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Biology subjects

Michie, A. M.

Publications and source records attributed to Michie, A. M..

6 recordsLinked to original sources

The chemical chaperone 4-phenylbutyric acid rescues molecular cell defects of COL3A1 mutations that cause vascular Ehlers Danlos Syndrome

PurposeVascular Ehlers Danlos Syndrome (vEDS) is a connective tissue disorder caused by COL3A1 mutations for which there are no treatments due to a limited understanding of underlying mechanisms. We aimed to address this critical knowledge gap, focusing on collagen folding, to establish if targeting protein folding represents a potential therapeutic approach. MethodsWe performed a mechanistic analysis of two novel COL3A1 glycine mutations, G189S and G906R, using primary patient fibroblast cultures, and performed pre-clinical proof-of-concept treatments using FDA-approved chemical chaperones targeting protein folding and/or degradation. ResultsCOL3A1 mutations caused secretion of misfolded collagen III and intracellular collagen retention, leading to matrix defects and endoplasmic reticulum (ER) stress, with increased severity for the more C-terminal mutation. Promoting ER protein folding capacity through the chemical chaperone 4-phenylbutyric acid rescued the ER stress, thermostability of secreted collagen, matrix defects and apoptosis. Optimising treatment duration and dosage helped overcome allele-dependent treatment efficacy. In contrast, protein degradation alone or combined with targeting protein folding did not increase efficacy. ConclusionER stress is a molecular mechanism in vEDS that can be influenced by the position of COL3A1 mutation, and promoting protein folding is a putative mechanism-based therapeutic approach that can rescue intra- and extracellular defects.

genetics↗

MYC-NFATC2 axis maintains cell cycle and mitochondrial function in AML cells

Acute myeloid leukaemia (AML) is a clonal haematological malignancy affecting the myeloid lineage with generally poor patient outcomes, owing to the lack of targeted therapies. The histone lysine demethylase 4A (KDM4A) has been established as a novel therapeutic target in AML, due to its selective oncogenic role within leukaemic cells. We identify that the transcription factor NFATC2 is a novel binding and transcriptional target of KDM4A in the human AML THP-1 cell line. Further, cytogenetically diverse AML cell lines, including THP-1, were dependent on NFATC2 for colony formation in vitro, highlighting a putative novel mechanism of AML oncogenesis. Our study demonstrates that NFATC2 maintenance of cell cycle progression in human AML cells was driven primarily by CCND1. Through RNA-seq and ChIP-seq, NFATC2 was shown to bind to the promoter region of genes involved in oxidative phosphorylation and subsequently regulate their gene expression in THP-1 cells. Furthermore, our data show that NFATC2 shares transcriptional targets with the transcription factor c-MYC, with MYC knockdown phenocopying NFATC2 knockdown. These data suggest a novel co-ordinated role for NFATC2 and MYC in the maintenance of THP-1 cell function, indicative of a potential means of therapeutic targeting in human AML. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=194 HEIGHT=200 SRC="FIGDIR/small/567209v1_ufig1.gif" ALT="Figure 1"> View larger version (50K): org.highwire.dtl.DTLVardef@10b4dc5org.highwire.dtl.DTLVardef@11ed9aaorg.highwire.dtl.DTLVardef@6457a9org.highwire.dtl.DTLVardef@1b722b4_HPS_FORMAT_FIGEXP M_FIG C_FIG Acute myeloid leukaemia (AML) cells of diverse cytogenetic backgrounds are dependent on NFATC2 for survival; in THP-1 cells, NFATC2 is downstream of the epigenetic regulator KDM4A. NFATC2 promotes G1/S phase transition in the cell cycle and oxidative phosphorylation. In addition, NFATC2 functions downstream of transcription factor MYC, and maintains CCND1 expression.

cancer biology↗

IL-27 maintains cytotoxic Ly6C+ gamma delta T cells that arise from immature precursors

In mice, {gamma}{delta} T cells that express the co-stimulatory molecule, CD27, are committed to the IFN{gamma}-producing lineage in the thymus, and in the periphery, these cells play a critical role in host defence and anti-tumor immunity. Unlike {beta} T cells that rely on MHC-presented peptides to drive their terminal differentiation, it is unclear whether MHC-unrestricted {gamma}{delta} T cells undergo further functional maturation after exiting the thymus. Here, we provide evidence of phenotypic and functional diversity within peripheral IFN{gamma}-producing {gamma}{delta} T cells. We found that immature CD27+Ly6C-- cells convert into mature CD27+Ly6C+ cells, and these mature cells control cancer progression while the immature cells cannot. The gene signatures of these two subsets were highly analogous to human immature and mature {gamma}{delta} T cells, indicative of conservation across species. We show that IL-27 supports the cytotoxic phenotype and function of mouse CD27+Ly6C+ cells and human V{delta}2+ cells, while IL-27 is dispensable for mouse CD27+Ly6C-- cells and human V{delta}1+ cells. These data reveal increased complexity within IFN{gamma}-producing {gamma}{delta} T cells, comprising of immature and terminally differentiated subsets, that offer new insights into unconventional T cell biology.

immunology↗

PKCβ facilitates leukemogenesis in chronic lymphocytic leukaemia by promoting constitutive BCR-mediated signaling

B cell antigen receptor (BCR) signaling competence is critical for pathogenesis of chronic lymphocytic leukemia (CLL). Defining key proteins that facilitate these networks aid in the identification of targets for therapeutic exploitation. We previously demonstrated that reduced PKC function in mouse hematopoietic stem/progenitor cells (HPSCs) resulted in PKC{beta}II upregulation and generation of a poor-prognostic CLL-like disease. Here, prkcb knockdown in HSPCs leads to reduced survival of PKC-KR-expressing CLL-like cells, concurrent with reduced expression of the leukemic markers CD5 and CD23. SP1 promotes elevated expression of prkcb in PKC-KR expressing cells enabling leukemogenesis. Global gene analysis revealed an upregulation of genes associated with B cell activation in PKC-KR expressing cells, coincident with upregulation of PKC{beta}II: supported by activation of key signaling hubs proximal to the BCR and elevated proliferation. Ibrutinib (BTK inhibitor) or enzastaurin (PKC{beta}II inhibitor) treatment of PKC-KR expressing cells and primary CLL cells showed similar patterns of Akt/mTOR pathway inhibition, supporting the role for PKC{beta}II in maintaining proliferative signals in our CLL mouse model. Ibrutinib or enzastaurin treatment also reduced PKC-KR-CLL cell migration towards CXCL12. Overall, we demonstrate that PKC{beta} expression facilitates leukemogenesis and identify that BCR-mediated signaling is a key driver of CLL development in the PKC-KR model. Statement of SignificancePKC{beta} facilitates leukemogenesis of CLL, driven through an SP1-regulated transcriptional program and promotes BCR signaling. Thus far, PKC{beta} is the only kinase within the BCR signaling pathway, a key pathway in driving CLL pathogenesis, implicated in the generation of neoplastic B lineage cells.

cancer biology↗

mTORC1 activity is essential for disease progression in chronic lymphocytic leukemia

The precise role of mechanistic target of rapamycin complex 1 (mTORC1) during chronic lymphocytic leukemia (CLL) pathogenesis remains to be elucidated. Targeted deletion of mTORC1 component Raptor in adult mice reveals that mTORC1 function is essential for initiation and maintenance of CLL. Raptor-deficient bone marrow-derived PKC-KR transduced haemopoietic progenitors failed to generate a CLL-like disease in vitro, due to an inability to overcome the mTORC1-mediated block in B cell lineage commitment. Induction of Raptor-deficiency in NSG mice transplanted with Mx1-Raptor BM-derived PKC-KR transduced cells after disease was established, revealed a reduced CLL-like disease load and a significant increase in survival in the mice. Interestingly in mice transplanted with an aggressive CLL-like disease, rapamycin treatment reduced disease burden more effectively than AZD2014 (dual mTORC1/2 inhibitor), indicating a skew towards mTORC1 sensitivity with more aggressive leukemic disease. Rapamycin efficiently targeted the translation elongation axis eEF2/eEF2K downstream of mTORC1, resulting in eEF2 inactivation through induction of eEF2T56 phosphorylation. Rapamycin treatment of primary CLL cells halted proliferation, modulated eEF2K/eEF2 phosphorylation and inhibited MCL1 expression. Our studies demonstrate that mTORC1 plays an essential role in leukemia progression in vitro and in vivo in our CLL mouse model, with evidence for increased rapamycin sensitivity in aggressive secondary CLL transplants. Furthermore, the suppression of translation elongation through inactivation of eEF2 may offer a novel therapeutic target for blocking CLL progression.

cancer biology↗

A KDM4A-PAF1-mediated epigenomic network is essential for acute myeloid leukemia cell self-renewal and survival

Epigenomic dysregulation is a common pathological feature in human hematological malignancies. H3K9me3 emerges as an important epigenomic marker in acute myeloid leukemia (AML). Its associated methyltransferases, such as SETDB1, suppress AML leukemogenesis, whilst H3K9me3 demethylases KDM4C is required for mixed lineage leukemia rearranged AML. However, the specific role and molecular mechanism of action of another member of KDM4 family, KDM4A has not previously been clearly defined. In this study, we delineated and functionally validated the epigenomic network regulated by KDM4A. We show that selective loss of KDM4A is sufficient to induce apoptosis in a broad spectrum of human AML cells. This detrimental phenotype results from a global accumulation of H3K9me3 and H3K27me3 at KDM4A targeted genomic loci thereby causing down-regulation of a KDM4A-PAF1 controlled transcriptional program essential for leukemogenesis, distinct from that of KDM4C. From this regulatory network, we further extracted a KDM4A-9 gene signature enriched with leukemia stem cell activity; the KDM4A-9 score alone or in combination with the known LSC17 score, effectively stratifies high-risk AML patients. Together, these results establish the essential and unique role of KDM4A for AML self-renewal and survival, supporting further investigation of KDM4A and its targets as a potential therapeutic vulnerability in AML.

cancer biology↗