An activating mutation of GNB1 is associated with resistance to tyrosine kinase inhibitors in ETV6-ABL1-positive leukemia.

Leukemias harboring the ETV6-ABL1 fusion represent a rare subset of hematological malignancies with unfavorable outcomes. The constitutively active chimeric Etv6-Abl1 tyrosine kinase can be specifically inhibited by tyrosine kinase inhibitors (TKIs). Although TKIs represent an important therapeutic tool, so far, the mechanism underlying the potential TKI resistance in ETV6-ABL1-positive malignancies has not been studied in detail. To address this issue, we established a TKI-resistant ETV6-ABL1-positive leukemic cell line through long-term exposure to imatinib. ETV6-ABL1-dependent mechanisms (including fusion gene/protein mutation, amplification, enhanced expression or phosphorylation) and increased TKI efflux were excluded as potential causes of resistance. We showed that TKI effectively inhibited the Etv6-Abl1 kinase activity in resistant cells, and using short hairpin RNA (shRNA)-mediated silencing, we confirmed that the resistant cells became independent from the ETV6-ABL1 oncogene. Through analysis of the genomic and proteomic profiles of resistant cells, we identified an acquired mutation in the GNB1 gene, K89M, as the most likely cause of the resistance. We showed that cells harboring mutated GNB1 were capable of restoring signaling through the phosphoinositide-3-kinase (PI3K)/Akt/mTOR and mitogen-activated protein kinase (MAPK) pathways, whose activation is inhibited by TKI. This alternative GNB1K89M-mediated pro-survival signaling rendered ETV6-ABL1-positive leukemic cells resistant to TKI therapy. The mechanism of TKI resistance is independent of the targeted chimeric kinase and thus is potentially relevant not only to ETV6-ABL1-positive leukemias but also to a wider spectrum of malignancies treated by kinase inhibitors.

Efficacy and safety of human mesenchymal stromal cells in healing of critical-size bone defects in immunodeficient rats.

To evaluate the preclinical efficacy and safety of human mesenchymal stem cells (hMSC) rapidly expanded in growth medium for clinical use with human serum and recombinant growth factors, we conducted a controlled, randomized trial of plasma clots with hMSC vs. plasma clots only in critical segmental femoral defects in rnu/rnu immunodeficient rats. X-ray, microCT and histomorphometrical evaluation were performed at 8 and 16 weeks. MSC were obtained from healthy volunteers and patients with lymphoid malignancy. Human MSC survived in the defect for the entire duration of the trial. MSC from healthy volunteers, in contrast to hMSC from cancer patients, significantly improved bone healing at 8, but not 16 weeks. However, at 16 weeks, hMSC significantly improved vasculogenesis in residual defect. We conclude that hMSC from healthy donors significantly contributed to the healing of bone defects at 8 weeks and to the vascularisation of residual connective tissue for up to 16 weeks. We found the administration of hMSC to be safe, as no adverse reaction to human cells at the site of implantation and no evidence of migration of hMSC to distant organs was detected.

Flow cytometric immunobead assay for fast and easy detection of PML-RARA fusion proteins for the diagnosis of acute promyelocytic leukemia.

The PML-RARA fusion protein is found in approximately 97% of patients with acute promyelocytic leukemia (APL). APL can be associated with life-threatening bleeding complications when undiagnosed and not treated expeditiously. The PML-RARA fusion protein arrests maturation of myeloid cells at the promyelocytic stage, leading to the accumulation of neoplastic promyelocytes. Complete remission can be obtained by treatment with all-trans-retinoic acid (ATRA) in combination with chemotherapy. Diagnosis of APL is based on the detection of t(15;17) by karyotyping, fluorescence in situ hybridization or PCR. These techniques are laborious and demand specialized laboratories. We developed a fast (performed within 4-5 h) and sensitive (detection of at least 10% malignant cells in normal background) flow cytometric immunobead assay for the detection of PML-RARA fusion proteins in cell lysates using a bead-bound anti-RARA capture antibody and a phycoerythrin-conjugated anti-PML detection antibody. Testing of 163 newly diagnosed patients (including 46 APL cases) with the PML-RARA immunobead assay showed full concordance with the PML-RARA PCR results. As the applied antibodies recognize outer domains of the fusion protein, the assay appeared to work independently of the PML gene break point region. Importantly, the assay can be used in parallel with routine immunophenotyping for fast and easy diagnosis of APL.