Deletion of histone deacetylase 3 in adult beta cells improves glucose tolerance via increased insulin secretion

Objective: Histone deacetylases are epigenetic regulators known to control gene transcription in various tissues. A member of this family, histone deacetylase 3 (HDAC3), has been shown to regulate metabolic genes. Cell culture studies with HDAC-specific inhibitors and siRNA suggest that HDAC3 plays a role in pancreatic β-cell function, but a recent genetic study in mice has been contradictory. Here we address the functional role of HDAC3 in β-cells of adult mice. Methods: An HDAC3 β-cell specific knockout was generated in adult MIP-CreERT transgenic mice using the Cre-loxP system. Induction of HDAC3 deletion was initiated at 8 weeks of age with administration of tamoxifen in corn oil (2 mg/day for 5 days). Mice were assayed for glucose tolerance, glucose-stimulated insulin secretion, and islet function 2 weeks after induction of the knockout. Transcriptional functions of HDAC3 were assessed by ChIP-seq as well as RNA-seq comparing control and β-cell knockout islets. Results: HDAC3 β-cell specific knockout (HDAC3βKO) did not increase total pancreatic insulin content or β-cell mass. However, HDAC3βKO mice demonstrated markedly improved glucose tolerance. This improved glucose metabolism coincided with increased basal and glucose-stimulated insulin secretion in vivo as well as in isolated islets. Cistromic and transcriptomic analyses of pancreatic islets revealed that HDAC3 regulates multiple genes that contribute to glucose-stimulated insulin secretion. Conclusions: HDAC3 plays an important role in regulating insulin secretion in vivo, and therapeutic intervention may improve glucose homeostasis. Author Video: Author Video Watch what authors say about their articles Keywords: HDAC3, Glucose tolerance, Insulin secretio

Genetic disruption of N-RasG12D palmitoylation perturbs hematopoiesis and prevents myeloid transformation in mice.

Oncogenic RAS mutations pose substantial challenges for rational drug discovery. Sequence variations within the hypervariable region of Ras isoforms underlie differential posttranslational modification and subcellular trafficking, potentially resulting in selective vulnerabilities. Specifically, inhibiting the palmitoylation/depalmitoylation cycle is an appealing strategy for treating NRAS mutant cancers, particularly as normal tissues would retain K-Ras4b function for physiologic signaling. The role of endogenous N-RasG12D palmitoylation in signal transduction, hematopoietic differentiation, and myeloid transformation is unknown, and addressing these key questions will inform efforts to develop mechanism-based therapies. To evaluate the palmitoylation/depalmitoylation cycle as a candidate drug target in an in vivo disease-relevant model system, we introduced a C181S mutation into a conditional NrasG12D "knock-in" allele. The C181S second-site amino acid substitution abrogated myeloid transformation by NrasG12D, which was associated with mislocalization of the nonpalmitoylated N-Ras mutant protein, reduced Raf/MEK/ERK signaling, and alterations in hematopoietic stem and progenitor populations. Furthermore, hematologic malignancies arising in NrasG12D/G12D,C181S compound heterozygous mice invariably acquired revertant mutations that restored cysteine 181. Together, these studies validate the palmitoylation cycle as a promising therapeutic target in NRAS mutant cancers

Assigning functionality to cysteines by base editing of cancer dependency genes

Covalent chemistry represents an attractive strategy for expanding the ligandability of the proteome, and chemical proteomics has revealed numerous electrophile-reactive cysteines on diverse human proteins. Determining which of these covalent binding events affect protein function, however, remains challenging. Here we describe a base-editing strategy to infer the functionality of cysteines by quantifying the impact of their missense mutation on cancer cell proliferation. The resulting atlas, which covers more than 13,800 cysteines on more than 1,750 cancer dependency proteins, confirms the essentiality of cysteines targeted by covalent drugs and, when integrated with chemical proteomic data, identifies essential, ligandable cysteines in more than 160 cancer dependency proteins. We further show that a stereoselective and site-specific ligand targeting an essential cysteine in TOE1 inhibits the nuclease activity of this protein through an apparent allosteric mechanism. Our findings thus describe a versatile method and valuable resource to prioritize the pursuit of small-molecule probes with high function-perturbing potential

ABHD17 regulation of plasma membrane palmitoylation and N-Ras-dependent cancer growth

Multiple Ras proteins, including N-Ras, depend on a palmitoylation/depalmitoylation cycle to regulate their subcellular trafficking and oncogenicity. General lipase inhibitors such as Palmostatin M (Palm M) block N-Ras depalmitoylation, but lack specificity and target several enzymes displaying depalmitoylase activity. Here, we describe ABD957, a potent and selective covalent inhibitor of the ABHD17 family of depalmitoylases, and show that this compound impairs N-Ras depalmitoylation in human acute myeloid leukemia (AML) cells. ABD957 produced partial effects on N-Ras palmitoylation compared with Palm M, but was much more selective across the proteome, reflecting a plasma membrane-delineated action on dynamically palmitoylated proteins. Finally, ABD957 impaired N-Ras signaling and the growth of NRAS-mutant AML cells in a manner that synergizes with MAP kinase kinase (MEK) inhibition. Our findings uncover a surprisingly restricted role for ABHD17 enzymes as regulators of the N-Ras palmitoylation cycle and suggest that ABHD17 inhibitors may have value as targeted therapies for NRAS-mutant cancers