An mTORC1 to HRI signaling axis promotes cytotoxicity of proteasome inhibitors in multiple myeloma.

Multiple myeloma (MM) causes approximately 20% of deaths from blood cancers. Notwithstanding significant therapeutic progress, such as with proteasome inhibitors (PIs), MM remains incurable due to the development of resistance. mTORC1 is a key metabolic regulator, which frequently becomes dysregulated in cancer. While mTORC1 inhibitors reduce MM viability and synergize with other therapies in vitro, clinically, mTORC1 inhibitors are not effective for MM. Here we show that the inactivation of mTORC1 is an intrinsic response of MM to PI treatment. Genetically enforced hyperactivation of mTORC1 in MM was sufficient to compromise tumorigenicity in mice. In vitro, mTORC1-hyperactivated MM cells gained sensitivity to PIs and hypoxia. This was accompanied by increased mitochondrial stress and activation of the eIF2α kinase HRI, which initiates the integrated stress response. Deletion of HRI elevated the toxicity of PIs in wt and mTORC1-activated MM. Finally, we identified the drug PMA as a robust inducer of mTORC1 activity, which synergized with PIs in inducing MM cell death. These results help explain the clinical inefficacy of mTORC1 inhibitors in MM. Our data implicate mTORC1 induction and/or HRI inhibition as pharmacological strategies to enhance MM therapy by PIs.

Endoplasmic Reticulum Homeostasis Regulates TLR4 Expression and Signaling in Mast Cells.

The endoplasmic reticulum (ER) is a dynamic organelle that responds to demand in secretory proteins by undergoing expansion. The mechanisms that control the homeostasis of ER size and function involve the activation of the unfolded protein response (UPR). The UPR plays a role in various effector functions of immune cells. Mast cells (MCs) are highly granular tissue-resident cells and key drivers of allergic inflammation. Their diverse secretory functions in response to activation through the high-affinity receptor for IgE (FcεRI) suggest a role for the UPR in their function. Using human cord blood-derived MCs, we found that FcεRI triggering elevated the expression level and induced activation of the UPR transducers IRE1α and PERK, accompanied by expansion of the ER. In mouse bone marrow-derived MCs and peritoneal MCs, the ER underwent a more moderate expansion, and the UPR was not induced following MC activation. The deletion of IRE1α in mouse MCs did not affect proliferation, survival, degranulation, or cytokine stimulation following FcεRI triggering, but it did diminish the surface expression of TLR4 and the consequent response to LPS. A similar phenotype was observed in human MCs using an IRE1α inhibitor. Our data indicate that the ER of MCs, primarily of humans, undergoes a rapid remodeling in response to activation that promotes responses to TLR4. We suggest that IRE1α inhibition can be a strategy for inhibiting the hyperactivation of MCs by LPS over the course of allergic responses.

Short Onset and Enhanced Analgesia Following Nasal Administration of Non-Controlled Drugs in Nanovesicular Systems.

Nasal nanovesicular delivery systems (NVS) containing the noncontrolled analgesic drugs Ketoprofen, Butorphanol or Tramadol, incorporated in a phospholipid nanovesicular carrier, were designed and investigated. The systems were first characterized for their physicochemical properties. Due to their composition, comprising propylene glycol as a lipid bilayers fluidizer, these systems contain soft vesicles. Pharmacokinetic profiles of Tramadol in plasma and brain and of Ketoprofen in plasma were also assessed. The analgesic effect of each of the three tested drugs was evaluated in the acetic acid mice model for pain. One important result obtained in this work is that the concentration of Tramadol in rats' plasma and brain increased rapidly after administration, reaching a peak value 10 min after administration with a Cmax of 2 to 5 folds greater than that for the oral or nasal non-vesicular treatments, respectively. In the case of Ketoprofen, the peak of the drug level in plasma was measured 10 min post nasal administration in NVS. The Cmax was three-fold higher relative to oral administration of this drug. In the experiment testing analgesia, a rapid and improved analgesia was observed for the tested drugs when delivered nasally in the nanocarrier. On the other hand, a weaker analgesic effect was observed for oral and nasal control systems. This new approach suggests that nasal delivery of non-controlled drugs in soft nanovesicles may open the way for better and noninvasive treatment of severe pain.

Aripiprazole Cytotoxicity Coincides with Activation of the Unfolded Protein Response in Human Hepatic Cells.

Schizophrenia is a mental disease that results in decreased life expectancy and well-being by promoting obesity and sedentary lifestyles. Schizophrenia is treated by antipsychotic drugs. Although the second-generation antipsychotics (SGA), Olanzapine and Aripiprazole, are more effective in treating schizophrenia, they display a higher risk of metabolic side effects, mostly by development of diabetes and insulin resistance, weight gain, and dyslipidemia. Endoplasmic reticulum (ER) stress is induced when ER homeostasis of lipid biosynthesis and protein folding is impaired. This leads to the activation of the unfolded protein response (UPR), a signaling cascade that aims to restore ER homeostasis or initiate cell death. Chronic conditions of ER stress in the liver are associated with diabetes and perturbed lipid metabolism. These metabolic dysfunctions resemble the pharmacological side effects of SGAs. We therefore investigated whether SGAs promote the UPR in human and mouse hepatocytes. We observed full-fledged activation of ER stress by Aripiprazole not by Olanzapine. This occurred at low micromolar concentrations and to variable intensities in different cell types, such as hepatocellular carcinoma, melanoma, and glioblastoma. Mechanistically, Aripiprazole caused depletion of ER calcium, leading to activation of inositol-requiring enzyme 1 (IRE1)and protein kinase R (PKR)-like endoplasmic reticulum kinase (PERK), two major transducers of the UPR. Cells underwent apoptosis with Aripiprazole treatment, which coincided with UPR induction, and this effect was reduced by adding glutathione without affecting UPR itself. Deletion of IRE1 from HepG2, a human liver cancer cell line, protected cells from Aripiprazole toxicity. Our study reveals for the first time a cytotoxic effect of Aripiprazole that involves the induction of ER stress. SIGNIFICANCE STATEMENT: The antischizophrenic drug Aripiprazole exerts cytotoxic properties at high concentrations. This study shows that this cytotoxicity is associated with the induction of endoplasmic reticulum (ER) stress and IRE1 activation, mechanisms involved in diet-induced obesity. Aripiprazole induced ER stress and calcium mobilization from the ER in human and mouse hepatocytes. Our study highlights a new mechanism of Aripiprazole that is not related to its effect on dopamine signaling.

Pharmacological induction of selective endoplasmic reticulum retention as a strategy for cancer therapy.

The integrated stress response (ISR) converges on eIF2α phosphorylation to regulate protein synthesis. ISR is activated by several stress conditions, including endoplasmic reticulum (ER) stress, executed by protein kinase R-like endoplasmic reticulum kinase (PERK). We report that ER stress combined with ISR inhibition causes an impaired maturation of several tyrosine kinase receptors (RTKs), consistent with a partial block of their trafficking from the ER to the Golgi. Other proteins mature or are secreted normally, indicating selective retention in the ER (sERr). sERr is relieved upon protein synthesis attenuation and is accompanied by the generation of large mixed disulfide bonded complexes, including ERp44. sERr was pharmacologically recapitulated by combining the HIV-protease inhibitor nelfinavir with ISRIB, an experimental drug that inhibits ISR. Nelfinavir/ISRIB combination is highly effective to inhibit the growth of RTK-addicted cell lines and hepatocellular (HCC) cells in vitro and in vivo. Thus, pharmacological sERr can be utilized as a modality for cancer treatment.