De novo UBE2A mutations are recurrently acquired during chronic myeloid leukemia progression and interfere with myeloid differentiation pathways

Despite the advent of tyrosine kinase inhibitors, a proportion of chronic myeloid leukemia patients in chronic phase fails to respond to Imatinib or to second generation inhibitors and progress to blast crisis. Limited improvements in the understanding of the molecular mechanisms responsible for chronic myeloid leukemia transformation from chronic phase to the aggressive blast crisis were achieved until now. We present here a massive parallel sequencing analysis of 10 blast crisis samples and of the corresponding autologous chronic phase controls which reveals, for the first time, recurrent mutations affecting the ubiquitin-conjugating enzyme E2A gene (UBE2A, formerly RAD6A). Additional analyses on a cohort of 24 blast crisis, 41 chronic phase as well as 40 acute myeloid leukemia and 38 atypical chronic myeloid leukemia patients at onset confirmed that UBE2A mutations are specifically acquired during chronic myeloid leukemia progression with a frequency of 16.7% in advanced phases. In vitro studies show that the mutations here described cause a decrease in UBE2A activity, leading to an impairment of myeloid differentiation in chronic myeloid leukemia cells

ERG Deregulation Induces PIM1 Over-Expression and Aneuploidy in Prostate Epithelial Cells

The ERG gene belongs to the ETS family of transcription factors and has been found to be involved in atypical chromosomal rearrangements in several cancers. To gain insight into the oncogenic activity of ERG, we compared the gene expression profile of NIH-3T3 cells stably expressing the coding regions of the three main ERG oncogenic fusions: TMPRSS2/ERG (tERG), EWS/ERG and FUS/ERG. We found that all three ERG fusions significantly up-regulate PIM1 expression in the NIH-3T3 cell line. PIM1 is a serine/threonine kinase frequently over-expressed in cancers of haematological and epithelial origin. We show here that tERG expression induces PIM1 in the non-malignant prostate cell line RWPE-1, strengthening the relation between tERG and PIM1 up-regulation in the initial stages of prostate carcinogenesis. Silencing of tERG reversed PIM1 induction. A significant association between ERG and PIM1 expression in clinical prostate carcinoma specimens was found, suggesting that such a mechanism may be relevant in vivo. Chromatin Immunoprecipitation experiments showed that tERG directly binds to PIM1 promoter in the RWPE-1 prostate cell line, suggesting that tERG could be a direct regulator of PIM1 expression. The up-regulation of PIM1 induced by tERG over-expression significantly modified Cyclin B1 levels and increased the percentage of aneuploid cells in the RWPE-1 cell line after taxane-based treatment. Here we provide the first evidence for an ERG-mediated PIM1 up-regulation in prostate cells in vitro and in vivo, suggesting a direct effect of ERG transcriptional activity in the alteration of genetic stability

Design, Synthesis, and Biological Activity of Urea Derivatives as Anaplastic Lymphoma Kinase Inhibitors

In anaplastic large-cell lymphomas, chromosomal translocations involving the kinase domain of anaplastic lymphoma kinase (ALK), generally fused to the 5' part of the nucleophosmin gene, produce highly oncogenic ALK fusion proteins that deregulate cell cycle, apoptosis, and differentiation in these cells. Other fusion oncoproteins involving ALK, such as echinoderm microtubule-associated protein-like 4-ALK, were recently found in patients with non-small-cell lung, breast, and colorectal cancers. Recent research has focused on the development of inhibitors for targeted therapy of these ALK-positive tumors. Because kinase inhibitors that target the inactive conformation are thought to be more specific than ATP-targeted inhibitors, we investigated the possibility of using two known inhibitors, doramapimod and sorafenib, which target inactive kinases, to design new urea derivatives as ALK inhibitors. We generated a homology model of ALK in its inactive conformation complexed with doramapimod or sorafenib in its active site. The results elucidated why doramapimod is a weak inhibitor and why sorafenib does not inhibit ALK. Virtual screening of commercially available compounds using the homology model of ALK yielded candidate inhibitors, which were tested using biochemical assays. Herein we present the design, synthesis, biological activity, and structure-activity relationships of a novel series of urea compounds as potent ALK inhibitors. Some compounds showed inhibition of purified ALK in the high nanomolar range and selective antiproliferative activity on ALK-positive cells

Concomitant BCORL1 and BRAF Mutations in Vemurafenib-Resistant Melanoma Cells

BRAF is the most frequently mutated gene in melanoma. Constitutive activation of mutant BRAFV600E leads to aberrant Ras-independent MAPK signaling and cell transformation. Inhibition of mutant BRAF is a current frontline therapy for such cases, with improved survival compared with chemotherapy. Unfortunately, reactivation of MAPK signaling by several mechanisms has been shown to cause drug resistance and disease recurrence. In this work, we describe the co-occurrence of an in-frame deletion within an amplified BRAFV600E locus and a missense point mutation of the transcriptional repressor BCORL1 in vemurafenib-resistant A375 melanoma cells. Functional data confirmed that truncated p47BRAFV600E and mutant BCORL1Q1076H both contribute to resistance. Interestingly, either endogenous BCORL1 silencing or ectopic BCORL1Q1076H expression mimicked the effects of a CRISPR/Cas9-edited BCORL1Q1076H locus, suggesting a complex mixture of loss- and gain-of-function effects caused by the mutation. Transcriptomic data confirmed this hypothesis. Finally, we show that the pan-RAF inhibitor sorafenib is not affected by expression of BRAF deletion variant and effectively synergizes with vemurafenib to block resistant cells, suggesting a possible intervention for this class of mutants

Discovery of Novel α-Carboline Inhibitors of the Anaplastic Lymphoma Kinase

[Image: see text] The anaplastic lymphoma kinase (ALK) is abnormally expressed and hyperactivated in a number of tumors and represents an ideal therapeutic target. Despite excellent clinical responses to ALK inhibition, drug resistance still represents an issue and novel compounds that overcome drug-resistant mutants are needed. We designed, synthesized, and evaluated a large series of azacarbazole inhibitors. Several lead compounds endowed with submicromolar potency were identified. Compound 149 showed selective inhibition of native and mutant drug-refractory ALK kinase in vitro as well as in a Ba/F3 model and in human ALK+ lymphoma cells. The three-dimensional (3D) structure of a 149:ALK-KD cocrystal is reported, showing extensive interaction through the hinge region and the catalytic lysine 1150

Sensitivity of RWPE-1_tERG cells to chemotherapeutic drugs.

<p><b>a</b>) Proliferative potential of tERG over-expressing and control (empty) cells measured by thymidine uptake assay up to 72 hrs growth in standard conditions. <b>b–c</b>) 72 hours treatment of RWPE-1 transfectants with taxol or idarubicin-hydrochloride. Proliferative potential was measured by thymidine uptake assay. <b>d–e</b>) Re-growth of RWPE-1 transfectants after 10 nM taxol (<b>d</b>) or 0.5 µM idarubcicin-hydrochloride (<b>e</b>): following 24-hrs treatment, drugs were removed and the cells allowed to grow for additional 72 hours. Proliferation of cells collected at the time of wash-out is set as 100%. * = p<0.05.</p

Validation of gene expression deregulation through quantitative Real-Time PCR in NIH-3T3 transfectants.

<p>Significant up- (↑) or down- (↓) regulation obtained by microarray analysis is shown alongside quantitative Real-Time PCR fold change values for a direct comparison. n = no change.</p

ERG dependent PIM1 induction in RWPE-1 cells.

<p><b>a–b</b>) PIM1 over-expression in RWPE-1 transfected with HA-tagged TMPRSS2/ERG coding region (RWPE-1_tERG) as evidenced by immunoblot (<b>a</b>) and Real-Time quantitative PCR (<b>b</b>). Actin is shown as a loading control. ERG and PIM1 fold changes were normalized against empty-vector cells. <b>c</b>) siRNA knockdown of ERG (siERG) in RWPE-1_tERG induces a decrease in PIM1 levels compared to non-targeting siRNA (siNT). <b>d</b>) Sequence of the 194 bp-long PIM1 promoter region amplified in ChIP analysis. The 7 bp putative ERG binding site identified by Transcription Element Search System (TESS) is shown in italic. The distances from the ATG starting site are presented. <b>e–f</b>) Chromatin Immunoprecipitation (ChIP) showed a significant enrichment of HA-tERG binding to PIM1 promoter compared to IgG control in RWPE-1_tERG. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) promoter was used as a negative control. ** = p<0.01.</p

The tERG/PIM1 axis favours aneuploidy.

<p><b>a</b>) Immunoblot assay demonstrates Cyclin B1 increase in RWPE-1_tERG cells. siRNA knockdown of PIM1 (siPIM1) reduces this effect compared to non-targeting siRNA (siNT). The same amount of whole cell lysates were loaded in two different polyacrilamide gels. The tERG_siPIM1 sample for the HA immunoblot was loaded in a different position compared to the gel for PIM1, Actin and Cyclin B1 blot <b>b</b>) Flow cytometric analysis of non-synchronized propidium iodide stained cells. Treatment with taxol for 15/24/42 hours induces an important increase in the >4n fraction of RWPE-1_tERG compared to empty cells. <b>c</b>) Percentage of >4n cells after taxol treatment obtained from the average of at least two independent experiments. ** = p<0.01, *** = p<0.001. <b>d</b>) Annexin-V assay was carried out to evaluate apoptosis after 42 hrs of 10 nM taxol treatment for RWPE-1 transfectants. The percentage of cells in each quadrant is shown.</p