OXPHOS Inhibition via LUC7L2 as a Target for SF3B1-Mutant Myelodysplastic Syndrome

SF3B1 gene mutations are the most common spliceosome mutations seen in myelodysplastic syndrome (MDS) patients. Though it is well known SF3B1 mutations cause downstream changes in erythroid differentiation and the cell cycle, which leads to malignancy, metabolic changes arising from this mutation are unknown. We conducted RNA sequencing from SF3B1-mutant MDS patient samples and found several genes related to metabolism were alternatively spliced. Of these, LUC7L2 was selected as our target as previous studies show its involvement in promoting oxidative phosphorylation (OXPHOS) via various downstream mechanisms when knocked down.We show that OXPHOS is increased in MOLM-13 myeloid malignant cells when LUC7L2 is inhibited. The results suggested that this gene, which is alternatively spliced and shows lower expression in SF3B1-mutant MDS, increases myeloid malignant dependence on OXPHOS

Myogenic carbohydrate transcriptome in murine C2C12 cells

Murine C2C12 cells, derived from adult dystrophic mouse thigh muscle simulate in vivo myogenesis (Yaffe and Saxel, 1977). They undergo three distinct stages of C2C12 myogenesis: proliferative myoblasts (day 0); cell cycle withdrawal and fusion to become early myotubes (day 4); and, finally spontaneously contracting late myotubes (day 9). The present study analyzed the changes in the transcriptome of the enzymes involved in carbohydrate metabolism during the three different stages of myogenic development of C2C12 cells. A carbohydrate metabolism qRT-PCR array was used to examine the mRNA levels of 84 different enzymes involved in carbohydrate metabolism (n=4 per stage). A total of 64 genes showed changes in mRNA levels that were statistically significant (ANOVA, p\u3c0.05) from the control group (day 0). Of these 64 genes, 29 genes increased or decreased by two (2.0) fold or higher and were considered to be biologically significant changes. Eleven genes were involved in the TCA cycle, 5 genes were involved in glycolysis, 7 genes were involved in glycogen metabolism, 4 genes were involved in the pentose phosphate pathway and two genes were involved in gluconeogenesis. Isoenzyme mRNA level shifts were also present during the three stages of myogenesis. In addition, the enzymatic activities of citrate synthase (CS) and creatine kinase (CK) were measured (n=6) during each stage. CS activity was 112, 204, and 271 nmol/min/mg total protein and 37, 62 and 105 nmol/min/mg total DNA respective to day 0, 4, and 9. CK activity was 86, 342, and 532 nmol/min/mg total protein and 29, 105, 661 nmol/min/mg total DNA respective to day 0, 4, and 9. These data indicate that the TCA cycle, glycolysis and glycogen metabolism pathways were significantly altered during myogenesis to meet the energetic and synthetic needs of developing cells

KRAS insertion mutations are oncogenic and exhibit distinct functional properties.

Oncogenic KRAS mutations introduce discrete amino acid substitutions that reduce intrinsic Ras GTPase activity and confer resistance to GTPase-activating proteins (GAPs). Here we discover a partial duplication of the switch 2 domain of K-Ras encoding a tandem repeat of amino acids G60_A66dup in a child with an atypical myeloproliferative neoplasm. K-Ras proteins containing this tandem duplication or a similar five amino acid E62_A66dup mutation identified in lung and colon cancers transform the growth of primary myeloid progenitors and of Ba/F3 cells. Recombinant K-Ras(G60_A66dup) and K-Ras(E62_A66dup) proteins display reduced intrinsic GTP hydrolysis rates, accumulate in the GTP-bound conformation and are resistant to GAP-mediated GTP hydrolysis. Remarkably, K-Ras proteins with switch 2 insertions are impaired for PI3 kinase binding and Akt activation, and are hypersensitive to MEK inhibition. These studies illuminate a new class of oncogenic KRAS mutations and reveal unexpected plasticity in oncogenic Ras proteins that has diagnostic and therapeutic implications

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

AMPK/FIS1-Mediated Mitophagy Is Required for Self-Renewal of Human AML Stem Cells

Leukemia stem cells (LSCs) are thought to drive the genesis of acute myeloid leukemia (AML) as well as relapse following chemotherapy. Because of their unique biology, developing effective methods to eradicate LSCs has been a significant challenge. In the present study, we demonstrate that intrinsic overexpression of the mitochondrial dynamics regulator FIS1 mediates mitophagy activity that is essential for primitive AML cells. Depletion of FIS1 attenuates mitophagy and leads to inactivation of GSK3, myeloid differentiation, cell cycle arrest, and a profound loss of LSC self-renewal potential. Further, we report that the central metabolic stress regulator AMPK is also intrinsically activated in LSC populations and is upstream of FIS1. Inhibition of AMPK signaling recapitulates the biological effect of FIS1 loss. These data suggest a model in which LSCs co-opt AMPK/FIS1-mediated mitophagy as a means to maintain stem cell properties that may be otherwise compromised by the stresses induced by oncogenic transformation

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

AMPK/FIS1-Mediated Mitophagy Is Required for Self-Renewal of Human AML Stem Cells

Leukemia stem cells (LSCs) are thought to drive the genesis of acute myeloid leukemia (AML) as well as relapse following chemotherapy. Because of their unique biology, developing effective methods to eradicate LSCs has been a significant challenge. In the present study, we demonstrate that intrinsic overexpression of the mitochondrial dynamics regulator FIS1 mediates mitophagy activity that is essential for primitive AML cells. Depletion of FIS1 attenuates mitophagy and leads to inactivation of GSK3, myeloid differentiation, cell cycle arrest, and a profound loss of LSC self-renewal potential. Further, we report that the central metabolic stress regulator AMPK is also intrinsically activated in LSC populations and is upstream of FIS1. Inhibition of AMPK signaling recapitulates the biological effect of FIS1 loss. These data suggest a model in which LSCs co-opt AMPK/FIS1-mediated mitophagy as a means to maintain stem cell properties that may be otherwise compromised by the stresses induced by oncogenic transformation