Clonal and microclonal mutational heterogeneity in high hyperdiploid acute lymphoblastic leukemia.

High hyperdiploidy (HD), the most common cytogenetic subtype of B-cell acute lymphoblastic leukemia (B-ALL), is largely curable but significant treatment-related morbidity warrants investigating the biology and identifying novel drug targets. Targeted deep-sequencing of 538 cancer-relevant genes was performed in 57 HD-ALL patients lacking overt KRAS and NRAS hotspot mutations and lacking common B-ALL deletions to enrich for discovery of novel driver genes. One-third of patients harbored damaging mutations in epigenetic regulatory genes, including the putative novel driver DOT1L (n=4). Receptor tyrosine kinase (RTK)/Ras/MAPK signaling pathway mutations were found in two-thirds of patients, including novel mutations in ROS1, which mediates phosphorylation of the PTPN11-encoded protein SHP2. Mutations in FLT3 significantly co-occurred with DOT1L (p=0.04), suggesting functional cooperation in leukemogenesis. We detected an extraordinary level of tumor heterogeneity, with microclonal (mutant allele fraction <0.10) KRAS, NRAS, FLT3, and/or PTPN11 hotspot mutations evident in 31/57 (54.4%) patients. Multiple KRAS and NRAS codon 12 and 13 microclonal mutations significantly co-occurred within tumor samples (p=4.8x10-4), suggesting ongoing formation of and selection for Ras-activating mutations. Future work is required to investigate whether tumor microheterogeneity impacts clinical outcome and to elucidate the functional consequences of epigenetic dysregulation in HD-ALL, potentially leading to novel therapeutic approaches

Genomic characterization of chronic lymphocytic leukemia (CLL) in radiation-exposed Chornobyl cleanup workers

Background Chronic lymphocytic leukemia (CLL) was the predominant leukemia in a recent study of Chornobyl cleanup workers from Ukraine exposed to radiation (UR-CLL). Radiation risks of CLL significantly increased with increasing bone marrow radiation doses. Current analysis aimed to clarify whether the increased risks were due to radiation or to genetic mutations in the Ukrainian population. Methods A detailed characterization of the genomic landscape was performed in a unique sample of 16 UR-CLL patients and age- and sex-matched unexposed general population Ukrainian-CLL (UN-CLL) and Western-CLL (W-CLL) patients (n = 28 and 100, respectively). Results Mutations in telomere-maintenance pathway genes POT1 and ATM were more frequent in UR-CLL compared to UN-CLL and W-CLL (both p < 0.05). No significant enrichment in copy-number abnormalities at del13q14, del11q, del17p or trisomy12 was identified in UR-CLL compared to other groups. Type of work performed in the Chornobyl zone, age at exposure and at diagnosis, calendar time, and Rai stage were significant predictors of total genetic lesions (all p < 0.05). Tumor telomere length was significantly longer in UR-CLL than in UN-CLL (p = 0.009) and was associated with the POT1 mutation and survival. Conclusions No significant enrichment in copy-number abnormalities at CLL-associated genes was identified in UR-CLL compared to other groups. The novel associations between radiation exposure, telomere maintenance and CLL prognosis identified in this unique case series provide suggestive, though limited data and merit further investigation

Behavioral Defects in Chaperone-Deficient Alzheimer's Disease Model Mice

Molecular chaperones protect cells from the deleterious effects of protein misfolding and aggregation. Neurotoxicity of amyloid-beta (Aβ) aggregates and their deposition in senile plaques are hallmarks of Alzheimer's disease (AD). We observed that the overall content of αB-crystallin, a small heat shock protein molecular chaperone, decreased in AD model mice in an age-dependent manner. We hypothesized that αB-crystallin protects cells against Aβ toxicity. To test this, we crossed αB-crystallin/HspB2 deficient (CRYAB-/-HSPB2-/-) mice with AD model transgenic mice expressing mutant human amyloid precursor protein. Transgenic and non-transgenic mice in chaperone-sufficient or deficient backgrounds were examined for representative behavioral paradigms for locomotion and memory network functions: (i) spatial orientation and locomotion was monitored by open field test; (ii) sequential organization and associative learning was monitored by fear conditioning; and (iii) evoked behavioral response was tested by hot plate method. Interestingly, αB-crystallin/HspB2 deficient transgenic mice were severely impaired in locomotion compared to each genetic model separately. Our results highlight a synergistic effect of combining chaperone deficiency in a transgenic mouse model for AD underscoring an important role for chaperones in protein misfolding diseases

Hot plate test.

<p>The duration of time to elicit a thermo-sensitive reflex response in mice is shown on the left axis (black bars). The temperature at which the response was recorded is shown on the right axis (grey bars). Mean values ± SEM are plotted. Data for different groups were compared by t-tests. Both the time and temperature required elicit a response in KOTg were significantly higher than those for KO (p = 0.022 and 0.031, respectively), WT (p = 0.001 and 0.001, respectively) and WTTg (p = 0.05 and 0.05, respectively). All other groups were statistically similar. WT is <i>CRYAB^+/+, Tg^0/0</i>; WTTg is <i>CRYAB^+/+, Tg^+/0</i>; KO is <i>CRYAB^-/-, Tg^0/0</i> and KOTg is <i>CRYAB^-/-, Tg^+/0</i>. (n = 7 for KOTg; n = 15 for KO; n = 7 for WTTg and n = 9 for WT).</p

Generation of the required genotypes of mice.

<p>(A) Schematic diagram showing the mouse crosses that lead to the required genotypes. (B) Immunoblots showing αB-crystallin and Hsp27 expression in two sets of mice at 7 months of age. WT is <i>CRYAB^+/+HspB2^+/+, Tg^0/0</i>; WTTg is <i>CRYAB^+/+HspB2^+/+, Tg^+/0</i>; KO is <i>CRYAB^-/-HspB2^-/-, Tg^0/0</i> and KOTg is <i>CRYAB^-/-HspB2^-/-, Tg^+/0</i>.</p

Chaperone levels in AD model mice.

<p>(A) Immunoblots showing age-dependence of αB-crystallin expression in mice expressing mutant human APP transgene. Brain lysates were prepared from two individual non-transgenic (NTg1 & NTg2) or transgenic (Tg1 & Tg2) mice each at 3 months or 7 months of age. Samples were immunoblotted for αB-crystallin, Hsp70, Hsp27, Aβ and actin. 40 µg of total protein was analyzed. Similar levels of Hsp70, Hsp27 and actin in all samples show equal protein loading. (B) Densitometric analysis of band intensities in A. Intensities were compared by two-sample Student's t-test for statistical signifcance. APP expression in Tg mice were significantly greater than NTg mice as expected (p = 0.006 at 3 months, p = 0.017 at 7 months). αB-crystallin expression was significantly lower at 7-months compared to 3-months old transgenic mice brain (p = 0.044). αB-crystallin levels in 7-month old transgenic mice was also significantly lower than non-transgenic mice at similar age (p = 0.018). All other comparisons showed no significant differences.</p

Fear conditioning test.

<p>Percent freezing of mice over time during Training (Top), Cued test (middle) and Contextual test (bottom). Mean values ± SEM are plotted. Data were analyzed by two-way repeated measure ANOVA. Differences between groups were not significant for training and cued tests. In contextual tests, WT performed significantly better than WTTg (p = 0.003); difference between WTTg and KOTg was significant (p = 0.014); other groups were not significantly different. Blue hatched boxes represent two separate auditory signals (80dB) of 30 seconds each during training and a single auditory signal for 2 minutes during cued test followed by 2 sec foot shock. Symbol representations are - KOTg (red circle), KO (orange circle), WTTg (yellow triangle) and WT (green triangle). (n = 7 for KOTg; n = 15 for KO; n = 7 for WTTg and n = 9 for WT).</p

Longer genotypically-estimated leukocyte telomere length is associated with increased adult glioma risk

Telomere maintenance has emerged as an important molecular feature with impacts on adult glioma susceptibility and prognosis. Whether longer or shorter leukocyte telomere length (LTL) is associated with glioma risk remains elusive and is often confounded by the effects of age and patient treatment. We sought to determine if genotypically-estimated LTL is associated with glioma risk and if inherited single nucleotide polymorphisms (SNPs) that are associated with LTL are glioma risk factors. Using a Mendelian randomization approach, we assessed differences in genotypically-estimated relative LTL in two independent glioma case-control datasets from the UCSF Adult Glioma Study (652 patients and 3735 controls) and The Cancer Genome Atlas (478 non-overlapping patients and 2559 controls). LTL estimates were based on a weighted linear combination of subject genotype at eight SNPs, previously associated with LTL in the ENGAGE Consortium Telomere Project. Mean estimated LTL was 31bp (5.7%) longer in glioma patients than controls in discovery analyses (P = 7.82x10-8) and 27bp (5.0%) longer in glioma patients than controls in replication analyses (1.48x10-3). Glioma risk increased monotonically with each increasing septile of LTL (O.R.=1.12; P = 3.83x10-12). Four LTL-associated SNPs were significantly associated with glioma risk in pooled analyses, including those in the telomerase component genes TERC (O.R.=1.14; 95% C.I.=1.03-1.28) and TERT (O.R.=1.39; 95% C.I.=1.27-1.52), and those in the CST complex genes OBFC1 (O.R.=1.18; 95% C.I.=1.05-1.33) and CTC1 (O.R.=1.14; 95% C.I.=1.02-1.28). Future work is needed to characterize the role of the CST complex in gliomagenesis and further elucidate the complex balance between ageing, telomere length, and molecular carcinogenesis