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.

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.

Deficiency of the ribosome biogenesis gene Sbds in hematopoietic stem and progenitor cells causes neutropenia in mice by attenuating lineage progression in myelocytes.

Shwachman-Diamond syndrome is a congenital bone marrow failure disorder characterized by debilitating neutropenia. The disease is associated with loss-of-function mutations in the SBDS gene, implicated in ribosome biogenesis, but the cellular and molecular events driving cell specific phenotypes in ribosomopathies remain poorly defined. Here, we established what is to our knowledge the first mammalian model of neutropenia in Shwachman-Diamond syndrome through targeted downregulation of Sbds in hematopoietic stem and progenitor cells expressing the myeloid transcription factor CCAAT/enhancer binding protein α (Cebpa). Sbds deficiency in the myeloid lineage specifically affected myelocytes and their downstream progeny while, unexpectedly, it was well tolerated by rapidly cycling hematopoietic progenitor cells. Molecular insights provided by massive parallel sequencing supported cellular observations of impaired cell cycle exit and formation of secondary granules associated with the defect of myeloid lineage progression in myelocytes. Mechanistically, Sbds deficiency activated the p53 tumor suppressor pathway and induced apoptosis in these cells. Collectively, the data reveal a previously unanticipated, selective dependency of myelocytes and downstream progeny, but not rapidly cycling progenitors, on this ubiquitous ribosome biogenesis protein, thus providing a cellular basis for the understanding of myeloid lineage biased defects in Shwachman-Diamond syndrome.

Mesenchymal Inflammation Drives Genotoxic Stress in Hematopoietic Stem Cells and Predicts Disease Evolution in Human Pre-leukemia.

Mesenchymal niche cells may drive tissue failure and malignant transformation in the hematopoietic system, but the underlying molecular mechanisms and relevance to human disease remain poorly defined. Here, we show that perturbation of mesenchymal cells in a mouse model of the pre-leukemic disorder Shwachman-Diamond syndrome (SDS) induces mitochondrial dysfunction, oxidative stress, and activation of DNA damage responses in hematopoietic stem and progenitor cells. Massive parallel RNA sequencing of highly purified mesenchymal cells in the SDS mouse model and a range of human pre-leukemic syndromes identified p53-S100A8/9-TLR inflammatory signaling as a common driving mechanism of genotoxic stress. Transcriptional activation of this signaling axis in the mesenchymal niche predicted leukemic evolution and progression-free survival in myelodysplastic syndrome (MDS), the principal leukemia predisposition syndrome. Collectively, our findings identify mesenchymal niche-induced genotoxic stress in heterotypic stem and progenitor cells through inflammatory signaling as a targetable determinant of disease outcome in human pre-leukemia.