The DNA Methyltransferase 1 (DNMT1) Controls the Shape and Dynamics of Migrating POA-Derived Interneurons Fated for the Murine Cerebral Cortex.

The proliferative niches in the subpallium generate a rich cellular variety fated for diverse telencephalic regions. The embryonic preoptic area (POA) represents one of these domains giving rise to the pool of cortical GABAergic interneurons and glial cells, in addition to striatal and residual POA cells. The migration from sites of origin within the subpallium to the distant targets like the cerebral cortex, accomplished by the adoption and maintenance of a particular migratory morphology, is a critical step during interneuron development. To identify factors orchestrating this process, we performed single-cell transcriptome analysis and detected Dnmt1 expression in murine migratory GABAergic POA-derived cells. Deletion of Dnmt1 in postmitotic immature cells of the POA caused defective migration and severely diminished adult cortical interneuron numbers. We found that DNA methyltransferase 1 (DNMT1) preserves the migratory shape in part through negative regulation of Pak6, which stimulates neuritogenesis at postmigratory stages. Our data underline the importance of DNMT1 for the migration of POA-derived cells including cortical interneurons.

Modulation of actin dynamics as potential macrophage subtype-targeting anti-tumour strategy.

Tumour-associated macrophages mainly comprise immunosuppressive M2 phenotypes that promote tumour progression besides anti-tumoural M1 subsets. Selective depletion or reprogramming of M2 may represent an innovative anti-cancer strategy. The actin cytoskeleton is central for cellular homeostasis and is targeted for anti-cancer chemotherapy. Here, we show that targeting G-actin nucleation using chondramide A (ChA) predominantly depletes human M2 while promoting the tumour-suppressive M1 phenotype. ChA reduced the viability of M2, with minor effects on M1, but increased tumour necrosis factor (TNF)α release from M1. Interestingly, ChA caused rapid disruption of dynamic F-actin filaments and polymerization of G-actin, followed by reduction of cell size, binucleation and cell division, without cellular collapse. In M1, but not in M2, ChA caused marked activation of SAPK/JNK and NFκB, with slight or no effects on Akt, STAT-1/-3, ERK-1/2, and p38 MAPK, seemingly accounting for the better survival of M1 and TNFα secretion. In a microfluidically-supported human tumour biochip model, circulating ChA-treated M1 markedly reduced tumour cell viability through enhanced release of TNFα. Together, ChA may cause an anti-tumoural microenvironment by depletion of M2 and activation of M1, suggesting induction of G-actin nucleation as potential strategy to target tumour-associated macrophages in addition to neoplastic cells.

BCR-ABL- and Ras-independent activation of Raf as a novel mechanism of Imatinib resistance in CML.

Although the BCR-ABL tyrosine kinase inhibitor Imatinib has undoubtedly revolutionized the therapy of chronic myeloid leukaemia (CML), acquired drug resistance remains a common problem in CML therapy. Resistance often arises from second-line mutations in BCR-ABL or overexpression of the BCR-ABL protein but in ~20% of CML cases resistance mechanisms do not involve altered BCR-ABL function. Imatinib-resistant CML cell lines have been widely used for comparative proteome/genome-wide expression screens in order to decipher resistance mechanisms but a clearcut molecular mechanism or molecular player in BCR-ABL-independent resistance to Imatinib has not yet evolved from those studies. Here, we report the identification of a novel mechanism for Imatinib resistance in CML cells with unaltered BCR-ABL function. Pharmacological analysis evidenced a constitutive, Imatinib-insensitive activation of the Erk-MAPK pathway in resistant cells. A systematic analysis of pathway constituents illustrated that Ras-GTP accumulation remained fully sensitive to Imatinib but c-Raf activity from serum-fed cultures was largely resistant to the drug's action. Sequencing excluded mutations in either B-Raf or c-Raf as the origin of resistance, indicating that a functional alteration in the regulation of c-Raf activity was responsible for this effect. Collectively, these findings highlight a novel mechanism of acquired Imatinib resistance based on the BCR-ABL and Ras-independent constitutive activation of the Erk-MAPK pathway through activated c-Raf, which could prove helpful for a better functional classification of the causes of Imatinib resistance in CML.

Cytogenetic characterisation and proteomic profiling of the Imatinib-resistant cell line KCL22-R.

Approximately 30% of chronic myeloid leukemia patients show initially no response to Imatinib, a potent inhibitor of BCR-ABL. This intrinsic resistance may be due to BCR-ABL-independent cell growth. Here we analysed the cytogenetic anomalies and the proteomic profiling in KCL22-S and KCL22-R, two Imatinib-sensitive and -resistant derivative cell lines of KCL22. A tetrasomy 8 and a non-reciprocal translocation +der(6)t(6;13)(p11.1;q12) were found only in KCL22-R as new evolved anomalies. Chromosome der(6)t(6;13) showed four variants differing in the chromatin content of 13q14-13qter including the retinoblastoma gene. Due to these sub-clones, approximately 65-79% of the Imatinib-treated KCL22-R cells showed a disomy and 21-35% a monosomy for 13q14. Imatinib removal reduced the main clone to approximately 20% in the benefit of the monosomic sub-clones. This was accompanied by an increased apoptosis rate and was revertible by Imatinib re-treatment. This effect may be connected with genes located in 13q14-qter. Proteomic profiling of the cell lines performed with ProteinChip technology (SELDI) revealed several differentially expressed proteins (n=45). In summary, we demonstrate here the complex changes on the cytogenetic and proteomic level which could be caused by Imatinib and the resistance resulting from it.