Utilizing insights of DNA repair machinery to discover MMEJ deletions and novel mechanisms.

We developed Del-read, an algorithm targeting medium-sized deletions (6-100 bp) in short-reads, which are challenging for current variant callers relying on alignment. Our focus was on Micro-Homolog mediated End Joining deletions (MMEJ-dels), prevalent in myeloid malignancies. MMEJ-dels follow a distinct pattern, occurring between two homologies, allowing us to generate a comprehensive list of MMEJ-dels in the exome.

Targeting mutations in leukemia with RKI-1447: A strategy to impair cellular division and nuclear structure.

Spliceosome machinery mutations are common early mutations in myeloid malignancies; however, effective targeted therapies against them are still lacking. In the current study, we used an high-throughput drug screen among four different isogenic cell lines and identified RKI-1447, a Rho-associated protein kinase inhibitor, as selective cytotoxic effector of mutant cells. RKI-1447 targeted mutated primary human samples in xenografts models.

Age-related micro-environmental changes as drivers of clonal hematopoiesis.

Purpose Of Review: Both aging and reduced diversity at the hematopoietic stem cells (HSCs) level are ubiquitous. What remains unclear is why some individuals develop clonal hematopoiesis (CH), and why does CH due to specific mutations occur in specific individuals. Much like aging, reduced diversity of HSCs is a complex phenotype shaped by numerous factors (germline & environment).

The role of RNF149 in the pre-emptive quality control substrate ubiquitination.

Protein quality control is a process in which a protein's folding status is constantly monitored. Mislocalized proteins (MLP), are processed by the various quality control pathways, as they are often misfolded due to inappropriate cellular surroundings. Polypeptides that fail to translocate into the ER due to an inefficient signal peptide, mutations or ER stress are recognized by the pre-emptive ER associated quality control (pEQC) pathway and degraded by the 26 S proteasome.

Clonal hematopoiesis in the donor does not adversely affect long-term outcomes following allogeneic hematopoietic stem cell transplantation: result from a 13-year follow-up.

Donor clonal hematopoiesis may be transferred to the recipient through allogeneic hematopoietic stem cell transplantation (HSCT), but the potential for adverse long-term impact on transplant outcomes remains unknown. A total of 744 samples from 372 recipients who received HSCT and the corresponding donors were included. Bar-coded error-corrected sequencing using a modified molecular inversion probe capture protocol was performed, which targeted 33 genes covering mutations involved in clonal hematopoiesis with indeterminate potential (CHIP) and other acute myeloid leukemia-related mutations.

Pseudo-mutant P53 is a unique phenotype of -mutated pre-leukemia.

Pre-leukemic clones carrying DNMT3A mutations have a selective advantage and an inherent chemoresistance, however the basis for this phenotype has not been fully elucidated. Mutations affecting the gene TP53 occur in pre-leukemic hematopoietic stem/progenitor cells (preL-HSPC) and lead to chemoresistance. Many of these mutations cause a conformational change and some of them were shown to enhance self-renewal capacity of preL-HSPC.

An improved molecular inversion probe based targeted sequencing approach for low variant allele frequency.

Deep targeted sequencing technologies are still not widely used in clinical practice due to the complexity of the methods and their cost. The Molecular Inversion Probes (MIP) technology is cost effective and scalable in the number of targets, however, suffers from low overall performance especially in GC rich regions. In order to improve the MIP performance, we sequenced a large cohort of healthy individuals ( = 4417), with a panel of 616 MIPs, at high depth in duplicates.

Recurrent deletions in clonal hematopoiesis are driven by microhomology-mediated end joining.

The mutational mechanisms underlying recurrent deletions in clonal hematopoiesis are not entirely clear. In the current study we inspect the genomic regions around recurrent deletions in myeloid malignancies, and identify microhomology-based signatures in CALR, ASXL1 and SRSF2 loci. We demonstrate that these deletions are the result of double stand break repair by a PARP1 dependent microhomology-mediated end joining (MMEJ) pathway.

Dasatinib response in acute myeloid leukemia is correlated with FLT3/ITD, PTPN11 mutations and a unique gene expression signature.

Novel targeted therapies demonstrate improved survival in specific subgroups (defined by genetic variants) of acute myeloid leukemia (AML) patients, validating the paradigm of molecularly targeted therapy. However, identifying correlations between AML molecular attributes and effective therapies is challenging. Recent advances in high-throughput in vitro drug sensitivity screening applied to primary AML blasts were used to uncover such correlations; however, these methods cannot predict the response of leukemic stem cells (LSCs).

The Median Eminence, A New Oligodendrogenic Niche in the Adult Mouse Brain.

The subventricular zone (SVZ) of the lateral ventricles and the subgranular zone (SGZ) of the dentate gyrus in the hippocampus are known as neurogenic niches. We show that the median eminence (ME) of the hypothalamus comprises BrdU newly proliferating cells co-expressing NG2 (oligodendrocyte progenitors) and RIP (pre-myelinating oligodendrocytes), suggesting their differentiation toward mature oligodendrocytes (OLs). ME cells can generate neurospheres (NS) in vitro, which differentiate mostly to OLs compared with SVZ-NS that typically generate neurons.

Antagonistic Center-Surround Mechanisms for Direction Selectivity in the Retina.

An antagonistic center-surround receptive field is a key feature in sensory processing, but how it contributes to specific computations such as direction selectivity is often unknown. Retinal On-starburst amacrine cells (SACs), which mediate direction selectivity in direction-selective ganglion cells (DSGCs), exhibit antagonistic receptive field organization: depolarizing to light increments and decrements in their center and surround, respectively. We find that a repetitive stimulation exhausts SAC center and enhances its surround and use it to study how center-surround responses contribute to direction selectivity.

Circulating Picomolar Levels of CCL2 Downregulate Ongoing Chronic Experimental Autoimmune Encephalomyelitis by Induction of Regulatory Mechanisms.

Multiple sclerosis is an inflammatory disease of the CNS characterized by neurologic impairment resulting from primary demyelination and axonal damage. The pathogenic mechanisms of disease development include Ag-specific T cell activation and Th1 differentiation, followed by T cell and macrophage migration into the CNS. CCL2 is a chemokine that induces migration of monocytes, memory T cells, and dendritic cells.

Daily Onset of Light and Darkness Differentially Controls Hematopoietic Stem Cell Differentiation and Maintenance.

Hematopoietic stem and progenitor cells (HSPCs) tightly couple maintenance of the bone marrow (BM) reservoir, including undifferentiated long-term repopulating hematopoietic stem cells (LT-HSCs), with intensive daily production of mature leukocytes and blood replenishment. We found two daily peaks of BM HSPC activity that are initiated by onset of light and darkness providing this coupling. Both peaks follow transient elevation of BM norepinephrine and TNF secretion, which temporarily increase HSPC reactive oxygen species (ROS) levels.

Small Molecules Co-targeting CKIα and the Transcriptional Kinases CDK7/9 Control AML in Preclinical Models.

CKIα ablation induces p53 activation, and CKIα degradation underlies the therapeutic effect of lenalidomide in a pre-leukemia syndrome. Here we describe the development of CKIα inhibitors, which co-target the transcriptional kinases CDK7 and CDK9, thereby augmenting CKIα-induced p53 activation and its anti-leukemic activity. Oncogene-driving super-enhancers (SEs) are highly sensitive to CDK7/9 inhibition.

Inhomogeneous Encoding of the Visual Field in the Mouse Retina.

Stimulus characteristics of the mouse's visual field differ above and below the skyline. Here, we show for the first time that retinal ganglion cells (RGCs), the output neurons of the retina, gradually change their functional properties along the ventral-dorsal axis to allow better representation of the different stimulus characteristics. We conducted two-photon targeted recordings of transient-Offα-RGCs and found that they gradually became more sustained along the ventral-dorsal axis, revealing >5-fold-longer duration responses in the dorsal retina.

CCR2 Regulates the Immune Response by Modulating the Interconversion and Function of Effector and Regulatory T Cells.

Chemokines and chemokine receptors establish a complex network modulating immune cell migration and localization. These molecules were also suggested to mediate the differentiation of leukocytes; however, their intrinsic, direct regulation of lymphocyte fate remained unclear. CCR2 is the main chemokine receptor inducing macrophage and monocyte recruitment to sites of inflammation, and it is also expressed on T cells.

Pathways and gene networks mediating the regulatory effects of cannabidiol, a nonpsychoactive cannabinoid, in autoimmune T cells.

Background: Our previous studies showed that the non-psychoactive cannabinoid, cannabidiol (CBD), ameliorates the clinical symptoms in mouse myelin oligodendrocyte glycoprotein (MOG)35-55-induced experimental autoimmune encephalomyelitis model of multiple sclerosis (MS) as well as decreases the memory MOG35-55-specific T cell (TMOG) proliferation and cytokine secretion including IL-17, a key autoimmune factor. The mechanisms of these activities are currently poorly understood.

Methods: Herein, using microarray-based gene expression profiling, we describe gene networks and intracellular pathways involved in CBD-induced suppression of these activated memory TMOG cells.

Post-CNS-inflammation expression of CXCL12 promotes the endogenous myelin/neuronal repair capacity following spontaneous recovery from multiple sclerosis-like disease.

Background: Demyelination and axonal degeneration, hallmarks of multiple sclerosis (MS), are associated with the central nervous system (CNS) inflammation facilitated by C-X-C motif chemokine 12 (CXCL12) chemokine. Both in MS and in experimental autoimmune encephalomyelitis (EAE), the deleterious CNS inflammation has been associated with upregulation of CXCL12 expression in the CNS. We investigated the expression dynamics of CXCL12 in the CNS with progression of clinical EAE and following spontaneous recovery, with a focus on CXCL12 expression in the hippocampal neurogenic dentate gyrus (DG) and in the corpus callosum (CC) of spontaneously recovered mice, and its potential role in promoting the endogenous myelin/neuronal repair capacity.

Anti-inflammatory effects of the cannabidiol derivative dimethylheptyl-cannabidiol - studies in BV-2 microglia and encephalitogenic T cells.

Background: Dimethylheptyl-cannabidiol (DMH-CBD), a non-psychoactive, synthetic derivative of the phytocannabinoid cannabidiol (CBD), has been reported to be anti-inflammatory in RAW macrophages. Here, we evaluated the effects of DMH-CBD at the transcriptional level in BV-2 microglial cells as well as on the proliferation of encephalitogenic T cells.

Methods: BV-2 cells were pretreated with DMH-CBD, followed by stimulation with the endotoxin lipopolysaccharide (LPS).

CD151 Regulates T-Cell Migration in Health and Inflammatory Bowel Disease.

The continuous recirculation of mature lymphocytes and their entry into the peripheral lymph nodes are crucial for the development of an immune response to foreign antigens. Occasionally, the entry and the subsequent response of T lymphocytes in these sites lead to severe inflammation and pathological conditions. Here, we characterized the tetraspanin molecule, CD151, as a regulator of T cell motility in health and in models of inflammatory bowel disease.

HU-446 and HU-465, Derivatives of the Non-psychoactive Cannabinoid Cannabidiol, Decrease the Activation of Encephalitogenic T Cells.

Cannabidiol (CBD), the non-psychoactive cannabinoid, has been previously shown by us to decrease peripheral inflammation and neuroinflammation in mouse experimental autoimmune encephalomyelitis (EAE) model of multiple sclerosis (MS). Here we have studied the anti-inflammatory effects of newly synthesized derivatives of natural (-)-CBD ((-)-8,9-dihydro-7-hydroxy-CBD; HU-446) and of synthetic (+)-CBD ((+)-8,9-dihydro-7-hydroxy-CBD; HU-465) on activated myelin oligodendrocyte glycoprotein (MOG)35-55-specific mouse encephalitogenic T cells (T(MOG) ) driving EAE/MS-like pathologies. Binding assays followed by molecular modeling revealed that HU-446 has negligible affinity toward the cannabinoid CB1 and CB2 receptors while HU-465 binds to both CB1 and CB2 receptors at the high nanomolar concentrations (Ki = 76.

Role of a Novel Human Leukocyte Antigen-DQA1*01:02;DRB1*15:01 Mixed Isotype Heterodimer in the Pathogenesis of "Humanized" Multiple Sclerosis-like Disease.

Gene-wide association and candidate gene studies indicate that the greatest effect on multiple sclerosis (MS) risk is driven by the HLA-DRB1*15:01 allele within the HLA-DR15 haplotype (HLA-DRB1*15:01-DQA1*01:02-DQB1*0602-DRB5*01:01). Nevertheless, linkage disequilibrium makes it difficult to define, without functional studies, whether the functionally relevant effect derives from DRB1*15:01 only, from its neighboring DQA1*01:02-DQB1*06:02 or DRB5*01:01 genes of HLA-DR15 haplotype, or from their combinations or epistatic interactions. Here, we analyzed the impact of the different HLA-DR15 haplotype alleles on disease susceptibility in a new "humanized" model of MS induced in HLA-transgenic (Tg) mice by human oligodendrocyte-specific protein (OSP)/claudin-11 (hOSP), one of the bona fide potential primary target antigens in MS.

Cannabidiol, a non-psychoactive cannabinoid, leads to EGR2-dependent anergy in activated encephalitogenic T cells.

Background: Cannabidiol (CBD), the main non-psychoactive cannabinoid, has been previously shown by us to ameliorate clinical symptoms and to decrease inflammation in myelin oligodendrocyte glycoprotein (MOG)35-55-induced mouse experimental autoimmune encephalomyelitis model of multiple sclerosis as well as to decrease MOG35-55-induced T cell proliferation and IL-17 secretion. However, the mechanisms of CBD anti-inflammatory activities are unclear.

Methods: Here we analyzed the effects of CBD on splenocytes (source of accessory T cells and antigen presenting cells (APC)) co-cultured with MOG35-55-specific T cells (TMOG) and stimulated with MOG35-55.