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Acute myeloid leukemias (AML) harboring a constitutively active internal tandem duplication (ITD) mutation in the FMS-like kinase tyrosine kinase (FLT3) receptor are associated with poor patient prognosis. Despite initial clinical responses to FLT3 kinase inhibitors, patients eventually relapse. Mechanisms of resistance include the acquisition of secondary FLT3 mutations and protective stromal signaling within the bone marrow niche. Here we show that LAM-003, a prodrug of the heat shock protein 90 inhibitor LAM-003A, has cytotoxic activity against AML cell lines and primary samples harboring FLT3-ITD. LAM-003 regressed tumors in an MV-4-11 xenograft mouse model and extended survival in a MOLM-13 systemic model. LAM-003 displayed synergistic activity with chemotherapeutic drugs and FLT3 inhibitors, with the most robust synergy being obtained with venetoclax, a BCL-2 inhibitor. This finding was verified in a MOLM-13 systemic survival model in which the combination significantly prolonged survival compared with the single agents. Importantly, LAM-003 exhibited equipotent activity against FLT3 inhibitor-resistant mutants of FLT3, such as D835 or F691, in cytotoxic and FLT3 degradation assays. LAM-003 also retained potency in AML cells grown in stromal-conditioned media that were resistant to FLT3 inhibitors. Lastly, a genome-wide CRISPR screen revealed epigenetic regulators, including KDM6A, as determinants of LAM-003 sensitivity in AML cell lines, leading to the discovery of synergy with an EZH2 inhibitor. Collectively, these preclinical findings support the use of LAM-003 in FLT3-ITD patients with AML who no longer respond to FLT3 inhibitor therapy either as a single agent or in combination with drugs known to be active in AML.

We identified apilimod as an antiproliferative compound by high-throughput screening of clinical-stage drugs. Apilimod exhibits exquisite specificity for phosphatidylinositol-3-phosphate 5-kinase (PIKfyve) lipid kinase and has selective cytotoxic activity in B-cell non-Hodgkin lymphoma (B-NHL) compared with normal cells. Apilimod displays nanomolar activity in vitro, and in vivo studies demonstrate single-agent efficacy as well as synergy with approved B-NHL drugs. Using biochemical and knockdown approaches, and discovery of a kinase domain mutation conferring resistance, we demonstrate that apilimod-mediated cytotoxicity is driven by PIKfyve inhibition. Furthermore, a critical role for lysosome dysfunction as a major factor contributing to apilimod's cytotoxicity is supported by a genome-wide CRISPR screen. In the screen, TFEB (master transcriptional regulator of lysosomal biogenesis) and endosomal/lysosomal genes CLCN7, OSTM1, and SNX10 were identified as important determinants of apilimod sensitivity. These findings thus suggest that disruption of lysosomal homeostasis with apilimod represents a novel approach to treat B-NHL.

Lamin B1 is a component of the nuclear lamina and plays a critical role in maintaining nuclear architecture, regulating gene expression and modulating chromatin positioning. We have previously shown that LMNB1 gene duplications cause autosomal dominant leukodystrophy (ADLD), a fatal adult onset demyelinating disease. The mechanisms by which increased LMNB1 levels cause ADLD are unclear. To address this, we used a transgenic mouse model where Lamin B1 overexpression is targeted to oligodendrocytes. These mice showed severe vacuolar degeneration of the spinal cord white matter together with marked astrogliosis, microglial infiltration, and secondary axonal damage. Oligodendrocytes in the transgenic mice revealed alterations in histone modifications favoring a transcriptionally repressed state. Chromatin changes were accompanied by reduced expression of genes involved in lipid synthesis pathways, many of which are known to play important roles in myelin regulation and are preferentially expressed in oligodendrocytes. Decreased lipogenic gene expression resulted in a significant reduction in multiple classes of lipids involved in myelin formation. Many of these gene expression changes and lipid alterations were observed even before the onset of the phenotype, suggesting a causal role. Our findings establish, for the first time, a link between LMNB1 and lipid synthesis in oligodendrocytes, and provide a mechanistic framework to explain the age dependence and white matter involvement of the disease phenotype. These results have implications for disease pathogenesis and may also shed light on the regulation of lipid synthesis pathways in myelin maintenance and turnover. SIGNIFICANCE STATEMENT Autosomal dominant leukodystrophy (ADLD) is fatal neurological disorder caused by increased levels of the nuclear protein, Lamin B1. The disease is characterized by an age-dependent loss of myelin, the fatty sheath that covers nerve fibers. We have studied a mouse model where Lamin B1 level are increased in oligodendrocytes, the cell type that produces myelin in the CNS. We demonstrate that destruction of myelin in the spinal cord is responsible for the degenerative phenotype in our mouse model. We show that this degeneration is mediated by reduced expression of lipid synthesis genes and the subsequent reduction in myelin enriched lipids. These findings provide a mechanistic framework to explain the age dependence and tissue specificity of the ADLD disease phenotype.

International audience; Emerging and strengthening evidence suggests an importantrole of myelin in plasticity and axonal survival. However, themechanisms regulating progression from oligodendrocyteprogenitor cells (OPCs) to myelinating oligodendrocytes remainonly partially understood. A series of overlapping yet distinctepigenetic events occur as a proliferating OPC exits the cellcycle, initiates differentiation, and becomes a myelin-formingoligodendrocyte that wraps axons. Here we discuss recentadvances towards understanding the epigenetic control ofoligodendrocyte development that integrates environmentalstimuli. We suggest that OPCs are directly responsive toextrinsic signals due to predominantly euchromatic nuclei,while the heterochromatic nuclei render differentiating andmyelinating cells less susceptible to signals modulating theepigenome.

We identified the PIKFYVE inhibitor apilimod as a potent and selective cytotoxic agent against B-cell non-Hodgkin lymphoma (B-NHL). Our data robustly establish PIKFYVE as the target through which apilimod kills B-NHL cells and show that apilimod-induced death in B-NHL is mediated by broad disruption of lysosome homeostasis characterized by lysosomal swelling, TFEB nuclear translocation, impaired maturation of lysosomal enzymes and incomplete autophagosome clearance. Furthermore, through genome-wide CRISPR knockout screening, we identified specific lysosomal genes (TFEB, CLCN7, OSTM1 and SNX10) as critical determinants of apilimod-induced cytotoxicity. Together these data highlight disruption of lysosome homeostasis through PIKFYVE inhibition as a novel anticancer mechanism in B-NHL and potentially other cancers.

The recent years have been characterized by a surge of studies on the role of transcription factors and histone modifications in regulating the progression of progenitors into oligodendrocytes. This review summarizes this body of evidence and presents an integrated view of transcriptional networks and epigenetic regulators defining proliferating progenitors and their differentiation along the oligodendrocyte lineage. We suggest that transcription factors in proliferating progenitors have direct access to DNA, due to predominantly euchromatic nuclei. As progenitors differentiate, however, transcriptional competence is modulated by the formation of heterochromatin, which modifies the association of DNA with nucleosomal histones and renders the access of transcription factors dependent on the activity of epigenetic modulators. These concepts are delineated within the context of development, and the potential functional implications are discussed.

β-Arrestin–mediated signaling downstream of seven transmembrane receptors (7TMRs) is a relatively new paradigm for signaling by these receptors. We examined changes in protein phosphorylation occurring when HEK293 cells expressing the angiotensin II type 1A receptor (AT1aR) were stimulated with the β-arrestin–biased ligand Sar 1 , Ile 4 , Ile 8 -angiotensin (SII), a ligand previously found to signal through β-arrestin–dependent, G protein-independent mechanisms. Using a phospho-antibody array containing 46 antibodies against signaling molecules, we found that phosphorylation of 35 proteins increased upon SII stimulation. These SII-mediated phosphorylation events were abrogated after depletion of β-arrestin 2 through siRNA-mediated knockdown. We also performed an MS-based quantitative phosphoproteome analysis after SII stimulation using a strategy of stable isotope labeling of amino acids in cell culture (SILAC). We identified 1,555 phosphoproteins (4,552 unique phosphopeptides), of which 171 proteins (222 phosphopeptides) showed increased phosphorylation, and 53 (66 phosphopeptides) showed decreased phosphorylation upon SII stimulation of the AT1aR. This study identified 38 protein kinases and three phosphatases whose phosphorylation status changed upon SII treatment. Using computational approaches, we performed system-based analyses examining the β-arrestin–mediated phosphoproteome including construction of a kinase-substrate network for β-arrestin–mediated AT1aR signaling. Our analysis demonstrates that β-arrestin–dependent signaling processes are more diverse than previously appreciated. Notably, our analysis identifies an AT1aR-mediated cytoskeletal reorganization network whereby β-arrestin regulates phosphorylation of several key proteins, including cofilin and slingshot. This study provides a system-based view of β-arrestin–mediated phosphorylation events downstream of a 7TMR and opens avenues for research in a rapidly evolving area of 7TMR signaling.