Transcriptional up-regulation of PHLDA1 by 17β-estradiol in MCF-7 breast cancer cells

Most breast cancer risk factors are associated with prolonged exposure of the mammary gland to high levels of estrogens. The actions of estrogens are predominantly mediated by two receptors, ER&#945; and ER&#946;, which act as transcription factors binding with high affinity to estrogen response elements in the promoter region of target genes. However, most target genes do not contain the consensus estrogen response elements, but rather degenerated palindromic sequences showing one or more mutations and other ER-binding sites such as AP-1 and SP-1. Using the differential display reverse transcription-polymerase chain reaction technique, our group identified several genes differentially expressed in normal tissue and in ER-positive and ER-negative primary breast tumors. One of the genes shown to be down-regulated in breast tumors compared to normal breast tissue was the PHLDA1 (Pleckstrin homology-like domain, family A, member 1). In the present study, we investigated the potential of PHLDA1 to be regulated by estrogen via ER in MCF-7 breast cancer cells. The promoter region of PHLDA1 shows an imperfect palindrome, an AP-1- and three SP-1-binding sites potentially regulated by estrogens. We also assessed the effects of 17&#946;-estradiol on PHLDA1 mRNA expression in MCF-7 breast cancer cells. MCF-7 cells exposed to 10 nM 17&#946;-estradiol showed more than 2-fold increased expression of the PHLDA1 transcripts compared to control cells (P = 0.05). The anti-estrogen ICI 182,780 (1 µM) inhibited PHLDA1 mRNA expression and completely abolished the effect of 10 nM 17&#946;-estradiol on PHLDA1 expression (P < 0.05), suggesting that PHLDA1 is regulated by estrogen via ER.FAPESPCNP

Germline mutations in mitochondrial complex I reveal genetic and targetable vulnerability in IDH1-mutant acute myeloid leukaemia

The interaction of germline variation and somatic cancer driver mutations is underinvestigated. Here we describe the genomic mitochondrial landscape in adult acute myeloid leukaemia (AML) and show that rare variants affecting the nuclear- and mitochondriallyencoded complex I genes show near-mutual exclusivity with somatic driver mutations affecting isocitrate dehydrogenase 1 (IDH1), but not IDH2 suggesting a unique epistatic relationship. Whereas AML cells with rare complex I variants or mutations in IDH1 or IDH2 all display attenuated mitochondrial respiration, heightened sensitivity to complex I inhibitors including the clinical-grade inhibitor, IACS-010759, is observed only for IDH1-mutant AML. Furthermore, IDH1 mutant blasts that are resistant to the IDH1-mutant inhibitor, ivosidenib, retain sensitivity to complex I inhibition. We propose that the IDH1 mutation limits the flexibility for citrate utilization in the presence of impaired complex I activity to a degree that is not apparent in IDH2 mutant cells, exposing a mutation-specific metabolic vulnerability. This reduced metabolic plasticity explains the epistatic relationship between the germline complex I variants and oncogenic IDH1 mutation underscoring the utility of genomic data in revealing metabolic vulnerabilities with implications for therapy.Mahmoud A. Bassal, Saumya E. Samaraweera, Kelly Lim, Brooks A. Bernard, Sheree Bailey, Satinder Kaur, Paul Leo, John Toubia, Chloe Thompson-Peach, Tran Nguyen, Kyaw Ze Ya Maung, Debora A. Casolari, Diana G. Iarossi, Ilaria S. Pagani, Jason Powell, Stuart Pitson, Siria Natera, Ute Roessner, Ian D. Lewis, Anna L. Brown, Daniel G. Tenen, Nirmal Robinson, David M. Ross, Ravindra Majeti, Thomas J. Gonda, Daniel Thomas, Richard J. D, Andre

Genetic drivers of kidney defects in the digeorge syndrome

BACKGROUND The DiGeorge syndrome, the most common of the microdeletion syndromes, affects multiple organs, including the heart, the nervous system, and the kidney. It is caused by deletions on chromosome 22q11.2; the genetic driver of the kidney defects is unknown. METHODS We conducted a genomewide search for structural variants in two cohorts: 2080 patients with congenital kidney and urinary tract anomalies and 22,094 controls. We performed exome and targeted resequencing in samples obtained from 586 additional patients with congenital kidney anomalies. We also carried out functional studies using zebrafish and mice. RESULTS We identified heterozygous deletions of 22q11.2 in 1.1% of the patients with congenital kidney anomalies and in 0.01% of population controls (odds ratio, 81.5; P = 4.5×1014). We localized the main drivers of renal disease in the DiGeorge syndrome to a 370-kb region containing nine genes. In zebrafish embryos, an induced loss of function in snap29, aifm3, and crkl resulted in renal defects; the loss of crkl alone was sufficient to induce defects. Five of 586 patients with congenital urinary anomalies had newly identified, heterozygous protein-Altering variants, including a premature termination codon, in CRKL. The inactivation of Crkl in the mouse model induced developmental defects similar to those observed in patients with congenital urinary anomalies. CONCLUSIONS We identified a recurrent 370-kb deletion at the 22q11.2 locus as a driver of kidney defects in the DiGeorge syndrome and in sporadic congenital kidney and urinary tract anomalies. Of the nine genes at this locus, SNAP29, AIFM3, and CRKL appear to be critical to the phenotype, with haploinsufficiency of CRKL emerging as the main genetic driver

Transcriptional up-regulation of PHLDA1 by 17β-estradiol in MCF-7 breast cancer cells

Most breast cancer risk factors are associated with prolonged exposure of the mammary gland to high levels of estrogens. The actions of estrogens are predominantly mediated by two receptors, ERα and ERβ, which act as transcription factors binding with high affinity to estrogen response elements in the promoter region of target genes. However, most target genes do not contain the consensus estrogen response elements, but rather degenerated palindromic sequences showing one or more mutations and other ER-binding sites such as AP-1 and SP-1. Using the differential display reverse transcription-polymerase chain reaction technique, our group identified several genes differentially expressed in normal tissue and in ER-positive and ER-negative primary breast tumors. One of the genes shown to be down-regulated in breast tumors compared to normal breast tissue was the PHLDA1 (Pleckstrin homology-like domain, family A, member 1). In the present study, we investigated the potential of PHLDA1 to be regulated by estrogen via ER in MCF-7 breast cancer cells. The promoter region of PHLDA1 shows an imperfect palindrome, an AP-1- and three SP-1-binding sites potentially regulated by estrogens. We also assessed the effects of 17β-estradiol on PHLDA1 mRNA expression in MCF-7 breast cancer cells. MCF-7 cells exposed to 10 nM 17β-estradiol showed more than 2-fold increased expression of the PHLDA1 transcripts compared to control cells (P = 0.05). The anti-estrogen ICI 182,780 (1 µM) inhibited PHLDA1 mRNA expression and completely abolished the effect of 10 nM 17β-estradiol on PHLDA1 expression (P < 0.05), suggesting that PHLDA1 is regulated by estrogen via ER

Efficacy of combined CDK9/13ET inhibition in preclinical models of MLL-rearranged acute leukemia

Abstract not availableHannah McCalmont, Ka Leung Li, Luke Jones, John Toubia, Sarah C. Bray, Debora A. Casolari ... at al

Novel modes of MPL activation in triple-negative myeloproliferative neoplasms

Available online: 5 August 2022The identification of a somatic mutation associated with myeloid malignancy is of diagnostic importance in myeloproliferative neoplasms (MPNs). Individuals with no mutation detected in common screening tests for variants in JAK2, CALR, and MPL are described as ‘triple-negative’ and pose a diagnostic challenge if there is no other evidence of a clonal disorder. To identify potential drivers that might explain the clinical phenotype, we used an extended sequencing panel to characterise a cohort of 44 previously diagnosed triple-negative MPN patients for canonical mutations in JAK2, MPL and CALR at low variant allele frequency (found in 4/44 patients), less common variants in the JAK-STAT signalling pathway (12 patients), or other variants in recurrently mutated genes from myeloid malignancies (18 patients), including hotspot variants of potential clinical relevance in eight patients. In one patient with thrombocytosis we identified biallelic germline MPL variants. Neither MPL variant was activating in cell proliferation assays, and one of the variants was not expressed on the cell surface, yet co-expression of both variants led to thrombopoietin hypersensitivity. Our results highlight the clinical value of extended sequencing including germline variant analysis and illustrate the need for detailed functional assays to determine whether rare variants in JAK2 or MPL are pathogenic.Saumya E. Samaraweera, Tatjana Geukens, Debora A. Casolari, Tran Nguyen, Caitlyn Sun, Sheree Bailey, Sarah Moore, Jinghua Feng, Andreas W. Schreiber, Wendy T. Parker, Anna L. Brown, Carolyn Butcher, Peter G. Bardy, Michael Osborn, Hamish S. Scott, Dipti Talaulikar, Carolyn S. Grove, Christopher N. Hahn, Richard J. D'Andrea, David M. Ros

Barley starch

This thesis examined barley amylopectin structure and looked for correlations between the structure and physical properties of starch. The structure of amylopectin and gelatinisation and retrogradation of starch were studied in 10 different barley cultivars/breeding lines with differing genetic background. Amylopectin is built up of thousands of chains of glucose monomers, organised into clusters. The detailed fine structure of amylopectin was studied by isolating clusters of amylopectin and their building blocks, which are the tightly branched units building up the clusters. Barley cultivars/breeding lines possessing the amo1 mutation had fewer long chains of DP≥38 in amylopectin and more large building blocks. The structure of building blocks was rather conserved between the different barley cultivars/breeding lines studied and was categorized into different size groups. These different building blocks were shown to be randomly distributed in the amylopectin molecule. The C-chains in amylopectin can be of any length and are a category of chains different from the B-chains. The backbone in amylopectin consists of a special type of B-chains which, when cleaved by α-amylase, become chains of a similar type to C-chains. Gelatinisation and retrogradation (recrystallisation of gelatinised starch) of barley starch was studied by differential scanning calorimetry. The amo1 mutation resulted in a broader gelatinisation temperature range and a higher enthalpy of retrogradation. Other structural features were also found to influence the physical properties of starch. Small clusters and denser structure of the building blocks resulted in higher gelatinisation temperature. Fast retrogradation was observed in barley which had amylopectin with shorter chains and many large building blocks consisting of many chains. Amylopectin structure was also studied in developing barley kernels. Three barley cultivars/breeding lines were grown in a phytotron and kernels were harvested at 9, 12 and 24 days after flowering. The results showed that amylopectin synthesized at later stages of development had a more tightly branched structure. Expression of the enzymes involved in starch biosynthesis is also known to change during endosperm development

Rare variants in Fanconi anemia genes are enriched in acute myeloid leukemia

10.1038/s41408-018-0090-7Blood Cancer Journal865