Hyper-activation of Aurora kinase a-polo-like kinase 1-FOXM1 axis promotes chronic myeloid leukemia resistance to tyrosine kinase inhibitors.

BACKGROUND: Chronic myeloid leukemia (CML) is a myeloproliferative disease caused by the constitutive tyrosine kinase (TK) activity of the BCR-ABL1 fusion protein. Accordingly, TK inhibitors have drastically changed the disease prognosis. However, persistence of the transformed hematopoiesis even in patients who achieved a complete response to TK inhibitors and the disease relapse upon therapy discontinuation represent a major obstacle to CML cure. METHODS: Thiostrepton, Danusertib and Volasertib were used to investigate the effects of FOXM1, AKA and Plk1 inhibition in K562-S and K562-R cells. Apoptotic cell death was quantified by annexin V/propidium iodide staining and flow cytometry. Quantitative reverse transcription (RT)-PCR was used to assess BCR-ABL1, FOXM1, PLK1 and AURKA expression. Protein expression and activation was assessed by Western Blotting (WB). Clonogenic assay were performed to confirm K562-R resistance to Imatinib and to evaluate cells sensitivity to the different drugs. RESULTS: Here we proved that BCR-ABL1 TK-dependent hyper-activation of Aurora kinase A (AURKA)-Polo-like kinase 1 (PLK1)-FOXM1 axis is associated with the outcome of Imatinib (IM) resistance in an experimental model (K562 cell line) and bone marrow hematopoietic cells. Notably, such a biomolecular trait was detected in the putative leukemic stem cell (LSC) compartment characterized by a CD34+ phenotype. Constitutive phosphorylation of FOXM1 associated with BCR-ABL1 TK lets FOXM1 binding with ß-catenin enables ß-catenin nuclear import and recruitment to T cell factor/lymphoid enhancer-binding factor (TCF/LEF) transcription complex, hence supporting leukemic cell proliferation and survival. Lastly, the inhibition of single components of AURKA-PLK1-FOXM1 axis in response to specific drugs raises the expression of growth factor/DNA damage-inducible gene a (GADD45a), a strong inhibitor of AURKA and, as so, a critical component whose induction may mediate the eradication of leukemic clone. CONCLUSIONS: Our conclusion is that AURKA, PLK1 and FOXM1 inhibition may be considered as a promising therapeutic approach to cure CML.

SETD2 and histone H3 lysine 36 methylation deficiency in advanced systemic mastocytosis.

The molecular basis of advanced systemic mastocytosis (SM) is not fully understood and despite novel therapies the prognosis remains dismal. Exome sequencing of an index-patient with mast cell leukemia (MCL) uncovered biallelic loss-of-function mutations in the SETD2 histone methyltransferase gene. Copy-neutral loss-of-heterozygosity at 3p21.3 (where SETD2 maps) was subsequently found in SM patients and prompted us to undertake an in-depth analysis of SETD2 copy number, mutation status, transcript expression and methylation levels, as well as functional studies in the HMC-1 cell line and in a validation cohort of 57 additional cases with SM, including MCL, aggressive SM and indolent SM. Reduced or no SETD2 protein expression-and consequently, H3K36 trimethylation-was found in all cases and inversely correlated with disease aggressiveness. Proteasome inhibition rescued SETD2 expression and H3K36 trimethylation and resulted in marked accumulation of ubiquitinated SETD2 in SETD2-deficient patients but not in patients with near-normal SETD2 expression. Bortezomib and, to a lesser extent, AZD1775 alone or in combination with midostaurin induced apoptosis and reduced clonogenic growth of HMC-1 cells and of neoplastic mast cells from advanced SM patients. Our findings may have implications for prognostication of SM patients and for the development of improved treatment approaches in advanced SM.

Lipid content in higher plants under osmotic stress.

In this work, we performed investigations on the lipid content of higher plants (spinach) under hyperosmotic stress, by means of thin layer chromatography (TLC) and mass spectrometry. In particular, the experiments have been performed at different plant organization levels: whole leaves, freshly prepared protoplast suspension and mesophyll cells obtained by reformation of the cell wall from protoplast suspension. The results obtained showed that hyperosmotic stress induces changes in the phospholipid content depending on the different plant organization levels studied. All phospholipids showed an increment of their content in stressed whole leaves. In particular, phosphatidylglycerol (PG) redoubles its content by 1 h of osmotic shock. Different responses to hyperosmotic stress were reported for the other systems. In the case of protoplasts, an increment of PG, phosphatidylcholine (PC) and phosphatidylinositol (PI) together with biphosphatidylglycerol (BPG) and phosphatidylethanolamine (PE) content decreasing were observed in stressed sample. For PG, identified as PG (34:4) by elecrospray ionization mass spectrometry, the increment was of about 30%. In the case of cells, conversely, a decrease of PG content under osmotic stress was recorded. The results suggest an important role of phospholipids, in particular of PG, in the osmotic stress response.