Discovery of 3,5-Diamino-1,2,4-triazole Ureas as Potent Anaplastic Lymphoma Kinase Inhibitors

A series of novel 3,5-diamino-1,2,4-triazole benzyl ureas was identified as having potent anaplastic lymphoma kinase (ALK) inhibition exemplified by 15a, 20a, and 23a, which exhibited antiproliferative IC50 values of 70, 40, and 20 nM in Tel-ALK transformed Ba/F3 cells, respectively. Moreover, 15a and 23a potently inhibited the growth and survival of NPM-ALK positive anaplastic large cell lymphoma cell (SU-DHL-1) and neuroblastoma cell lines (KELLY, SH-SY5Y) containing the F1174L ALK mutation. These compounds provide novel leads for the development of small-molecule ALK inhibitors for cancer therapy

Molecular rationale for the use of PI3K/AKT/mTOR pathway inhibitors in combination with crizotinib in ALK-mutated neuroblastoma

Mutations in the ALK tyrosine kinase receptor gene represent important therapeutic targets in neuroblastoma, yet their clinical translation has been challenging. The ALKF1174L mutation is sensitive to the ALK inhibitor crizotinib only at high doses and mediates acquired resistance to crizotinib in ALK-translocated cancers. We have shown that the combination of crizotinib and an inhibitor of downstream signaling induces a favorable response in transgenic mice bearing ALKF1174L/MYCN-positive neuroblastoma. Here, we investigated the molecular basis of this effect and assessed whether a similar strategy would be effective in ALK-mutated tumors lacking MYCN overexpression. We show that in ALK-mutated, MYCN-amplified neuroblastoma cells, crizotinib alone does not affect mTORC1 activity as indicated by persistent RPS6 phosphorylation. Combined treatment with crizotinib and an ATP-competitive mTOR inhibitor abrogated RPS6 phosphorylation, leading to reduced tumor growth and prolonged survival in ALKF1174L/MYCN-positive models compared to single agent treatment. By contrast, this combination, while inducing mTORC1 downregulation, caused reciprocal upregulation of PI3K activity in ALK-mutated cells expressing wild-type MYCN. Here, an inhibitor with potency against both mTOR and PI3K was more effective in promoting cytotoxicity when combined with crizotinib. Our findings should enable a more precise selection of molecularly targeted agents for patients with ALK-mutated tumors

CDK7 Inhibition Suppresses Super-Enhancer-Linked Oncogenic Transcription in MYCN-Driven Cancer

The MYC oncoproteins are thought to stimulate tumor cell growth and proliferation through amplification of gene transcription, a mechanism that has thwarted most efforts to inhibit MYC function as potential cancer therapy. Using a covalent inhibitor of cyclin-dependent kinase 7 (CDK7) to disrupt the transcription of amplified MYCN in neuroblastoma cells, we demonstrate downregulation of the oncoprotein with consequent massive suppression of MYCN-driven global transcriptional amplification. This response translated to significant tumor regression in a mouse model of high-risk neuroblastoma, without the introduction of systemic toxicity. The striking treatment selectivity of MYCN-overexpressing cells correlated with preferential downregulation of super-enhancer-associated genes, including MYCN and other known oncogenic drivers in neuroblastoma. These results indicate that CDK7 inhibition, by selectively targeting the mechanisms that promote global transcriptional amplification in tumor cells, may be useful therapy for cancers that are driven by MYC family oncoproteins.United States. National Institutes of Health (R01CA148688)United States. National Institutes of Health (R01CA148688S1)United States. National Institutes of Health (R01CA179483-01)United States. National Institutes of Health (CA109901)United States. National Institutes of Health (HG002668)United States. National Institutes of Health (R21HG006778)American Cancer Society (RSG-12-247-TBG)United States. Department of Defense (PR120741A)Friends for Life Neuroblastoma Foundatio

Expression of the neuron-specific protein CHD5 is an independent marker of outcome in neuroblastoma

<p>Abstract</p> <p>Background</p> <p>The chromodomain, helicase DNA-binding protein 5 (CHD5) is a potential tumor suppressor gene located on chromosome 1p36, a region recurrently deleted in high risk neuroblastoma (NB). Previous data have shown that <it>CHD5 </it>mRNA is present in normal neural tissues and in low risk NB, nevertheless, the distribution of CHD5 protein has not been explored. The aim of this study was to investigate CHD5 protein expression as an immunohistochemical marker of outcome in NB. With this purpose, CHD5 protein expression was analyzed in normal neural tissues and neuroblastic tumors (NTs). <it>CHD5 </it>gene and protein expression was reexamined after induction chemotherapy in a subset of high risk tumors to identify potential changes reflecting tumor response.</p> <p>Results</p> <p>We provide evidence that CHD5 is a neuron-specific protein, absent in glial cells, with diverse expression amongst neuron types. Within NTs, CHD5 immunoreactivity was found restricted to differentiating neuroblasts and ganglion-like cells, and absent in undifferentiated neuroblasts and stromal Schwann cells. Correlation between protein and mRNA levels was found, suggesting transcriptional regulation of <it>CHD5</it>. An immunohistochemical analysis of 90 primary NTs highlighted a strong association of CHD5 expression with favorable prognostic variables (age at diagnosis <12 months, low clinical stage, and favorable histology; P < 0.001 for all), overall survival (OS) (P < 0.001) and event-free survival (EFS) (P < 0.001). Multivariate analysis showed that CHD5 prognostic value is independent of other clinical and biologically relevant parameters, and could therefore represent a marker of outcome in NB that can be tested by conventional immunohistochemistry. The prognostic value of CHD5 was confirmed in an independent, blinded set of 32 NB tumors (P < 0.001).</p> <p>Reactivation of <it>CHD5 </it>expression after induction chemotherapy was observed mainly in those high risk tumors with induced tumor cell differentiation features. Remarkably, these NB tumors showed good clinical response and prolonged patient survival.</p> <p>Conclusions</p> <p>The neuron-specific protein CHD5 may represent a marker of outcome in NB that can be tested by conventional immunohistochemistry. Re-establishment of CHD5 expression induced by chemotherapy could be a surrogate marker of treatment response.</p

The relationship of small vessel disease burden on cerebral and regional brain atrophy rates and cognitive performance over one year of follow-up after transient ischemic attack

BackgroundStroke, even when minor, increases the risk of dementia. We aimed to determine whether patients with transient ischaemic attack (TIA) exhibit higher rates of cerebral and regional atrophy 1-year after first stroke symptoms and evaluate the relationship with small vessel disease and cognitive performance.MethodsTIA patients and controls without cognitive symptoms underwent high-resolution T1-weighted MRI and cognitive testing at baseline and 1-year. Percent brain volume change (PBVC) was measured, and the location of regional atrophy and small vessel disease (CSVD) burden was evaluated. Neuropsychological testing assessed memory, processing speed, and executive function.ResultsA total of 76 TIA patients and 53 controls of mean age 67 (SD = 8) and 68 years (SD = 8) were recruited. TIA patients demonstrated greater improvement of visual memory and executive function at 1-year. TIA patients had greater median PBVC/year compared to controls (−0.79% [(−1.22)-(−0.38)] vs. -0.41% [(−0.62)-0.19]; p &lt; 0.001), and higher rates of volume loss (ml/year) in subcortical gray (−0.53 [(−1.09)-(−0.06)] vs. -0.13 [(−0.61)-0.31]; p &lt; 0.05) and white matter (−2.21 [−5.47, 0.40] vs. -0.93 [(−3.43)-2.10]; p &lt; 0.05). Linear regression showed that TIA, age, and systolic blood pressure (SBP) were associated with greater cerebral volume loss over 1-year. There was no significant relationship between PBVC and 1-year cognition.ConclusionA near two-fold increase in rate of cerebral atrophy 1-year after TIA is associated with higher SBP emphasizing the need for improved treatment of SBP. Cerebral and regional atrophy rates may be used to select patients for vascular risk reduction trials or novel therapeutics in future dementia prevention trials

Genome-Wide Analysis of Neuroblastomas using High-Density Single Nucleotide Polymorphism Arrays

BACKGROUND: Neuroblastomas are characterized by chromosomal alterations with biological and clinical significance. We analyzed paired blood and primary tumor samples from 22 children with high-risk neuroblastoma for loss of heterozygosity (LOH) and DNA copy number change using the Affymetrix 10K single nucleotide polymorphism (SNP) array. FINDINGS: Multiple areas of LOH and copy number gain were seen. The most commonly observed area of LOH was on chromosome arm 11q (15/22 samples; 68%). Chromosome 11q LOH was highly associated with occurrence of chromosome 3p LOH: 9 of the 15 samples with 11q LOH had concomitant 3p LOH (P = 0.016). Chromosome 1p LOH was seen in one-third of cases. LOH events on chromosomes 11q and 1p were generally accompanied by copy number loss, indicating hemizygous deletion within these regions. The one exception was on chromosome 11p, where LOH in all four cases was accompanied by normal copy number or diploidy, implying uniparental disomy. Gain of copy number was most frequently observed on chromosome arm 17q (21/22 samples; 95%) and was associated with allelic imbalance in six samples. Amplification of MYCN was also noted, and also amplification of a second gene, ALK, in a single case. CONCLUSIONS: This analysis demonstrates the power of SNP arrays for high-resolution determination of LOH and DNA copy number change in neuroblastoma, a tumor in which specific allelic changes drive clinical outcome and selection of therapy

Platelet-Associated CD40/CD154 Mediates Remote Tissue Damage after Mesenteric Ischemia/Reperfusion Injury

Several innate and adaptive immune cell types participate in ischemia/reperfusion induced tissue injury. Amongst them, platelets have received little attention as contributors in the process of tissue damage after ischemia reperfusion (I/R) injury. It is currently unknown whether platelets participate through the immunologically important molecules including, CD40 and when activated, CD154 (CD40L), in the pathogenesis of I/R injury. We hypothesized that constitutive expression of CD40 and activation-induced expression of CD154 on platelets mediate local mesenteric and remote lung tissue damage after I/R injury. Wild type (WT; C57BL/6J), CD40 and CD154 deficient mice underwent mesenteric ischemia for 30 minutes followed by reperfusion for 3 hours. WT mice subjected to mesenteric I/R injury displayed both local intestinal and remote lung damage. In contrast, there was significantly less intestinal damage and no remote lung injury in CD40 and CD154 deficient mice when compared to WT mice. Platelet-depleted WT mice transfused with platelets from CD40 or CD154 deficient mice failed to reconstitute remote lung damage. In contrast, when CD40 or CD154 deficient mice were transfused with WT platelets lung tissue damage was re-established. Together, these findings suggest that multiple mechanisms are involved in local and remote tissue injury and also identify platelet-expressed CD40 and/or CD154 as mediators of remote tissue damage

Increased Serum and Musculotendinous Fibrogenic Proteins following Persistent Low-Grade Inflammation in a Rat Model of Long-Term Upper Extremity Overuse.

We examined the relationship between grip strength declines and muscle-tendon responses induced by long-term performance of a high-repetition, low-force (HRLF) reaching task in rats. We hypothesized that grip strength declines would correlate with inflammation, fibrosis and degradation in flexor digitorum muscles and tendons. Grip strength declined after training, and further in weeks 18 and 24, in reach limbs of HRLF rats. Flexor digitorum tissues of reach limbs showed low-grade increases in inflammatory cytokines: IL-1β after training and in week 18, IL-1α in week 18, TNF-α and IL-6 after training and in week 24, and IL-10 in week 24, with greater increases in tendons than muscles. Similar cytokine increases were detected in serum with HRLF: IL-1α and IL-10 in week 18, and TNF-α and IL-6 in week 24. Grip strength correlated inversely with IL-6 in muscles, tendons and serum, and TNF-α in muscles and serum. Four fibrogenic proteins, TGFB1, CTGF, PDGFab and PDGFbb, and hydroxyproline, a marker of collagen synthesis, increased in serum in HRLF weeks 18 or 24, concomitant with epitendon thickening, increased muscle and tendon TGFB1 and CTGF. A collagenolytic gelatinase, MMP2, increased by week 18 in serum, tendons and muscles of HRLF rats. Grip strength correlated inversely with TGFB1 in muscles, tendons and serum; with CTGF-immunoreactive fibroblasts in tendons; and with MMP2 in tendons and serum. Thus, motor declines correlated with low-grade systemic and musculotendinous inflammation throughout task performance, and increased fibrogenic and degradative proteins with prolonged task performance. Serum TNF-α, IL-6, TGFB1, CTGF and MMP2 may serve as serum biomarkers of work-related musculoskeletal disorders, although further studies in humans are needed

Tracking the international spread of SARS-CoV-2 lineages B.1.1.7 and B.1.351/501Y-V2

Publisher Copyright: © 2021 O'Toole Á et al.Late in 2020, two genetically-distinct clusters of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) with mutations of biological concern were reported, one in the United Kingdom and one in South Africa. Using a combination of data from routine surveillance, genomic sequencing and international travel we track the international dispersal of lineages B.1.1.7 and B.1.351 (variant 501Y-V2). We account for potential biases in genomic surveillance efforts by including passenger volumes from location of where the lineage was first reported, London and South Africa respectively. Using the software tool grinch (global report investigating novel coronavirus haplotypes), we track the international spread of lineages of concern with automated daily reports, Further, we have built a custom tracking website (cov-lineages.org/global_report.html) which hosts this daily report and will continue to include novel SARS-CoV-2 lineages of concern as they are detected.Peer reviewe