Carfilzomib Enhances the Suppressive Effect of Ruxolitinib in Myelofibrosis

As the first FDA-approved tyrosine kinase inhibitor for treatment of patients with myelofibrosis (MF), ruxolitinib improves clinical symptoms but does not lead to eradication of the disease or significant reduction of the mutated allele burden. The resistance of MF clones against the suppressive action of ruxolitinib may be due to intrinsic or extrinsic mechanisms leading to activity of additional pro-survival genes or signalling pathways that function independently of JAK2/STAT5. To identify alternative therapeutic targets, we applied a pooled-shRNA library targeting ~5000 genes to a JAK2V617F-positive cell line under a variety of conditions, including absence or presence of ruxolitinib and in the presence of a bone marrow microenvironment-like culture medium. We identified several proteasomal gene family members as essential to HEL cell survival. The importance of these genes was validated in MF cells using the proteasomal inhibitor carfilzomib, which also enhanced lethality in combination with ruxolitinib. We also showed that proteasome gene expression is reduced by ruxolitinib in MF CD34+ cells and that additional targeting of proteasomal activity by carfilzomib enhances the inhibitory action of ruxolitinib in vitro. Hence, this study suggests a potential role for proteasome inhibitors in combination with ruxolitinib for management of MF patients.

Bortezomib inhibits lung fibrosis and fibroblast activation without proteasome inhibition

The FDA-approved proteasomal inhibitor bortezomib (BTZ) has attracted interest for its potential anti-fibrotic actions. However, neither its in vivo efficacy in lung fibrosis nor its dependence on proteasome inhibition has been conclusively defined. Herein, we identify that therapeutic administration of BTZ in a mouse model of pulmonary fibrosis diminished the severity of fibrosis without reducing proteasome activity in the lung. Under conditions designed to mimic this lack of proteasome inhibition in vitro, it reduced fibroblast proliferation, differentiation into myofibroblasts, and collagen synthesis. It promoted de-differentiation of myofibroblasts and overcame their characteristic resistance to apoptosis. Mechanistically, BTZ inhibited kinases important for fibroblast activation while inducing expression of dual-specificity phosphatase 1 or DUSP1, and knockdown of DUSP1 abolished its anti-fibrotic actions in fibroblasts. Our findings identify a novel proteasome-independent mechanism of anti-fibrotic actions for BTZ and support its therapeutic repurposing for pulmonary fibrosis.

Unconventional endocytosis and trafficking of Transferrin Receptor induced by iron

The post-translational regulation of transferrin receptor (TfR1) is largely unknown. We investigated whether iron availability affects TfR1 endocytic cycle and protein stability in HepG2 hepatoma cells exposed to ferric ammonium citrate (FAC). NH4Cl and bafilomycin A1, but not the proteasomal inhibitor MG132, prevented the FAC-mediated decrease in TfR1 protein levels, thus indicating lysosomal involvement. Knock-down experiments showed that TfR1 lysosomal degradation is independent of: 1) endocytosis mediated by the clathrin adaptor AP2; 2) Tf, which was suggested to facilitate TfR1 internalization; 3) H-Ferritin and 4) MARCH8, previously implicated in TfR1 degradation. Notably, FAC decreased the number of TfR1 molecules at the cell surface and increased the Tf endocytic rate. Colocalization experiments confirmed that, upon FAC treatment, TfR1 was endocytosed in an AP2- and Tf-independent pathway and trafficked to the lysosome for degradation. This unconventional endocytic regulatory mechanism aimed at reducing surface TfR1 may represent an additional post-translational control to prevent iron overload. Our results show that iron is a key regulator of the trafficking of TfR1, which has been widely used to study endocytosis often not considering its function in iron homeostasis.

Circadian regulation of muscle growth independent of locomotor activity

Muscle tissue shows circadian variation, but whether and how the intracellular circadian clock per se regulates muscle growth remains unclear. By measuring muscle growth over 12 h periods, here we show that muscle grows more during the day than at night. Inhibition of muscle contraction reduces growth to a similar extent in day and night, but does not ablate the circadian variation in growth. Muscle protein synthesis is higher during the day compared to night, whereas markers of protein degradation are higher at night. Mechanistically, the TORC1 inhibitor rapamycin inhibits the extra daytime growth, but no effect on muscle growth at night was detected. Conversely, the proteasomal inhibitor MG132 increases muscle growth at night, but has no effect during the day, irrespective of activity. Ablation of contractile activity rapidly reduces muscle protein synthesis both during the day and at night and leads to a gradual increase in Murf gene expression without ablating circadian variation in growth. Removal of circadian input by exposure to either permanent light or permanent darkness reduces muscle growth. We conclude that circadian variation in muscle growth is independent of the presence of, or changes in, physical activity and affects both protein synthesis and degradation in distinct circadian phases.

Autophagy Regulated by Gain of Function Mutant p53 Enhances Proteasomal Inhibitor-Mediated Cell Death through Induction of ROS and ERK in Lung Cancer Cells

Mutations in p53, especially gain of function (GOF) mutations, are highly frequent in lung cancers and are known to facilitate tumor aggressiveness. Yet, the links between mutant GOF-p53 and lung cancers are not well established. In the present study, we set to examine how we can better sensitize resistant GOF-p53 lung cancer cells through modulation of cellular protein degradation machineries, proteasome and autophagy. H1299 p53 null lung cancer cells were stably transfected with R273H mutant GOF-p53 or wild-type (wt) p53 or empty vectors. The presence of R273H-P53 conferred the cancer cells with drug resistance not only against the widely used chemotherapeutic agents like cisplatin (CDDP) or 5-flurouracil (5-FU) but also against potent alternative modes of therapy like proteasomal inhibition. Therefore, there is an urgent need for new strategies that can overcome GOF-p53 induced drug resistance and prolong patient survival following failure of standard therapies. We observed that the proteasomal inhibitor, peptide aldehyde N-acetyl-leu-leu-norleucinal (commonly termed as ALLN), caused an activation of cellular homeostatic machinery, autophagy in R273H-P53 cells. Interestingly, inhibition of autophagy by chloroquine (CQ) alone or in combination with ALLN failed to induce enhanced cell death in the R273H-P53 cells; however, in contrast, an activation of autophagy by serum starvation or rapamycin increased sensitivity of cells to ALLN-induced cytotoxicity. An activated autophagy was associated with increased ROS and ERK signaling and an inhibition of either ROS or ERK signaling resulted in reduced cytotoxicity. Furthermore, inhibition of GOF-p53 was found to enhance autophagy resulting in increased cell death. Our findings provide novel insights pertaining to mechanisms by which a GOF-p53 harboring lung cancer cell is better sensitized, which can lead to the development of advanced therapy against resistant lung cancer cells.

Prevention of Streptozotocin-Induced Diabetic Nephropathy by MG132: Possible Roles of Nrf2 and IκB

Our previous study showed that proteasomal inhibitor MG132 can prevent diabetic nephropathy (DN) along with upregulation of nuclear factor (erythroid-derived 2)-like 2 (Nrf2). The present study was to investigate whether MG132 can prevent DN in wild-type and Nrf2-KO mice. Type 1 diabetes was induced in wild-type and Nrf2-KO mice by multiple low doses of streptozotocin. Two weeks after streptozotocin injection, both wild-type and Nrf2-KO mice were randomly divided into four groups: control, MG132, DM, and DM/MG132. MG132 (10 μg/kg/day) or vehicle was administered intraperitoneally for 4 months. Renal function, morphology, and biochemical changes were measured after 4-month treatment with MG132. MG132 treatment suppressed proteasomal activity in the two genotypes. In wild-type mice, MG132 attenuated diabetes-induced renal dysfunction, fibrosis, inflammation, and oxidative damage along with increased Nrf2 and IκB expression. Deletion of Nrf2 gene resulted in a partial, but significant attenuation of MG132 renal protection in Nrf2-KO mice compared with wild-type mice. MG132-increased IκB expression was not different between wild-type and Nrf2-KO mice. This work indicates that MG132 inhibits diabetes-increased proteasomal activity, resulting in Nrf2 and IκB upregulation and renal protection, which could be used as a strategy to prevent diabetic nephropathy.