Identification of novel DNA-damage tolerance genes reveals regulation of translesion DNA synthesis by nucleophosmin

Cells cope with replication-blocking lesions via translesion DNA synthesis (TLS). TLS is carried out by low-fidelity DNA polymerases that replicate across lesions, thereby preventing genome instability at the cost of increased point mutations. Here we perform a twostage siRNA-based functional screen for mammalian TLS genes and identify 17 validated TLS genes. One of the genes, NPM1, is frequently mutated in acute myeloid leukaemia (AML). We show that NPM1 (nucleophosmin) regulates TLS via interaction with the catalytic core of DNA polymerase-eta (pol eta), and that NPM1 deficiency causes a TLS defect due to proteasomal degradation of pol eta. Moreover, the prevalent NPM1c+ mutation that causes NPM1 mislocalization in similar to 30% of AML patients results in excessive degradation of pol eta. These results establish the role of NPM1 as a key TLS regulator, and suggest a mechanism for the better prognosis of AML patients carrying mutations in NPM1

Identification of novel DNA-damage tolerance genes reveals regulation of translesion DNA synthesis by nucleophosmin

Cells cope with replication-blocking lesions via translesion DNA synthesis (TLS). TLS is carried out by low-fidelity DNA polymerases that replicate across lesions, thereby preventing genome instability at the cost of increased point mutations. Here we perform a twostage siRNA-based functional screen for mammalian TLS genes and identify 17 validated TLS genes. One of the genes, NPM1, is frequently mutated in acute myeloid leukaemia (AML). We show that NPM1 (nucleophosmin) regulates TLS via interaction with the catalytic core of DNA polymerase-eta (pol eta), and that NPM1 deficiency causes a TLS defect due to proteasomal degradation of pol eta. Moreover, the prevalent NPM1c+ mutation that causes NPM1 mislocalization in similar to 30% of AML patients results in excessive degradation of pol eta. These results establish the role of NPM1 as a key TLS regulator, and suggest a mechanism for the better prognosis of AML patients carrying mutations in NPM1

Unusual presentation of familial Mediterranean fever: role of genetic diagnosis

OBJECTIVE—To describe the role of molecular analysis in the diagnosis of an unusual presentation of familial Mediterranean fever (FMF).
CASE REPORT—Two patients presenting with prolonged fever without signs and symptoms of serositis are described. FMF was diagnosed by genetic analysis, which disclosed that both patients were homozygous for the M694V mutation of the Mediterranean fever (MEFV) gene.
CONCLUSION—Molecular analysis of FMF should complement the investigation of patients with fever of unknown origin. This test enables a definite diagnosis of the disease and may promote the diagnosis and treatment of patients with an unusual or incomplete clinical picture of FMF.


Quantitative contrast agent-free MRI of macrophage iron deposits.

<p>(a) Multi-gradient echo (MGE) MRI image (first-echo) showing variation of contrast with increasing concentration of iron(III) (0-0-0.25 mg g^-1). (b) Quantitative MRI measurements of signal relaxation rates R₂* (= 1/ T₂*) for the MGE image series as a function of iron(III) concentration (solid-line linear fit, dashed lines 95% confidence interval). (c) Parametric iron(III) maps generated for the standards, and H₂O (dashed circle). (d) Iron MRI maps of whole blood, transferrin (Tfn, ~20μg iron/mL), macrophage hemosiderin from mouse spleen, superparamagnetic iron oxide (SPIO) nanoparticles in aqueous solution (200μg/mL), and in vivo in mammary tumors, livers (no tumor), and spleens (no tumor) of the MMTV-PyMT breast cancer models. Upper scale bar, 1mm, lower scale bar 2mm. Expansions show high-iron pixel cluster regions indicative of hemosiderin deposition. Scale bar 200μm. High-iron(III) MRI clusters quantified automatically by (e) counting the total number of high iron pixel clusters in the iron MRI images (0.15–0.3 mg iron(III) g^-1 range), and (f) measuring the average cross-sectional areas of the clusters (mm²). Points are individual animals (mean±s.e.m.; n = 8 animals; n.s. not significant p>0.05, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 two-tailed unpaired t-test).</p

Imaging HLM deposits in brain metastasis.

<p>(a, left) Prussian blue iron(III) histology, and (right) immunofluorescence staining (DAPI, CD68, CD31) of brain metastasis from MMTV-PyMT mammary-brain model (intracardiac injection). Scale bar 1 mm. Expansion shows individual metastasis, and individual iron(III)⁺ HLM deposit (5x). Scale bar 250μm. (b) The total number of iron(III)⁺ HLMs and CD68⁺ macrophages per mm² brain metastasis measured by automated cell counting. Points are individual metastases. (mean±s.e.m.; n = 86 iron(III)⁺ and CD68⁺ metastasis; n = 16 brain sections, n = 5 animals; ****p<0.0001 Mann-Whitney test). (c) Iron(III) histochemistry beside single color channel and merged CD68, CD206, and AIF1 immunofluorescent markers. Scale bar 50μm. (d) In vivo iron(III) MRI maps measured in MMTV-PyMT mammary-brain metastasis models. Scale bar 2mm. Expansion shows high-iron(III) cluster regions and individual metastases. (e) High-iron(III) MRI clusters automatically counted in normal brain regions and the metastases, and (f) the average cross-sectional areas of the high-iron(III) clusters (mean±s.e.m.; n = 97 metastasis, n = 26 high-iron(III) clusters; n = 66 normal regions, n = 6 high-iron(III) clusters, n = 19 animals; **p<0.001 Mann-Whitney test). (g) Whole brain cross sections of the metastasis models stained for Prussian blue iron(III). The processed images show individual metastasis (outlines), HLM deposits (arrows), and MRI resolution-matched HLM clusters in the individual metastasis (filled regions). Scale bar 1mm. Expansion scale bar 400μm. Prussian blue iron(III) histology automatically assessed for (h) the average HLM cluster cross-sectional area measured per brain metastasis, and (i) number of iron(III)⁺ HLMs per cluster (mean±s.e.m.; n = 46 brain metastasis; n = 212 HLM clusters, n = 16 brain sections, n = 5 animals).</p

Quantitative contrast agent-free MRI of macrophage iron deposits.

<p>(a) Multi-gradient echo (MGE) MRI image (first-echo) showing variation of contrast with increasing concentration of iron(III) (0-0-0.25 mg g^-1). (b) Quantitative MRI measurements of signal relaxation rates R₂* (= 1/ T₂*) for the MGE image series as a function of iron(III) concentration (solid-line linear fit, dashed lines 95% confidence interval). (c) Parametric iron(III) maps generated for the standards, and H₂O (dashed circle). (d) Iron MRI maps of whole blood, transferrin (Tfn, ~20μg iron/mL), macrophage hemosiderin from mouse spleen, superparamagnetic iron oxide (SPIO) nanoparticles in aqueous solution (200μg/mL), and in vivo in mammary tumors, livers (no tumor), and spleens (no tumor) of the MMTV-PyMT breast cancer models. Upper scale bar, 1mm, lower scale bar 2mm. Expansions show high-iron pixel cluster regions indicative of hemosiderin deposition. Scale bar 200μm. High-iron(III) MRI clusters quantified automatically by (e) counting the total number of high iron pixel clusters in the iron MRI images (0.15–0.3 mg iron(III) g^-1 range), and (f) measuring the average cross-sectional areas of the clusters (mm²). Points are individual animals (mean±s.e.m.; n = 8 animals; n.s. not significant p>0.05, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 two-tailed unpaired t-test).</p

Combining epitope-distinct antibodies to HER2: cooperative inhibitory effects on invasive growth.

International audienceMonoclonal antibodies (mAbs) to HER2 are currently used to treat breast cancer, but low clinical efficacy, along with primary and acquired resistance to therapy, commonly limit clinical applications. We previously reported that combinations of antibodies directed at non-overlapping epitopes of HER2 are endowed with enhanced antitumor effects, probably due to accelerated receptor degradation. Here, we extend these observations to three-dimensional mammary cell models, and compare the effects of single mAbs with the effects of antibody combinations. Collectively, our in vitro assays and computational image analyses indicate that combining mAbs against different epitopes of HER2 better inhibits invasive growth. Importantly, while growth factors are able to reduce intraluminal apoptosis and induce an invasive phenotype, combinations of mAbs better than single mAbs can reverse the growth factor-induced phenotypes of HER2-overexpressing spheroids. In conclusion, our studies propose that mAb combinations negate the biological effects of growth factors on invasive growth of HER2-overexpressing cells. Hence, combining mAbs offers a therapeutic strategy, potentially able to enhance clinical efficacy of existing antireceptor immunotherapeutics