Mutations in the promoter of the human Telomerase Reverse Transcriptase (hTERT) gene are common and associated with its elevated expression in bladder cancer, melanoma, and glioblastoma. Though these mutations and TERT overexpression are associated with aggressive disease and poor outcome, an incomplete understanding of mutant TERT regulation limits treatment options directed at this gene. Herein, we unravel a signaling pathway that leads to upregulated hTERT expression resulting from the −124 bp promoter mutation, the most frequent variant across human cancer. We employed engineered bladder cancer cells that harbor a GFP insertion at the TSS region on −124 hTERT promoter for high-content screening drug discovery using a focused library of ~800 kinase inhibitors. Studies using in vitro and in vivo models prioritized AST-487, an inhibitor of the wild-type, and mutant RET (rearranged during transfection) proto-oncogene as a novel drug inhibitor of both wild-type and mutant promoter-driven hTERT expression. We also identified the RET kinase pathway, targeted by AST-487, as a novel regulator of mutant hTERT promoter-driven transcription in bladder cancer cells. Collectively, our work provides new potential precision medicine approaches for cancer patients with upregulated hTERT expression, perhaps, especially those harboring mutations in both the RET gene and the hTERT promoter, such as in thyroid cancer.

Pericentromeric heterochromatin largely comprises repeated DNA sequences prone to aberrant recombination during double-strand break (DSB) repair. Studies in Drosophila and mouse cells revealed that ‘safe’ homologous recombination (HR) repair of these sequences relies on the relocalization of repair sites to outside the heterochromatin domain before Rad51 recruitment. Relocalization requires a striking network of nuclear actin filaments (F-actin) and myosins generating directed motions. Understanding this pathway requires the ability to detect nuclear actin filaments that are significantly less abundant than cytoplasmic filaments, and to image and track repair sites for long time periods. Here we describe an optimized protocol for live cell imaging of nuclear F-actin in response to IR in Drosophila cells, and for repair focus tracking in mouse cells, including imaging setup, image processing approaches, and analytical methods. We emphasize approaches that can be applied to identify the most effective fluorescent markers for live cell imaging, strategies to minimize photobleaching and phototoxicity with a DeltaVision deconvolution microscope, and image processing and analysis methods using SoftWoRx and Imaris software. These approaches enable a deeper understanding of the spatial and temporal dynamics of heterochromatin repair and have broad applicability in the fields of nuclear architecture, nuclear dynamics, and DNA repair.

The deregulation of lineage control programs is often associated with the progression of haematological malignancies. The molecular regulators of lineage choices in the context of tyrosine kinase inhibitor (TKI) resistance remain poorly understood in chronic myeloid leukemia (CML). To find a potential molecular regulator contributing to lineage distribution and TKI resistance, we undertook an RNA-sequencing approach for identifying microRNAs (miRNAs). Following an unbiased screen, elevated miRNA182-5p levels were detected in Bcr-Abl-inhibited K562 cells (CML blast crisis cell line) and in a panel of CML patients. Earlier, miRNA182-5p upregulation was reported in several solid tumours and haematological malignancies. We undertook a strategy involving transient modulation and CRISPR/Cas9 (clustered regularly interspersed short palindromic repeats)-mediated knockout of the MIR182 locus in CML cells. The lineage contribution was assessed by methylcellulose colony formation assay. The transient modulation of miRNA182-5p revealed a biased phenotype. Strikingly, Δ182 cells (homozygous deletion of MIR182 locus) produced a marked shift in lineage distribution. The phenotype was rescued by ectopic expression of miRNA182-5p in Δ182 cells. A bioinformatic analysis and Hes1 modulation data suggested that Hes1 could be a putative target of miRNA182-5p. A reciprocal relationship between miRNA182-5p and Hes1 was seen in the context of TK inhibition. In conclusion, we reveal a key role for miRNA182-5p in restricting the myeloid development of leukemic cells. We propose that the Δ182 cell line will be valuable in designing experiments for next-generation pharmacological interventions.

The interplay between the Notch pathway and cell cycle modulators such as CCND2 is currently unexplored and might offer novel therapeutic interventions. We report that a range of constructs that mimic Notch1 pathway down regulated CCND2 reporter activity. Chromatin immuno-precipitation (CHIP) assay showed enrichment of the HES1 protein on CCND2 promoter in HES1 transfected 293T cells. Consistent with this data, meta-analysis of selected databases reveals reciprocal expression between HES1 and CCND2 in tumor cell lines and subsets of normal hematopoietic cells. Wild type HES1 but not the DNA binding mutant reduces the proliferative activity of Multiple Myeloma (MM) cells and subsequently induces apoptosis. Combining an RNA sequencing platform and QRT-PCR assay, we found an induction of HES1 and repression of CCND2 expression levels, block in cell proliferation and subsequent induction of apoptosis upon HDAC inhibitor treatment in MM cells. Overall our data shows that the Notch pathway can negatively modulate CCND2 via HES1 with concomitant specific cellular outcomes. We suggest that small molecules enhancing HES1 expression might have therapeutic role in subsets of MM patients.

Abstract MiR182 is an evolutionarily conserved miR, present in a cluster with miR183, and miR96 on human chromosome 7q32.2. This cluster is over-expressed in hESCs, and iPSCs. Developmentally, miR182 is over-expressed during erythropoiesis from CD34 cells. Erythroid differentiation of CML cells induced by Imatinib treatment provides an alternate mechanism of Imatinib escape, and also emphasizes the need to explore miR182 function in this context. In cancerous cells, miR182 regulates metastasis in melanoma cells by regulation of MITF and FOXO3, and DNA repair in breast cancer by targeting BRCA1. These reports provide some understanding of miR182 function by transient in-vitro assays. To refine functional importance, targeted deletion of MIR182 has been done on mice retinal cells where it is shown an over-expressed candidate by quantitative methods; nevertheless MIR182 deletion does not show any phenotypic change indicating challenges to study their functions along with better model systems. We focus to study K562 cells, CML blast crisis cell line with wild type Bcr-Abl tyrosine kinase, partially differentiated cells able to differentiate in different lineages upon induction. We exploit use of CRISPR/Cas9 mediated knock-out approach to find out miR182 function in K562 cells. We show that we have successfully deleted MIR182 loci in K562 cells by CRISPR. We hypothesize if miR182 regulates proliferation or differentiation in bi-potent K562 cells. Using phenotypic characterization, we studied MIR182 deleted cells. We show that myeloid differentiation is augmented by MIR182 deletion. Homozygous MIR182 deleted cells display decreased proliferation. We find striking inverse correlation with notch signalling genes in the context of Imatinib resistance. Notch signalling has been documented in cancer cells where its role has been shown in context dependent which require more explanation of its regulation. Hes1, one of main transcriptional regulator of notch signalling pathway, is targeted by miR182 shown by both bio-informatics, and in-vitro assays. Hes1 manipulation confirms it as down-stream target of miR182 in CML cells. We next explored Imatinib resistance phenotype in the context of miR182 given its expression in CD34 cells. We demonstrate miR182 is over-expressed in CML cells towards Imatinib treatment in ex-vivo, and in-vitro. We find miR182 essential for proliferation of K562 cells, Imatinib resistance. Taken together, our studies highlight miR182 targets notch signalling genes in CML cells implication of which we have shown in the context of Bcr-Abl independent Imatinib resistance mediated by differentiation deregulation. Citation Format: Deepak Arya, P. Sasikala, Shang Li, Dasaradhi Palakodeti, Cecil Ross, Sudhir Krishna. MiR182 mediated control over myeloid differentiation provides novel mechanism of Imatinib resistance in chronic myeloid leukemia. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1931.

OBJECTIVES:Based on previous screening results, the cytotoxic effect of the hexane (JDH) and ethyl acetate extracts (JDE) of the marine sponge Jaspis diastra were evaluated on HeLa cells and the present study aimed at determining their possible mechanism of cell death.METHODS:Nuclear staining, membrane potential change, flow cytometry analysis of cell cycle distribution and annexin V staining were undertaken to investigate the effects of JDE and JDH. Electrospray ionization mass spectrometry (ESI-MS) and nuclear magnetic resonance were used to characterize an isolated bioactive molecule.KEY FINDINGS:JDE displayed an IC50 25 times more significant than the JDH. Flow cytometry analysis revealed JDE induced apoptosis in HeLa cells accompanied by the collapse of mitochondrial membrane potential. Fractionation of JDE resulted in the isolation of the known cytotoxic cyclodepsipeptide, Jaspamide.CONCLUSIONS:Taking our results together suggest that JDE can be valuable for the development of anticancer drugs, especially for cervical cancer. Further investigations are currently in progress with the aim to determine and isolate other bioactive compounds from this extract.

As part of our ongoing studies on bioactive natural products from marine sponges, we investigated the cytotoxic potential of extracts from the new sponge Petrosia tuberosa sampled from Mauritius waters. Bioguided fractionation of the ethyl acetate extract by vacuum liquid chromatography (VLC) revealed two fractions, namely VLC (6-9) and (13-17) showing cell deaths of 86 ± 1% and 88 ± 4%, respectively, at 50 μg/mL on HeLa cells. At 10 μg/mL, only VLC (13-17) displayed a significant cell death (56 ± 7%) compared with VLC (6-9) (8 ± 1 %). The cytotoxic activity of VLC (13-17) was also determined on nine other human cancer cell lines. Clonogenic assay, mitochondrial membrane potential change, DNA fragmentation and microscopic analysis of fraction VLC (13-17) revealed distinct features of apoptosis on HeLa cells. Further fractionation and purification of this fraction by chromatographic techniques resulted in isolation of one known secondary metabolite, petrosynol. Its structure was determined by 1 H and 13 C-NMR analyses.

Marine sponges are considered as a gold mine of new natural products possessing numerous biological activities. We examined the cytotoxic properties of the ethyl acetate extract (JDE) of the previously unrecorded sponge, Jaspis sp. collected from Mauritius Waters. JDE displayed an interesting IC50 of 0.057±0.04μg/mL on HL-60 cells evaluated by MTS assay. Mitochondrial membrane potential change, microscopic analysis and DNA fragmentation assays also confirmed JDE induced apoptosis on HL-60 cells. Annexin V staining demonstrated that JDE induced apoptosis at different concentrations. Treatment with 100ng/mL of JDE led to an accumulation of cells in G2/M phase after 24 h, causing a significant increase of cells (24h: 5.84%; 48h: 13.41%) in sub-G1 phase suggesting that JDE can induce cell cycle arrest in G2/M phase.

Chronic Myeloid Leukaemia (CML) is a resultant of the 9:22 translocation event leading to the constitutive kinase activity of BCR-ABL. Imatinib is the drug used as the first line therapy in CML. We report a longitudinal case study for a CML patient under treatment with imatinib. The bone marrow aspirate of this CML patient was used for exome sequencing. Single nucleotide variants (SNVs) unique to the exome sequencing sample datasets were analysed with an emphasis on kinases. These mutations were mapped to the structure to further understand the significance of the SNVs in the context of its stability and kinase-drug interaction. Here we present a data filtering pipeline with examples for sequence to structure approach. This strategy can be used to filter kinases from next generation sequencing data relevant to cancers.

Abstract Background Genome sequencing has emerged recently as a technology that can be used to address questions regarding the clonal evolution of cancers. This has the potential to be translated into practical applications in a clinical setting. We have undertaken a case study by carrying out whole exome sequencing of a patient with CML since its clinical outcome varies amongst patients during the progression of disease. Our study is an attempt for a better understanding of why patients differ in their response to different dose of drug regimen and to determine its role in clinical outcome. Our case involves a patient with BCR–ABL positive CML. She has been responding to Imatinib, at 200 mg once a day, a dosage lower than the recommended 400mg /day. She has been taking 200 mg / day irregularly for two years and there were frequent interruptions of regular dosage schedule because of severe symptomatic cytopenias requiring blood transfusions. At the end of 2 years, there has been no progression of disease. Her bone marrow aspirate and biopsy have been normal and the BCR-ABL transcript has been below detectable levels. It was by serendipity that we happened to study the whole exome of this patient before we started her on Imatinib. As her dose response was erratic, we decided to do a longitudinal study. We examined the whole exome of the patient with CML at different points during the progression of disease. The aim of this study is to identify novel polymorphisms or variants which might be associated with the case, there by resisting the disease to progress. We were interested in the process of understanding the variability of the clinical outcome to standard treatment from a genomics perspective. Results The exome sequencing of the bone marrow aspirate was performed at the time of diagnosis and two years post treatment with Imatinib. Matched skin biopsy was used as a control. This study has been approved by the Institutional Ethical Review Board of St. John's Medical college and Hospital. The SNVs of the skin data were used as the control in this experiment to account for germ line mutations. We observe severe genome instability in terms of single nucleotide variants (SNVs) at the time of diagnosis. The counts of SNVs are observed to be drastically reduced in the treated bone marrow sample than at the time of diagnosis. No insertions or deletions were observed in this longitudinal case study. It was also observed that the SNV counts were not dominated in any particular chromosome. The SNVs picked up by exome sequencing were hence contrasted at the stage of diagnosis and post treatment. These SNVs were compared to dbSNP to retain only those which are novel and not previously reported. This finally gave rise to three discreet sets to consider: 1). The SNVs present in the genome of the patient at diagnosis 2). Post treatment, and 3). a subset common to both of them. The non-synonymous coding mutations with single base substitutions were 1,08,436 at the time of diagnosis and reduced to 163 in a stage of remission. The overlaps between these two categories were 169 in number. These hits in respective categories were further filtered based on the score of SNV call, relevance to the disease, presence within domain boundaries and final predicted impact on the function of the coded protein. We have identified novel mutations within the ABL, BCR, Kit and NOTCH genes with high probability of impact on function in the bone marrow sample at diagnosis. These will further be validated experimentally to be confirmed as a probable marker for screening patients. This study will be extended to screen a cohort of patients with similar prognosis as the patient in our case study. The novel mutations will be mapped on the protein structure to show which functional domains could be affected and to provide a structural basis for the deleterious effects of such mutations. We hence attempt to understand this clinical case with an integrated view involving basic experimental biology, bio-informatics and structural biology. These polymorphisms would provide insights on the evolution of CML and eventually allow us to use such readouts as a screen across CML patients to identify cases with better disease prognosis. With the distinct population groups and diverse spectrum of diseases, we believe that our work lays the foundation for larger studies in both CML and other diseases requiring such approaches. Disclosures: No relevant conflicts of interest to declare.