Atiqur Rahman

Protein Kinase CK2 (Casein Kinase 2 or CK2) is a constitutively active serine-threonine kinase overactive in human malignancies. Increased expression and activity of CK2 in Acute Myeloid Leukemia (AML) is associated with a poor outcome. CK2 promotes AML cell survival by impinging on multiple oncogenic signaling pathways. The selective small-molecule CK2 inhibitor CX-4945 has shown in vitro cytotoxicity in AML. Here, we report that CX-4945 has a strong in vivo therapeutic effect in preclinical models of AML. The analysis of genome-wide DNA-binding and gene expression in CX-4945 treated AML cells shows that one mechanism, by which CK2 inhibition exerts a therapeutic effect in AML, involves the revival of IKAROS tumor suppressor function. CK2 phosphorylates IKAROS and disrupts IKAROS’ transcriptional activity by impairing DNA-binding and association with chromatin modifiers. Here, we demonstrate that CK2 inhibition decreases IKAROS phosphorylation and restores IKAROS binding to DNA. Further functional experiments show that IKAROS negatively regulates the transcription of anti-apoptotic genes, including BCL-XL (B cell Lymphoma like–2 like 1, BCL2L1). CX-4945 restitutes the IKAROS-mediated repression of BCL-XL in vivo and sensitizes AML cells to apoptosis. Using CX-4945, alongside the cytotoxic chemotherapeutic drug daunorubicin, augments BCL-XL suppression and AML cell apoptosis. Overall, these results establish the in vivo therapeutic efficacy of CX-4945 in AML preclinical models and determine the role of CK2 and IKAROS in regulating apoptosis in AML. Furthermore, our study provides functional and mechanistic bases for the addition of CK2 inhibitors to AML therapy.

IKAROS, encoded by the IKZF1 gene, is a DNA-binding protein that functions as a tumor suppressor in T cell acute lymphoblastic leukemia (T-ALL). Recent studies have identified IKAROS’s novel function in the epigenetic regulation of gene expression in T-ALL and uncovered many genes that are likely to be directly regulated by IKAROS. Here, we report the transcriptional regulation of two genes, phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit delta (PIK3CD) and phosphoinositide kinase, FYVE-type zinc finger containing (PIKFYVE), by IKAROS in T-ALL. PIK3CD encodes the protein p110δ subunit of phosphoinositide 3-kinase (PI3K). The PI3K/AKT pathway is frequently dysregulated in cancers, including T-ALL. IKAROS binds to the promoter regions of PIK3CD and PIKFYVE and reduces their transcription in primary T-ALL. Functional analysis demonstrates that IKAROS functions as a transcriptional repressor of both PIK3CD and PIKFYVE. Protein kinase CK2 (CK2) is a pro-oncogenic kinase that is overexpressed in T-ALL. CK2 phosphorylates IKAROS, impairs IKAROS’s DNA-binding ability, and functions as a repressor of PIK3CD and PIKFYVE. CK2 inhibition results in increased IKAROS binding to the promoters of PIK3CD and PIKFYVE and the transcriptional repression of both these genes. Overall, the presented data demonstrate for the first time that in T-ALL, CK2 hyperactivity contributes to PI3K signaling pathway upregulation, at least in part, through impaired IKAROS transcriptional regulation of PIK3CD and PIKFYVE. Targeting CK2 restores IKAROS’s regulatory effects on the PI3K oncogenic signaling pathway.

Chronic obstructive pulmonary disease (COPD) and lung cancer are major lung diseases affecting millions worldwide. Both diseases have links to cigarette smoking and exert a considerable societal burden. People suffering from COPD are at higher risk of developing lung cancer than those without, and are more susceptible to poor outcomes after diagnosis and treatment. Lung cancer and COPD are closely associated, possibly sharing common traits such as an underlying genetic predisposition, epithelial and endothelial cell plasticity, dysfunctional inflammatory mechanisms including the deposition of excessive extracellular matrix, angiogenesis, susceptibility to DNA damage and cellular mutagenesis. In fact, COPD could be the driving factor for lung cancer, providing a conducive environment that propagates its evolution. In the early stages of smoking, body defences provide a combative immune/oxidative response and DNA repair mechanisms are likely to subdue these changes to a certain extent; however, in patients with COPD with lung cancer the consequences could be devastating, potentially contributing to slower postoperative recovery after lung resection and increased resistance to radiotherapy and chemotherapy. Vital to the development of new-targeted therapies is an in-depth understanding of various molecular mechanisms that are associated with both pathologies. In this comprehensive review, we provide a detailed overview of possible underlying factors that link COPD and lung cancer, and current therapeutic advances from both human and preclinical animal models that can effectively mitigate this unholy relationship.

Background: Undifferentiated thyroid carcinoma is the most aggressive type of thyroid carcinoma. It is refractory to most of the conventional therapies and its median overall survival is less than six months. Mutation in BRAF gene was found around 25% of undifferentiated thyroid carcinoma. Mutated BRAF particularly the V600E mutated BRAF auto-activates the BRAF-MEK proliferation-survival cell signalling pathway. The objectives of our current study were to inhibit the BRAF kinase and investigate its effect on undifferentiated thyroid carcinoma proliferation. Materials and methods: 8050C, a homozygous BRAF V600E mutate undifferentiated thyroid carcinoma cell line, was used to develop anti-BRAF shRNA stable cell line. Quantitative real time polymerase chain reaction (qPCR) and western blot were performed for ratifying the BRAF inhibition efficiency of stable cell line. Proliferation and colony formation assay were done to see the effect of BRAF inhibition on undifferentiated thyroid carcinoma proliferation. In addition, impact of BRAF inhibition on proliferation cell signalling pathways were checked through western blot analysis. Results: Efficiency of BRAF knockdown was around 87% which resulted in approximately 83% inhibition of BRAF kinase (p < 0.05). About 54% proliferation inhibition (p < 0.05) was observed in the BRAF inhibited group compare to control group in the first week. But the proliferation rate in the BRAF inhibited group increased over the subsequent weeks and we did not observed any sign of cell growth arrest. In addition, colony formation assay revealed approximately 13% reduction of colony formation ability (p < 0.05) in BRAF inhibited group compare to control group. Western blot analysis of signalling kinases discovered reduction of around 72% MEK kinase activity after BRAF kinase inhibition. As we observed increasing trend of proliferation after an initial sharp drop in cell proliferation after BRAF kinase inhibition, we hypothesized and analysed the alternative PI3K-AKT proliferation pathway. Surprisingly we found about 63% increase of AKT kinase activity in the BRAF inhibited group compare to control group (p < 0.05). Conclusions: Inhibition of BRAF kinase reduced cell proliferation initially, but this proliferation inhibition did not lead to cell growth arrest at the end. Increased AKT kinase activity counteracted the effect of BRAF kinase inhibition as AKT kinase has the ability to contribute in overall cell proliferation. Therefore, both kinases need to be inhibited simultaneously for cell growth arrest in undifferentiated thyroid carcinoma. Citation Format: Md. Atiqur Rahman, Ali Salajegheh, Haleh Vosgha, Hamidreza Maroof, Robert A. Smith, Alfred KY Lam. Inhibition of BRAF kinase alone in BRAF V600E-mutated undifferentiated thyroid carcinoma results in no growth arrest. [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 183.

The aim of our study was to inhibit BRAF kinase expression and investigate its effect on cellular functions in thyroid carcinomas. 8505C (BRAF^V600E/V600E) undifferentiated thyroid carcinoma cell line and B-CPAP (BRAF^V600E/V600E) papillary thyroid carcinoma cell line were used to develop doxycycline-inducible anti-BRAF shRNA stable cell lines. The inhibitions of BRAF expression in these cells were confirmed with qPCR and Western blot. Impacts of BRAF protein inhibition on cellular functions and signalling pathways were observed through Western blot, proliferation and colony formation assays. BRAF kinase expression was inhibited 83 % in undifferentiated thyroid carcinoma and 82 % in papillary thyroid carcinoma (p < 0.05). As a result of BRAF kinase inhibition, reduction in MEK kinase activity was seen (p < 0.05) in both thyroid cancer cell lines (72 and 75 %, respectively). Initially, big drop in proliferation (p < 0.05) was observed (52 and 54 %, respectively), but later an increasing proliferation trend was noticed in BRAF kinase-inhibited cell lines. In addition, reduction in colony formation (p < 0.05) was seen in BRAF kinase-inhibited carcinoma cells (13 and 15 %, respectively). On the other hand, increase in AKT kinase activity (63 and 70 %, respectively; p < 0.05) was discovered in both BRAF kinase-inhibited carcinoma cells. Increased activation of alternative proliferation pathways (as determined by the increase of AKT kinase activity) counteracts the effect of BRAF kinase inhibition in thyroid carcinomas. Thus, alternative proliferation pathways should be inhibited for therapeutic suppression of BRAF-induced proliferation in thyroid carcinomas.

miR-205 plays a crucial role in angiogenesis and has been found in association with several types of cancers. The aims of this study were to investigate the clinical and functional roles of miR-205 on as the major initiator and modulator of angiogenesis in thyroid cancer. 101 thyroid carcinomas, including 51 conventional and 37 follicular variants of papillary thyroid carcinomas, and 13 undifferentiated thyroid carcinomas in addition to 13 lymph nodes with metastatic thyroid carcinoma were recruited to be compared with 14 nodular goitre and seven normal thyroid tissues. Five thyroid carcinoma cell lines, of papillary and undifferentiated origin with and without history of metastasis, were also used. Expression of vascular endothelial growth factor A (VEGFA) and miR-205 were measured and exogenous miR-205 were transfected to observe the changes of VEGFA (by immunofluorescence and western blot techniques). Proliferation assay, cell cycle analysis and apoptosis assays were also used to evaluate the role of miR-205 in these events. Significant under-expression of miR-205 and over-expression of VEGFA mRNA and protein were noticed in thyroid cancer tissues and cell lines compared to normal thyroid control. Transfection of miR-205 into the cancer cell lines caused significant reduction of VEGFA protein and significant inhibition in cell proliferation, arrest in G0-G1 of the cell cycle and promotion of total apoptosis (PmiRNA-205 were demonstrated for the first time in thyroid cancer. The current experiments provided specific information on the functional consequences of VEGF manipulation via miRNA on cancer.