibitor sorafenib in addition to standard therapy. Here, we studied the sorafenib effect on proliferation in a panel of 13 FLT3-ITD− and FLT3-ITD+ AML cell lines. Sorafenib IC50 values ranged from 0.001 to 5.6 μm, whereas FLT3-ITD+ cells (MOLM-13, MV4-11) were found to be more sensitive to sorafenib than FLT3-ITD− cells. However, we identified two FLT3-ITD− cell lines (MONO-MAC-1 and OCI-AML-2) which were also sorafenib sensitive. Phosphoproteome analyses revealed that the affected pathways differed in sorafenib sensitive FLT3-ITD− and FLT3-ITD+ cells. In MV4-11 cells sorafenib suppressed mTOR signaling by direct inhibition of FLT3. In MONO-MAC-1 cells sorafenib inhibited the MEK/ERK pathway. These data suggest that the FLT3 status in AML patients might not be the only factor predicting response to treatment with sorafenib.

Polymorphisms of the murine mitochondrial ND4 , CYTB and COX3 genes impact hematopoiesis during aging

During aging, mitochondrial DNA (mtDNA) can accumulate mutations leading to increasing levels of reactive oxygen species (ROS). Increased ROS were described to activate formerly quiescent hematopoietic stem cells (HSC). Mutations in mtDNA were shown to enhance the risk for myelodysplastic syndrome and leukemia. However, the complex relationship between mtDNA variations, ROS and aging of the hematopoietic system is not fully understood. Herein, three mouse strains with mtDNA polymorphisms in genes of respiratory chain complexes I (ND4), III (CYTB) and IV (COX3) were compared to a reference strain during aging. Analysis focused on ROS and ATP levels, bone marrow composition and blood counts. Additionally, hematopoietic restoration capacity following cytotoxic stress was tested. Mice with polymorphisms in ND4 and CYTB gene had significantly decreasing ROS levels in bone marrow cells during aging, without effecting ATP levels. In addition, the frequency of stem and progenitor cells increased during aging but the amount of lymphocytes in the peripheral blood decreased during aging. In summary, the presence of mtDNA polymorphisms affecting the respiratory chain complexes I, III and IV was associated with altered ROS levels as well as changes in BM and peripheral blood composition during aging.

Characterization of the novel indolylmaleimides’ PDA-66 and PDA-377 effect on canine lymphoma cells

Protein kinase inhibitors are widely used in chemotherapeutic cancer regimens. Maleimide derivatives such as SB-216763 act as GSK-3 inhibitor targeting cell proliferation, cell death and cell cycle progression. Herein, the two arylindolylmaleimide derivatives PDA-66 and PDA-377 were evaluated as potential chemotherapeutic agents on canine B-cell lymphoma cell lines. Canine lymphoma represents a naturally occurring model closely resembling the human high-grade non-Hodgkins lymphoma (NHL). PDA-66 showed more pronounced effects on both cell lines. Application of 2.5μM PDA-66 resulted in a significant induction of apoptosis (approx. 11 %), decrease of the metabolic activity (approx. 95 %), anti-proliferative effect (approx. 85 %) and cell death within 48h. Agent induced mode of action was characterized by whole transcriptome sequencing, 12 h and 24 h post-agent exposure. Key PDA-66-modulated pathways identified were cell cycle, DNA replication and p53 signaling. Expression analyses indicated that the drug acting mechanism is mediated through DNA replication and cycle arrest involving the spindle assembly checkpoint. In conclusion, both PDA derivatives displayed strong anti-proliferation activity in canine B-cell lymphoma cells. The cell and molecular PDA-induced effect characterization and the molecular characterization of the agent acting mechanism provides the basis for further evaluation of a potential drug for canine lymphoma serving as model for human NHL.

Frequent and reliable engraftment of certain adult primary acute lymphoblastic leukemias in mice

Birgitta Christine Heckl , Michela Carlet , Binje Vick, Catrin Roolf, Ameera Alsadeq, Michaela Grunert, Wen-Hsin Liu, Andrea Liebl, Wolfgang Hiddemann, Rolf Marschalek, Denis Martin Schewe , Karsten Spiekermann, Christian Junghanss and Irmela Jeremias Research Unit Apoptosis in Hematopoietic Stem Cells (AHS), Helmholtz Center Munich, German Research Center for Environmental Health, Munich, Germany; German Cancer Consortium (DKTK), Partner Site, Munich, Germany; Department of Medicine III – Hematology, Oncology and Palliative Care, Rostock University Medical Center, Rostock, Germany; Pediatric Hematology/Oncology, ALL-BFM Study Group, Christian Albrechts University Kiel and University Hospital Schleswig-Holstein, Kiel, Germany; Department of Medicine III, University Hospital, LMU, Munich, Germany; Institute of Pharmaceutical Biology, Goethe University, Frankfurt, Germany; Department of Pediatrics, Dr. von Hauner Children’s Hospital, Ludwig Maximilians University (LMU), Munich, Germany

Decitabine demonstrates antileukemic activity in B cell precursor acute lymphoblastic leukemia with MLL rearrangements

BackgroundPromotor hypermethylation of CpG islands is common in B cell precursor acute lymphoblastic leukemia (BCP-ALL) with mixed lineage leukemia (MLL) gene rearrangements. Hypomethylating agents (HMA) such as azacitidine (AZA) and decitabine (DEC) reduce DNA hypermethylation by incorporation into DNA and were successfully introduced into the clinic for the treatment of myeloid neoplasias.MethodsHere, we investigated whether HMA induce comparable biological effects in MLL-positive BCP-ALL. Further, efficacy of HMA and concomitant application of cytostatic drugs (cytarabine and doxorubicin) were evaluated on established SEM and RS4;11 cell lines. In addition, promising approaches were studied on BCP-ALL cell line- and patient-derived xenograft models.ResultsIn general, DEC effects were stronger compared to AZA on MLL-positive BCP-ALL cells. DEC significantly reduced proliferation by induction of cell cycle arrest in G0/G1 phase and apoptosis. Most sensitive to HMA were SEM cells which are characterized by a fast cell doubling time. The combination of low-dose HMA and conventional cytostatic agents revealed a heterogeneous response pattern. The strongest antiproliferative effects were observed when ALL cells were simultaneously exposed to HMA and cytostatic drugs. Most potent synergistic effects of HMA were induced with cytarabine. Finally, the therapeutic potential of DEC was evaluated on BCP-ALL xenograft models. DEC significantly delayed leukemic proliferation in xenograft models as demonstrated longitudinally by non-invasive bioluminescence as well as 18F-FDG-PET/CT imaging. Unexpectedly, in vivo concomitant application of DEC and cytarabine did not enhance the antiproliferative effect compared to DEC monotherapy.ConclusionsOur data reveal that DEC is active in MLL-positive BCP-ALL and warrant clinical evaluation.