Bruton's Tyrosine Kinase Inhibitors with Distinct Binding Modes Reveal Differential Functional Impact on B-Cell Receptor Signaling.

Small molecule inhibitors of Bruton's tyrosine kinase (BTK) have been approved for the treatment of multiple B-cell malignancies and are being evaluated for autoimmune and inflammatory diseases. Various BTK inhibitors (BTKi) have distinct potencies, selectivity profiles, and binding modes within the ATP-binding site. On the basis of the latter feature, BTKis can be classified into those that occupy the back-pocket, H3 pocket, and the hinge region only.

Evaluation of the DLL3-targeting antibody-drug conjugate rovalpituzumab tesirine in preclinical models of neuroblastoma.

Neuroblastomas have neuroendocrine features and often show similar gene expression patterns to small cell lung cancer including high expression of delta-like ligand 3 (). Here we determine the efficacy of rovalpituzumab tesirine (Rova-T), an antibody drug conjugated (ADC) with a pyrrolobenzodiazepine (PBD) dimer toxin targeting DLL3, in preclinical models of human neuroblastoma. We evaluated DLL3 expression in RNA sequencing data sets and performed immunohistochemistry (IHC) on neuroblastoma patient derived xenograft (PDX), human neuroblastoma primary tumor and normal childhood tissue microarrays (TMAs).

Venetoclax-based Rational Combinations are Effective in Models of -amplified Neuroblastoma.

Venetoclax is a small molecule inhibitor of the prosurvival protein BCL-2 that has gained market approval in BCL-2-dependent hematologic cancers including chronic lymphocytic leukemia and acute myeloid leukemia. Neuroblastoma is a heterogenous pediatric cancer with a 5-year survival rate of less than 50% for high-risk patients, which includes nearly all cases with amplified We previously demonstrated that venetoclax is active in -amplified neuroblastoma but has limited single-agent activity in most models, presumably the result of other pro-survival BCL-2 family protein expression or insufficient prodeath protein mobilization. As the relative tolerability of venetoclax makes it amenable to combining with other therapies, we evaluated the sensitivity of -amplified neuroblastoma models to rational combinations of venetoclax with agents that have both mechanistic complementarity and active clinical programs.

An antibody-drug conjugate directed to the ALK receptor demonstrates efficacy in preclinical models of neuroblastoma.

Enthusiasm for the use of antibody-drug conjugates (ADCs) in cancer therapy has risen over the past few years. The success of this therapeutic approach relies on the identification of cell surface antigens that are widely and selectively expressed on tumor cells. Studies have shown that native ALK protein is expressed on the surface of most neuroblastoma cells, providing an opportunity for development of immune-targeting strategies.

Mitochondria-associated ER membranes (MAMs) and lysosomal storage diseases.

Lysosomal storage diseases (LSDs) comprise a large group of disorders of catabolism, mostly due to deficiency of a single glycan-cleaving hydrolase. The consequent endo-lysosomal accumulation of undigested or partially digested substrates in cells of virtually all organs, including the nervous system, is diagnostic of these diseases and underlies pathogenesis. A subgroup of LSDs, the glycosphingolipidoses, are caused by deficiency of glycosidases that process/degrade sphingolipids and glycosphingolipids (GSLs).

Dual and Inhibition Demonstrates Synergy against Neuroblastoma.

Anaplastic lymphoma kinase () is the most frequently mutated oncogene in the pediatric cancer neuroblastoma. We performed an screen for synergistic drug combinations that target neuroblastomas with mutations in to determine whether drug combinations could enhance antitumor efficacy. We screened combinations of eight molecularly targeted agents against 17 comprehensively characterized human neuroblastoma-derived cell lines.

Orphan Nuclear Receptor NR4A1 Binds a Novel Protein Interaction Site on Anti-apoptotic B Cell Lymphoma Gene 2 Family Proteins.

B cell lymphoma gene 2 (Bcl-2) family proteins are key regulators of programmed cell death and important targets for drug discovery. Pro-apoptotic and anti-apoptotic Bcl-2 family proteins reciprocally modulate their activities in large part through protein interactions involving a motif known as BH3 (Bcl-2 homology 3). Nur77 is an orphan member of the nuclear receptor family that lacks a BH3 domain but nevertheless binds certain anti-apoptotic Bcl-2 family proteins (Bcl-2, Bfl-1, and Bcl-B), modulating their effects on apoptosis and autophagy.

Exploitation of the Apoptosis-Primed State of MYCN-Amplified Neuroblastoma to Develop a Potent and Specific Targeted Therapy Combination.

Fewer than half of children with high-risk neuroblastoma survive. Many of these tumors harbor high-level amplification of MYCN, which correlates with poor disease outcome. Using data from our large drug screen we predicted, and subsequently demonstrated, that MYCN-amplified neuroblastomas are sensitive to the BCL-2 inhibitor ABT-199.

The ALK/ROS1 Inhibitor PF-06463922 Overcomes Primary Resistance to Crizotinib in ALK-Driven Neuroblastoma.

Unlabelled: Neuroblastomas harboring activating point mutations in anaplastic lymphoma kinase (ALK) are differentially sensitive to the ALK inhibitor crizotinib, with certain mutations conferring intrinsic crizotinib resistance. To overcome this clinical obstacle, our goal was to identify inhibitors with improved potency that can target intractable ALK variants such as F1174L. We find that PF-06463922 has high potency across ALK variants and inhibits ALK more effectively than crizotinib in vitro.

Crizotinib Synergizes with Chemotherapy in Preclinical Models of Neuroblastoma.

Purpose: The presence of an ALK aberration correlates with inferior survival for patients with high-risk neuroblastoma. The emergence of ALK inhibitors such as crizotinib has provided novel treatment opportunities. However, certain ALK mutations result in de novo crizotinib resistance, and a phase I trial of crizotinib showed a lack of response in patients harboring those ALK mutations.

ER stress-induced cell death mechanisms.

The endoplasmic-reticulum (ER) stress response constitutes a cellular process that is triggered by a variety of conditions that disturb folding of proteins in the ER. Eukaryotic cells have developed an evolutionarily conserved adaptive mechanism, the unfolded protein response (UPR), which aims to clear unfolded proteins and restore ER homeostasis. In cases where ER stress cannot be reversed, cellular functions deteriorate, often leading to cell death.

Interleukin-1 receptor-associated kinase-2 (IRAK2) is a critical mediator of endoplasmic reticulum (ER) stress signaling.

Endoplasmic reticulum (ER) stress occurs when unfolded proteins accumulate in the lumen of the organelle, triggering signal transduction events that contribute either to cellular adaptation and recovery or alternatively to cellular dysfunction and death. ER stress has been implicated in numerous diseases. To identify novel modulators of ER stress, we undertook a siRNA library screen of the kinome, revealing Interleukin-1 Receptor-Associated Kinase-2 (IRAK2) as a contributor to unfolded protein response (UPR) signaling and ER stress-induced cell death.

Endoplasmic reticulum protein BI-1 regulates Ca²⁺-mediated bioenergetics to promote autophagy.

Autophagy is a lysosomal degradation pathway that converts macromolecules into substrates for energy production during nutrient-scarce conditions such as those encountered in tumor microenvironments. Constitutive mitochondrial uptake of endoplasmic reticulum (ER) Ca²⁺ mediated by inositol triphosphate receptors (IP₃Rs) maintains cellular bioenergetics, thus suppressing autophagy. We show that the ER membrane protein Bax inhibitor-1 (BI-1) promotes autophagy in an IP₃R-dependent manner.

Synthetic triterpenoid cyano enone of methyl boswellate activates intrinsic, extrinsic, and endoplasmic reticulum stress cell death pathways in tumor cell lines.

We explored the effect of a novel synthetic triterpenoid compound cyano enone of methyl boswellates (CEMB) on various prostate cancer and glioma cancer cell lines. CEMB displayed concentration-dependent cytotoxic activity with submicromolar lethal dose 50% (LD(50)) values in 10 of 10 tumor cell lines tested. CEMB-induced cytotoxicity is accompanied by activation of downstream effector caspases (caspases 3 and 7) and by upstream initiator caspases involved in both the extrinsic (caspase 8) and intrinsic (caspase 9) apoptotic pathways.

Population analysis of the GLB1 gene in South Brazil.

Infantile GM1 gangliosidosis is caused by the absence or reduction of lysosomal beta-galactosidase activity. Studies conducted in Brazil have indicated that it is one of the most frequent lysosomal storage disorders in the southern part of the country. To assess the incidence of this disorder, 390 blood donors were tested for the presence of two common mutations (1622-1627insG and R59H) in the GLB1 gene.

High-throughput fluorescence assay for small-molecule inhibitors of autophagins/Atg4.

Autophagy is an evolutionarily conserved process for catabolizing damaged proteins and organelles in a lysosome-dependent manner. Dysregulation of autophagy may cause various diseases, such as cancer and neurodegeneration. However, the relevance of autophagy to diseases remains controversial because of the limited availability of chemical modulators.

GM1-ganglioside accumulation at the mitochondria-associated ER membranes links ER stress to Ca(2+)-dependent mitochondrial apoptosis.

Mitochondria-associated ER membranes, or MAMs, define the sites of endoplasmic reticulum/mitochondria juxtaposition that control Ca(2+) flux between these organelles. We found that in a mouse model of the human lysosomal storage disease GM1-gangliosidosis, GM1-ganglioside accumulates in the glycosphingolipid-enriched microdomain (GEM) fractions of MAMs, where it interacts with the phosphorylated form of IP3 receptor-1, influencing the activity of this channel. Ca(2+) depleted from the ER is then taken up by the mitochondria, leading to Ca(2+) overload in this organelle.

Chemokine-induced recruitment of genetically modified bone marrow cells into the CNS of GM1-gangliosidosis mice corrects neuronal pathology.

Bone marrow cells (BMCs) could correct some pathologic conditions of the central nervous system (CNS) if these cells would effectively repopulate the brain. One such condition is G(M1)-gangliosidosis, a neurodegenerative glycosphingolipidosis due to deficiency of lysosomal beta-galactosidase (beta-gal). In this disease, abnormal build up of G(M1)-ganglioside in the endoplasmic reticulum of brain cells results in calcium imbalance, induction of an unfolded protein response (UPR), and neuronal apoptosis.

G(M1)-ganglioside degradation and biosynthesis in human and murine G(M1)-gangliosidosis.

Background: Gangliosides are building blocks of cell membranes and their biosynthesis and degradation have been extensively studied in the past. Regulation of the metabolism of these glycolipids controls fundamental cell functions. G(M1)-gangliosidosis, a neurodegenerative glycosphingolipid storage disease, is caused by deficiency of lysosomal beta-galactosidase with consequent disruption of the normal degradative pathway of G(M1)-ganglioside.

GM1-ganglioside-mediated activation of the unfolded protein response causes neuronal death in a neurodegenerative gangliosidosis.

GM1-ganglioside (GM1) is a major sialoglycolipid of neuronal membranes that, among other functions, modulates calcium homeostasis. Excessive accumulation of GM1 due to deficiency of lysosomal beta-galactosidase (beta-gal) characterizes the neurodegenerative disease GM1-gangliosidosis, but whether the accumulation of GM1 is directly responsible for CNS pathogenesis was unknown. Here we demonstrate that activation of an unfolded protein response (UPR) associated with the upregulation of BiP and CHOP and the activation of JNK2 and caspase-12 leads to neuronal apoptosis in the mouse model of GM1-gangliosidosis.