FUBP3 regulates chronic myeloid leukaemia progression through PRC2 complex regulated PAK1-ERK signalling.

The development of resistance and heterogeneity in differential response towards tyrosine kinase inhibitors (TKI) in chronic myeloid leukaemia (CML) treatment has led to the exploration of factors independent of the Philadelphia chromosome. Among these are the association of deletions of genes on derivative (der) 9 chromosome with adverse outcomes in CML patients. However, the functional role of genes near the breakpoint on der (9) in CML prognosis and progression remains largely unexplored. Copy number variation and mRNA expression were evaluated for five genes located near the breakpoint on der (9). Our data showed a significant association between microdeletions of the FUBP3 gene and its reduced expression with poor prognostic markers and adverse response outcomes in CML patients. Further investigation using K562 cells showed that the decrease in FUBP3 protein was associated with an increase in proliferation and survival due to activation of the MAPK-ERK pathway. We have established a novel direct interaction of FUBP3 protein and PRC2 complex in the regulation of ERK signalling via PAK1. Our findings demonstrate the role of the FUBP3 gene located on der (9) in poor response and progression in CML with the identification of additional druggable targets such as PAK1 in improving response outcomes in CML patients.

Microarray analysis reveals distinct immune signatures in BCR-ABL positive and negative myeloproliferative neoplasms.

BACKGROUND: BCR-ABL mutation on the Philadelphia chromosome is the key driver of chronic myeloid leukemia (CML) pathogenesis. However, there are certain cases of myeloproliferative neoplasms (MPN) wherein no inherent driver mutation is detected resulting in clinical phenotype. It is important to identify key genes and pathways in driving the disease. The aim of the study was to use a gene-based omics approach to molecularly characterize these mutation-positive and negative cases to further strengthen diagnostics and precision medicine. METHODS: A microarray profiling was done on CD34 positive cells isolated from two BCR-ABL positive and five BCR-ABL negative samples. JAK2V617F mutation testing was also done to rule out the presence of any other mutation in the latter group. The fold change cut-off was taken as ±1.5 with p≤0.5 for significant genes. The gene network and pathway analysis were done using DAVID and STRING software. RESULTS: The genes upregulated in BCR-ABL negative samples were shown to be involved in immune regulation, signal transduction and T- and B-cell signalling. The protein-protein interaction network of upregulated genes in these samples were enriched for various immunomodulatory genes such as HLADP, HLADQ, IL7R, CCR7, CD3 subtypes. These genes further formed a network with signal transduction genes such as LCK, FYN, RAG1, DOCK1, AKT3, SMAD3, LEF1. CONCLUSION: The results suggested a modulation of immune response genes and its subsequent effect on oncogenic signalling in BCR-ABL negative samples as compared to BCR-ABL positive samples. The protein network analysis was enriched for genes involved in Src, TGF-beta and PI3K-AKT pathway contributing to the proliferation of neoplastic clone.

Gene expression profiling of CD34(+) cells from patients with myeloproliferative neoplasms.

Myeloproliferative neoplasms (MPN) are clonal disorders characterized by the increased proliferation of hematopoietic stem cell precursors and mature blood cells. Mutations of Janus kinase 2 (JAK2), Calreticulin (CALR) and MPL (myeloproliferative leukemia virus) are key driver mutations in MPN. However, the molecular profile of triple negative MPN has been a subject of ambiguity over the past few years. Mutations of, methylcytosine dioxygenase TET2, polycomb group protein ASXL1 and histone-lysine N-methyltransferase EZH2 genes have accounted for certain subsets of triple negative MPNs but the driving cause for majority of cases is still unexplored. The present study performed a microarray-based transcriptomic profile analysis of bone marrow-derived CD34(+) cells from seven MPN samples. A total of 21,448 gene signatures were obtained, which were further filtered into 472 upregulated and 202 downregulated genes. Gene ontology and protein-protein interaction (PPI) network analysis highlighted an upregulation of genes involved in cell cycle and chromatin modification in JAK2V617F negative vs. positive MPN samples. Out of the upregulated genes, seven were associated with the hematopoietic stem cell signature, while forty-seven were associated with the embryonic stem cell signature. The majority of the genes identified were under the control of NANOG and E2F4 transcription factors. The PPI network indicated a strong interaction between chromatin modifiers and cell cycle genes, such as histone-lysine N-methyltransferase SUV39H1, SWI/SNF complex subunit SMARCC2, SMARCE2, chromatin remodeling complex subunit SS18, tubulin ß (TUBB) and cyclin dependent kinase CDK1. Among the upregulated epigenetic markers, there was a ~10-fold increase in MYB expression in JAK2V617F negative samples. A significant increase in total CD34 counts in JAK2V617F negative vs. positive samples (P<0.05) was also observed. Overall, the present data showed a distinct pattern of expression in JAK2V617F negative vs. positive samples with upregulated genes involved in epigenetic modification.

High frequency of exon 20 S768I EGFR mutation detected in malignant pleural effusions: A poor prognosticator of NSCLC.

BACKGROUND: Lung cancer is the cause of a fourth of all cancer-related deaths. About a third of all lung adenocarcinoma tumours harbour mutations on exons 18 to 21 of the epidermal growth factor receptor (EGFR) gene. Detection of these mutations allows for targeted therapies in the form of EGFR Tyrosine kinase inhibitors. Recently, "liquid biopsies" have emerged as an alternative to conventional tissue mutation detection. AIM: In this pilot study, we attempted to optimize EGFR mutation detection from malignant pleural effusions (MPEs) as "liquid biopsies" when tissue biopsies were unavailable. Resulting mutations were then to be mapped on the EGFR gene and explored using cBioPortal, a public cancer genomic database. METHODS AND RESULTS: We first attempted a direct sequencing approach and showed that single nucleotide variants (SNVs) were likely to be missed in MPEs. We then switched to and optimized an EGFR mutant-specific quantitative polymerase chain reaction-based assay. This assay was piloted on n = 10 pleural effusion samples (one non-malignant pleural effusion as a negative control). 5/9 (55.55%) samples harboured EGFR mutations with 2/9 (22.22%) being exon 19 deletions and 3/9 (33.33%) the S768I mutation. The frequency of the S768I SNV in our study was significantly higher than that observed in other studies (~0.2%). Utilizing cBioPortal data, we report that patients with S768I have a shorter median survival time (6 months vs 38 months), progression-free survival time (8 months vs 44 months) and lower tumor mutation count compared to patients with other EGFR mutations. CONCLUSIONS: The shorter survival of patients with the S768I SNV predicts aggressive disease and poor prognosis as a result of this mutation. Studies in larger cohorts and/or animal models are necessary to confirm these findings.

Ally to adversary: mesenchymal stem cells and their transformation in leukaemia.

Mesenchymal stem cells (MSC) are the key regulators of hematopoiesis. Owing to their dynamic nature; MSC differentiate into various lineages that further constitute the niche which are required for maintenance of the hematopoietic stem cells (HSC). A plethora of growth factors and cytokines secreted by MSC are essential for regulating the homeostasis within the niche in terms of cycling and quiescence of HSC. Additionally, there is a strong evidence suggesting the role of MSC in transformation of the niche to favour survival of leukemic cells. Regulation of HSC by MSC via BMP, Wnt, Notch and Sonic Hedgehog signalling has been well elaborated, however the modulation of MSC by HSC/LSC is yet unresolved. The cross talk between the HSC and MSC via paracrine or autocrine mechanisms is essential for the transformation. There are some reports implicating cell adhesion molecules, growth factors and cytokines; in modulation of MSC function and differentiation. The role of exosome mediated modulation has also been reported in the context of MSC transformation however, much needs to be done to understand this phenomenon in the present context. Similarly, the role of circulating nucleic acids, a well-studied molecular phenomenon in other tumours, requires attention in their potential role in crosstalk between MSC and HSC. This review underlines the current understanding of the physiological and pathophysiological roles of MSC and its transformation in diseased state, laying stress on developing further understanding of MSC regulation for development of the latter as therapeutic targets.