Allosteric inhibition enhances the efficacy of ABL kinase inhibitors to target unmutated BCR-ABL and BCR-ABL-T315I

Background: Chronic myelogenous leukemia (CML) and Philadelphia chromosome-positive (Ph+) acute lymphatic leukemia (Ph + ALL) are caused by the t(9;22), which fuses BCR to ABL resulting in deregulated ABL-tyrosine kinase activity. The constitutively activated BCR/ABL-kinase "escapes" the auto-inhibition mechanisms of c-ABL, such as allosteric inhibition. The ABL-kinase inhibitors (AKIs) Imatinib, Nilotinib or Dasatinib, which target the ATP-binding site, are effective in Ph + leukemia. Another molecular therapy approach targeting BCR/ABL restores allosteric inhibition. Given the fact that all AKIs fail to inhibit BCR/ABL harboring the 'gatekeeper' mutation T315I, we investigated the effects of AKIs in combination with the allosteric inhibitor GNF2 in Ph + leukemia. Methods: The efficacy of this approach on the leukemogenic potential of BCR/ABL was studied in Ba/F3 cells, primary murine bone marrow cells, and untransformed Rat-1 fibroblasts expressing BCR/ABL or BCR/ABL-T315I as well as in patient-derived long-term cultures (PDLTC) from Ph + ALL-patients. Results: Here, we show that GNF-2 increased the effects of AKIs on unmutated BCR/ABL. Interestingly, the combination of Dasatinib and GNF-2 overcame resistance of BCR/ABL-T315I in all models used in a synergistic manner. Conclusions: Our observations establish a new approach for the molecular targeting of BCR/ABL and its resistant mutants using a combination of AKIs and allosteric inhibitors

Overcoming Bcr-Abl T315I mutation by combination of GNF-2 and ATP competitors in an Abl-independent mechanism

ABSTRACT: BACKGROUND: Philadelphia positive leukemias are characterized by the presence of Bcr-Abl fusion protein which exhibits an abnormal kinase activity. Selective Abl kinase inhibitors have been successfully established for the treatment of Ph (+) leukemias. Despite high rates of clinical response, Ph (+) patients can develop resistance against these kinase inhibitors mainly due to point mutations within the Abl protein. Of special interest is the 'gatekeeper' T315I mutation, which confers complete resistance to Abl kinase inhibitors. Recently, GNF-2, Abl allosteric kinase inhibitor, was demonstrated to possess cellular activity against Bcr-Abl transformed cells. Similarly to Abl kinase inhibitors (AKIs), GNF-2 failed to inhibit activity of mutated Bcr-Abl carrying the T315I mutation. METHODS: Ba/F3 cells harboring native or T315I mutated Bcr-Abl constructs were treated with GNF-2 and AKIs. We monitored the effect of GNF-2 with AKIs on the proliferation and clonigenicity of the different Ba/F3 cells. In addition, we monitored the auto-phosphorylation activity of Bcr-Abl and JAK2 in cells treated with GNF-2 and AKIs. RESULTS: In this study, we report a cooperation between AKIs and GNF-2 in inhibiting proliferation and clonigenicity of Ba/F3 cells carrying T315I mutated Bcr-Abl. Interestingly, cooperation was most evident between Dasatinib and GNF-2. Furthermore, we showed that GNF-2 was moderately active in inhibiting the activity of JAK2 kinase, and presence of AKIs augmented GNF-2 activity. CONCLUSIONS: Our data illustrated the ability of allosteric inhibitors such as GNF-2 to cooperate with AKIs to overcome T315I mutation by Bcr-Abl-independent mechanisms, providing a possibility of enhancing AKIs efficacy and overcoming resistance in Ph+ leukemia cells

The Third Intron of the Interferon Regulatory Factor-8 Is an Initiator of Repressed Chromatin Restricting Its Expression in Non-Immune Cells

<div><p>Interferon Regulatory Factor-8 (IRF-8) serves as a key factor in the hierarchical differentiation towards monocyte/dendritic cell lineages. While much insight has been accumulated into the mechanisms essential for its hematopoietic specific expression, the mode of restricting IRF-8 expression in non-hematopoietic cells is still unknown. Here we show that the repression of IRF-8 expression in restrictive cells is mediated by its 3^rd intron. Removal of this intron alleviates the repression of Bacterial Artificial Chromosome (BAC) IRF-8 reporter gene in these cells. Fine deletion analysis points to conserved regions within this intron mediating its restricted expression. Further, the intron alone selectively initiates gene silencing only in expression-restrictive cells. Characterization of this intron’s properties points to its role as an initiator of sustainable gene silencing inducing chromatin condensation with suppressive histone modifications. This intronic element cannot silence episomal transgene expression underlining a strict chromatin-dependent silencing mechanism. We validated this chromatin-state specificity of IRF-8 intron upon <i>in-vitro</i> differentiation of induced pluripotent stem cells (iPSCs) into cardiomyocytes. Taken together, the IRF-8 3^rd intron is sufficient and necessary to initiate gene silencing in non-hematopoietic cells, highlighting its role as a nucleation core for repressed chromatin during differentiation.</p></div

AdOx treatment alleviated both IRF-8 reporter gene and the endogenous IRF-8 in NIH3T3 restricting cells.

<p>NIH3T3 cells harboring BAC-IRF8.1-EGFP construct were either left untreated or treated with 25μM AdOx. EGFP (<b>A</b>) and IRF-8 (<b>B</b>) expression were measured using flow cytometry (for details see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0156812#sec002" target="_blank">Materials and Methods</a>). Cells exhibiting high EGFP expression were sorted and subjected to ChIP analysis (<b>C</b>) using monoclonal antibodies directed against H3K27me3 and fold enrichment was calculated as described under <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0156812#pone.0156812.g004" target="_blank">Fig 4</a>. Asterisks represent statistical significance (Students t-test, * <i>p</i><0.05).</p

IRF-8 3^rd intron is sufficient to repress reporter gene expression in restrictive cells.

<p>(<b>A</b>) NIH3T3 and RAW cells were transduced with constructs harboring either IRF-8 3^rd intron or GAPDH 2^nd intron upstream to the reporter gene as schematically illustrated (pMSCV-IRF8int3 and pMSCV-GAPDHint2, respectively). (<b>B</b>) Luciferase expression levels were determined 72 hrs post transduction. pMSCV-Luc Luciferase expression was determined as 1. Values are in relative light unites, means ± AVEDEV (n≥3). ChIP-qPCR analysis of H3K27me3 modification at the Luciferase reporter gene (<b>C</b>) and Puromycin gene (<b>D</b>) in NIH3T3 or RAW cells transduced with either pMSCV-IRF8int3 or pMSCV-GAPDHint2 constructs was performed using different three primer pairs from luciferase and Puromycin genes (Luciferase 1, 2,3, and Puromycin 1,2,3, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0156812#pone.0156812.s007" target="_blank">S1 Table</a>, respectively). Values are means ± AVEDEV (n = 3) and calculated as described under <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0156812#pone.0156812.g004" target="_blank">Fig 4</a>. Asterisk indicates P-values that are significant after FDR correction for multiple hypotheses testing with α = 0.05.</p

Deletion analysis using BAC-IRF-8 reporter constructs points to the role of the 3^rd intron in modulating lineage specific expression of IRF-8.

<p>Schematic illustrations of the various BAC-IRF-8 constructs, to which a cassette containing a fluorescent reporter (mCherry) and a selectable marker (Neo driven by the PKG promoter) was inserted, are shown. (<b>A</b>) BAC-IRF-8.1- the cassette was inserted to the first ATG. (<b>B</b>) BAC-IRF-8.2- the cassette was swapped with the entire IRF-8 coding region (CDS). (<b>C</b>) The reporter construct BAC-IRF-8.3 is similar to that illustrated in panel A except that the 2^nd intron was deleted. (<b>D</b>) The reporter construct BAC-IRF-8.4 is similar to that illustrated in panel A except that the 3^rd intron was deleted. Exons (black boxes) are numbered. RAW and NIH3T3 cells were transfected with the various BAC constructs and the fluorescence intensity of the reporter gene in three different RAW and NIH3T3 stable clones, harboring 1–2 copies of the BAC reporter construct, was visualized under fluorescent microscope and quantified as described under Materials and Methods. (Students t-test,** p<0.01). Additionally, RNA was extracted from 3 independent clones for each BAC construct before and following treatment with IFN-γ and the Fold of Induction levels of the reporter gene and the endogenous IRF-8 were determined by real-time qRT-PCR (ii section of each panel).</p

IRF-8 3^rd intron has no effect on expression level and chromatin state of the transduced Luciferase and Puromycin genes in naïve iPSCs.

<p>iPSCs were transduced with either pMSCV-IRF8int3 or pMSCV-GAPDHint2) reporter constructs. Subsequently, cells were further differentiated to cardiomyocytes (as detailed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0156812#sec002" target="_blank">Materials and Methods</a>). Luciferase levels were measured and relative Luciferase activity was calculated for the undifferentiated and differentiated iPSCs ((<b>A</b>) and (<b>D</b>), respectively). ChIP-qPCR analysis of H3K27me3 modification was performed on undifferentiated and differentiated iPSCs at the Luciferase reporter gene ((<b>B</b>) and <b>(E</b>), respectively) and the Puromycin gene ((<b>C</b>) and (<b>F</b>), respectively). Values are means ± AVEDEV (n = 3) and fold enrichment was calculated as described under <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0156812#pone.0156812.g004" target="_blank">Fig 4</a>.</p

Fine deletions of the CNSs within IRF-8 3^rd intron.

<p>(<b>A</b>) BAC-IRF-8.1 constructs harboring deletions in conserved regions were transfected to restricted NIH3T3 cells as described under <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0156812#pone.0156812.g001" target="_blank">Fig 1</a> and stable clones were isolated. Representative clones were plated and left either untreated or treated with IFN-γ (100 U/ml) for 16 hrs and EGFP fluorescence was observed by microscopy. Deletion of CNS1 (position 1–284, upper panel), deletion of CNS2 (position (680–860, middle panel) and deletion of CNS3 (Position1230-1730, lower panel) is indicated. (<b>B</b>) GFP fluorescence intensity was determined in NIH3T3 clones transfected with various BAC IRF-8 constructs harboring CNS deletions as described under <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0156812#pone.0156812.g001" target="_blank">Fig 1</a>. Values are mean ± AVEDEV (n = 3). Asterisk indicates P-values that are significant after FDR correction for multiple hypotheses testing with α = 0.05.</p

Differential nucleosome occupancy and histone PTM profile across the IRF-8 3^rd intron between RAW and NIH3T3 cells.

<p>(<b>A</b>) Differential nucleosome occupancy across the IRF-8 3^rd intron between RAW and NIH3T3 cells. RAW, NIH3T3 (<b>B</b>) as well as bone marrow derived GMP and BMDM cells (<b>C</b>) were subjected to ChIP-qPCR using histone modification monoclonal antibodies directed against H3K27ac (black bars) and H3K27me3 (gray bars). Values are means ± AVEDEV (n = 3) and calculated as fold enrichment compared with mock IP (normal IgG) and normalized for DNA quantity against fold enrichment at the GAPDH gene. Asterisks represent statistical significance (Students t-test, * <i>p</i><0.05, ** <i>p</i><0.01).</p