Consecutive epigenetically-active agent combinations act in ID1-RUNX3-TET2 and HOXA pathways for Flt3ITD+ve AML

Co-occurrence of Flt3ITD and TET2 mutations provoke an animal model of AML by epigenetic repression of Wnt pathway antagonists, including RUNX3, and by hyperexpression of ID1, encoding Wnt agonist. These affect HOXA over-expression and treatment resistance. A comparable epigenetic phenotype was identified among adult AML patients needing novel intervention. We chose combinations of targeted agents acting on distinct effectors, at the levels of both signal transduction and chromatin remodeling, in relapsed/refractory AML's, including Flt3ITD+ve, described with a signature of repressed tumor suppressor genes, involving Wnt antagonist RUNX3, occurring along with ID1 and HOXA over-expressions. We tracked patient response to combination of Flt3/Raf inhibitor, Sorafenib, and Vorinostat, pan-histone deacetylase inhibitor, without or with added Bortezomib, in consecutive phase I trials. A striking association of rapid objective remissions (near-complete, complete responses) was noted to accompany induced early pharmacodynamic changes within patient blasts in situ, involving these effectors, significantly linking RUNX3/Wnt antagonist de-repression (80%) and ID1 downregulation (85%), to a response, also preceded by profound HOXA9 repression. Response occurred in context of concurrent TET2 mutation/hypomorphy and Flt3ITD+ve mutation (83% of complete responses). Addition of Bortezomib to the combination was vital to attainment of complete response in Flt3ITD+ve cases exhibiting such Wnt pathway dysregulation

RNA-interference in rice against Rice tungro bacilliform virus results in its decreased accumulation in inoculated rice plants

Rice tungro is a viral disease seriously affecting rice production in South and Southeast Asia. Tungro is caused by the simultaneous infection in rice of Rice tungro bacilliform virus (RTBV), a double-stranded DNA virus and Rice tungro spherical virus (RTSV), a single-stranded RNA virus. To apply the concept of RNA-interference (RNAi) for the control of RTBV infection, transgenic rice plants expressing DNA encoding ORF IV of RTBV, both in sense as well as in anti-sense orientation, resulting in the formation of double-stranded (ds) RNA, were raised. RNA blot analysis of two representative lines indicated specific degradation of the transgene transcripts and the accumulation of small molecular weight RNA, a hallmark for RNA-interference. In the two transgenic lines expressing ds-RNA, different resistance responses were observed against RTBV. In one of the above lines (RTBV-O-Ds1), there was an initial rapid buildup of RTBV levels following inoculation, comparable to that of untransformed controls, followed by a sharp reduction, resulting in approximately 50-fold lower viral titers, whereas the untransformed controls maintained high levels of the virus till 40 days post-inoculation (dpi). In RTBV-O-Ds2, RTBV DNA levels gradually rose from an initial low to almost 60% levels of the control by 40 dpi. Line RTBV-O-Ds1 showed symptoms of tungro similar to the untransformed control lines, whereas line RTBV-O-Ds2 showed extremely mild symptoms

Transgenic expression of coat protein gene of Rice tungro bacilliform virus in rice reduces the accumulation of viral DNA in inoculated plants

Rice tungro, a devastating disease of rice in south and southeast Asia, is caused by the joint infection of Rice tungro bacilliform virus (RTBV) and Rice tungro spherical virus (RTSV). In order to obtain transgenic resistance against RTBV, indica rice cultivar Pusa Basmati-1 was transformed to express the coat protein (CP) gene of an Indian isolate of RTBV. Rice plants containing the transgene integrated in low copy numbers were obtained, in which the CP was shown to accumulate in the leaf tissue. The progenies representing three independent transformation events were challenged with Indian isolates of RTBV using viruliferous Green leafhoppers, and the viral titers in the inoculated plants were monitored using DNA dot-blot hybridization. As compared to non-transgenic controls, two independent transgenic lines showed significantly low levels of RTBV DNA, especially towards later stages of infection and a concomitant reduction of tungro symptoms

A water soluble parthenolide analogue suppresses in vivo tumor growth of two tobacco associated cancers, lung and bladder cancer, by targeting NF-κB and generating reactive oxygen species

Dimethylaminoparthenolide (DMAPT) is a water soluble parthenolide analogue with preclinical activity in hematologic malignancies. Using NSCLC cell lines (A549, H522) and an immortalized human bronchial epithelial cell line (BEAS2B) and TCC cell lines (UMUC-3, HT-1197, HT-1376) and a bladder papilloma (RT-4), we aimed to characterize DMAPT's anti-cancer activity in tobacco associated neoplasms. Flow cytometric, electrophorectic mobility gel shift assays (EMSA), and western blot studies measured generation of reactive oxygen species (ROS), inhibition of NFκB DNA binding, and changes in cell cycle distribution and apoptotic proteins. DMAPT generated ROS with subsequent JNK activation and also decreased NFκB DNA binding and anti-apoptotic proteins, TRAF-2 and XIAP. DMAPT induced apoptotic cell death and altered cell cycle distribution with upregulation of p21 and p73 levels in a cell type dependent manner. DMAPT suppressed cyclin D1 in BEAS2B. DMAPT retained NFκB and cell cycle inhibitory activity in the presence of the tobacco carcinogen nitrosamine ketone, 4(methylnitrosamino)-1-(3–pyridyl)-1-butanone (NNK). Using a BrdU accumulation assay, 5 to 20μM of DMAPT was shown to inhibit cellular proliferation of all cell lines by more than 95%. Oral dosing of DMAPT suppressed in vivo A549 and UMUC-3 subcutaneous xenograft growth by 54% (p=0.015) and 63% (p<0.01) respectively and A549 lung metastatic volume by 28% (p=0.043). In total this data demonstrates DMAPT's novel anti-cancer properties in both early and late stage tobacco associated neoplasms as well as its significant in vivo activity. The data provides support for the conduct of clinical trials in TCC and NSCLC

Kaposi Sarcoma Herpes Virus Latency Associated Nuclear Antigen Protein Release the G2/M Cell Cycle Blocks by Modulating ATM/ATR Mediated Checkpoint Pathway

The Kaposi's sarcoma-associated herpesvirus infects the human population and maintains latency stage of viral life cycle in a variety of cell types including cells of epithelial, mesenchymal and endothelial origin. The establishment of latent infection by KSHV requires the expression of an unique repertoire of genes among which latency associated nuclear antigen (LANA) plays a critical role in the replication of the viral genome. LANA regulates the transcription of a number of viral and cellular genes essential for the survival of the virus in the host cell. The present study demonstrates the disruption of the host G2/M cell cycle checkpoint regulation as an associated function of LANA. DNA profile of LANA expressing human B-cells demonstrated the ability of this nuclear antigen in relieving the drug (Nocodazole) induced G2/M checkpoint arrest. Caffeine suppressed nocodazole induced G2/M arrest indicating involvement of the ATM/ATR. Notably, we have also shown the direct interaction of LANA with Chk2, the ATM/ATR signalling effector and is responsible for the release of the G2/M cell cycle block

A hypothetical model shows the putative mechanisms for the bypassing of the nocodazole induced G2/M block by LANA.

<p>Nocodazole treatment reduces the level of phosphorylated Cdc2. The viral nuclear antigen LANA binds directly to Chk2, which may result in the phosphorylation of Cdc25c and sequester it in the cytoplasm. Thus, it may be unable to regulate the phosphorylation of nuclear Cdc2 resulting the activation of cyclin B-Cdc2 and progression through the G2/M phase, releasing the nocodozole induced block.</p

LANA disrupt the nocodazole induced G2/M cell cycle block through the ATM/ATR signaling pathway.

<p>(A) The release of nocodazole induced G2/M block by LANA is inhibited by caffeine. BJAB cells (transfected with the pA3M vector or pA3M-LANA) were treated with nocodazole (200 ng/mL) with and without caffeine (5 mM) for 24 hours. The cells were then harvested, stained with PI and their DNA cell cycle profiles were determined by FACS analysis. Results shown are percentages of cells in each phase of the cell cycle. The data represent the mean of 3 separate experiments. (B, C) The release of nocodazole induced G2/M block by LANA occurs through ATR. BJAB cells (control), ATM siRNA and ATR siRNA transfected BJAB cell were treated with nocodazole (200 ng/mL) in the presence and absence of LANA. The cells were then harvested stained with PI and their DNA cell cycle phases were determined by FACS. Results are shown as the percentages of cells in all cell cycle phases. Mean and standard deviations were derived from 3 independent experiments. +, present; −, absent.</p

Downregulation of Chk2 protein levels inhibits the ability of LANA to release the nocodazole G2/M block.

<p>(A) Western blot for Chk2 showed the specific effect of Chk2 siRNA on Chk2 protein levels. BJAB cells carrying pA3M vector (control) and pA3M-LANA were transfected with control and Chk2 siRNA for another 24 hours. The cells were harvested, whole cell lysates and subjected to western blot analysis. β-actin was used as loading control. The result shown is the representative figure of 3 independent experiments. (B) BJAB cells with pA3M vector and pA3M-LANA were treated with nocodazole and with and without Chk2 siRNA for 24 hours. The cells were then harvested, stained with PI and their cell cycle phases were determined by flow cytometry. Results in the untreated and treated cells are shown as the percentages of cells in various cell cycle phases. (C) BC3 cells transfected with control or Chk2 siRNA were treated with nocodazole for 24 hours and subjected cell cycle analysis. Results in the untreated and treated cells were shown, together with the percentages of cells in all cell cycle phases. Mean and standard deviations were derived from 3 independent experiments. +, present; −, absent.</p