Transcription factor Nrf1 regulates proteotoxic stress-induced autophagy.

Cells exposed to proteotoxic stress invoke adaptive responses aimed at restoring proteostasis. Our previous studies have established a firm role for the transcription factor Nuclear factor-erythroid derived-2-related factor-1 (Nrf1) in responding to proteotoxic stress elicited by inhibition of cellular proteasome. Following proteasome inhibition, Nrf1 mediates new proteasome synthesis, thus enabling the cells to mitigate the proteotoxic stress. Here, we report that under similar circumstances, multiple components of the autophagy-lysosomal pathway (ALP) were transcriptionally upregulated in an Nrf1-dependent fashion, thus providing the cells with an additional route to cope with proteasome insufficiency. In response to proteasome inhibitors, Nrf1-deficient cells displayed profound defects in invoking autophagy and clearance of aggresomes. This phenomenon was also recapitulated in NGLY1 knockout cells, where Nrf1 is known to be non-functional. Conversely, overexpression of Nrf1 induced ALP genes and endowed the cells with an increased capacity to clear aggresomes. Overall, our results significantly expand the role of Nrf1 in shaping the cellular response to proteotoxic stress.

Evaluation of the NRF1-proteasome axis as a therapeutic target in breast cancer.

Proteasomes are multi-subunit complexes that specialize in protein degradation. Cancer cells exhibit a heightened dependence on proteasome activity, presumably to support their enhanced proliferation and other cancer-related characteristics. Here, a systematic analysis of TCGA breast cancer datasets revealed that proteasome subunit transcript levels are elevated in all intrinsic subtypes (luminal, HER2-enriched, and basal-like/triple-negative) when compared to normal breast tissue. Although these observations suggest a pan-breast cancer utility for proteasome inhibitors, our further experiments with breast cancer cell lines and patient-derived xenografts (PDX) pointed to triple-negative breast cancer (TNBC) as the most sensitive subtype to proteasome inhibition. Finally, using TNBC cells, we extended our studies to in vivo xenograft experiments. Our previous work has firmly established a cytoprotective role for the transcription factor NRF1 via its ability to upregulate proteasome genes in response to proteasome inhibition. In further support of this notion, we show here that NRF1 depletion significantly reduced tumor burden in an MDA-MB-231 TNBC xenograft mouse model treated with carfilzomib. Taken together, our results point to TNBC as a particularly vulnerable breast cancer subtype to proteasome inhibition and provide a proof-of-principle for targeting NRF1 as a viable means to increase the efficacy of proteasome inhibitors in TNBC tumors.

BET Inhibitors Synergize with Carfilzomib to Induce Cell Death in Cancer Cells via Impairing Nrf1 Transcriptional Activity and Exacerbating the Unfolded Protein Response.

Currently, proteasome inhibitors bortezomib, carfilzomib, and ixazomib are successfully used in clinics to treat multiple myeloma. However, these agents show limited efficacy against solid tumors. Identification of drugs that can potentiate the action of proteasome inhibitors could help expand the use of this therapeutic modality to solid tumors. Here, we found that bromodomain extra-terminal (BET) family protein inhibitors such as JQ1, I-BET762, and I-BET151 synergize with carfilzomib in multiple solid tumor cell lines. Mechanistically, BET inhibitors attenuated the ability of the transcription factor Nrf1 to induce proteasome genes in response to proteasome inhibition, thus, impeding the bounce-back response of proteasome activity, a critical pathway by which cells cope with proteotoxic stress. Moreover, we found that treatment with BET inhibitors or depletion of Nrf1 exacerbated the unfolded protein response (UPR), signaling that was initiated by proteasome inhibition. Taken together, our work provides a mechanistic explanation behind the synergy between proteasome and BET inhibitors in cancer cell lines and could prompt future preclinical and clinical studies aimed at further investigating this combination.

Disabling the Protease DDI2 Attenuates the Transcriptional Activity of NRF1 and Potentiates Proteasome Inhibitor Cytotoxicity.

Proteasome inhibition is used therapeutically to induce proteotoxic stress and trigger apoptosis in cancer cells that are highly dependent on the proteasome. As a mechanism of resistance, inhibition of the cellular proteasome induces the synthesis of new, uninhibited proteasomes to restore proteasome activity and relieve proteotoxic stress in the cell, thus evading apoptosis. This evolutionarily conserved compensatory mechanism is referred to as the proteasome-bounce back response and is orchestrated in mammalian cells by nuclear factor erythroid derived 2-related factor 1 (NRF1), a transcription factor and master regulator of proteasome subunit genes. Upon synthesis, NRF1 is cotranslationally inserted into the endoplasmic reticulum (ER), then is rapidly retrotranslocated into the cytosol and degraded by the proteasome. In contrast, during conditions of proteasome inhibition or insufficiency, NRF1 escapes degradation, is proteolytically cleaved by the aspartyl protease DNA damage inducible 1 homolog 2 (DDI2) to its active form, and enters the nucleus as an active transcription factor. Despite these insights, the cellular compartment where the proteolytic processing step occurs remains unclear. Here we further probed this pathway and found that NRF1 can be completely retrotranslocated into the cytosol where it is then cleaved and activated by DDI2. Furthermore, using a triple-negative breast cancer cell line MDA-MB-231, we investigated the therapeutic utility of attenuating DDI2 function. We found that DDI2 depletion attenuated NRF1 activation and potentiated the cytotoxic effects of the proteasome inhibitor carfilzomib. More importantly, expression of a point-mutant of DDI2 that is protease-dead recapitulated these effects. Taken together, our results provide a strong rationale for a combinational therapy that utilizes inhibition of the proteasome and the protease function of DDI2. This approach could expand the repertoire of cancer types that can be successfully treated with proteasome inhibitors in the clinic.

Nrf1-mediated transcriptional regulation of the proteasome requires a functional TIP60 complex.

Inhibition of the proteasome leads to proteotoxic stress, which is characterized by the buildup of ubiquitinated proteins that cannot be degraded properly. The transcription factor Nrf1 (also called NFE2L1) counteracts proteotoxic stress by inducing transcription of proteasome subunit genes, resulting in the restoration of proteasome activity. Further understanding of the Nrf1 pathway is therefore of interest in both neurodegeneration, where proteasome activity could be enhanced, and cancer, where suppression of this pathway could potentiate the cell-killing effect mediated by proteasome inhibitor drugs. Here, to identify novel regulators of Nrf1, we performed an RNAi screen in an engineered cell line, reporting on Nrf1 transcriptional activity. In addition to validating known regulators, we discovered that the AAA+ ATPase RUVBL1 is necessary for Nrf1's transcriptional activity. Given that RUVBL1 is part of different multisubunit complexes that play key roles in transcription, we dissected this phenomenon further and found that the TIP60 chromatin-regulatory complex is essential for Nrf1-dependent transcription of proteasome genes. Consistent with these observations, Nrf1, RUVBL1, and TIP60 proteins were co-recruited to the promoter regions of proteasome genes after proteasome inhibitor treatments. More importantly, depletion of RUVBL1 or TIP60 in various cancer cells sensitized them to cell death induced by proteasome inhibition. Overall, our study provides a framework for manipulating the TIP60-Nrf1 axis to alter proteasome function in various human diseases, including cancer.

Inhibition of NGLY1 Inactivates the Transcription Factor Nrf1 and Potentiates Proteasome Inhibitor Cytotoxicity.

Proteasome inhibitors are used to treat blood cancers such as multiple myeloma (MM) and mantle cell lymphoma. The efficacy of these drugs is frequently undermined by acquired resistance. One mechanism of proteasome inhibitor resistance may involve the transcription factor Nuclear Factor, Erythroid 2 Like 1 (NFE2L1, also referred to as Nrf1), which responds to proteasome insufficiency or pharmacological inhibition by upregulating proteasome subunit gene expression. This "bounce-back" response is achieved through a unique mechanism. Nrf1 is constitutively translocated into the ER lumen, N-glycosylated, and then targeted for proteasomal degradation via the ER-associated degradation (ERAD) pathway. Proteasome inhibition leads to accumulation of cytosolic Nrf1, which is then processed to form the active transcription factor. Here we show that the cytosolic enzyme N-glycanase 1 (NGLY1, the human PNGase) is essential for Nrf1 activation in response to proteasome inhibition. Chemical or genetic disruption of NGLY1 activity results in the accumulation of misprocessed Nrf1 that is largely excluded from the nucleus. Under these conditions, Nrf1 is inactive in regulating proteasome subunit gene expression in response to proteasome inhibition. Through a small molecule screen, we identified a cell-active NGLY1 inhibitor that disrupts the processing and function of Nrf1. The compound potentiates the cytotoxicity of carfilzomib, a clinically used proteasome inhibitor, against MM and T cell-derived acute lymphoblastic leukemia (T-ALL) cell lines. Thus, NGLY1 inhibition prevents Nrf1 activation and represents a new therapeutic approach for cancers that depend on proteasome homeostasis.

Nrf1 can be processed and activated in a proteasome-independent manner.

In response to proteasome inhibition, the transcription factor Nrf1 facilitates de novo synthesis of proteasomes by inducing proteasome subunit (PSM) genes [1,2]. Previously, we showed that activation of the p120 form of Nrf1, a membrane-bound protein in the endoplasmic reticulum (ER) with the bulk of its polypeptide in the lumen, involves its retrotranslocation into the cytosol in a manner that depends on the AAA-ATPase p97/VCP [3]. This is followed by proteolytic processing and mobilization of the transcriptionally active p110 form of Nrf1 to the nucleus. A subsequent study suggested that site-specific proteolytic processing of Nrf1 by the proteasome yields an active 75 kDa fragment [4]. We show here that under conditions where all three active sites of the proteasome are completely blocked, p120 Nrf1 can still be proteolytically cleaved to the p110 form, which is translocated to the nucleus to activate transcription of PSM genes. Thus, our results indicate that a proteasome-independent pathway can promote the release of active p110 Nrf1 from the ER membrane.

Two new sesquiterpenoids from the rhizomes of Nardostachys jatamansi.

Phytochemical investigation of CHCl(3):MeOH (1:1) extract from the rhizomes of Nardostachys jatamansi led to the isolation of two new sesquiterpenoids (5 and 6), along with six known compounds (1-4, 7, and 8). The structures of two new compounds were established using IR, MS, 1D, and 2D NMR techniques. In addition, all the isolates were tested for their cytotoxicities against the A549 (lung cancer), DU-145 (prostate cancer), MCF-7 (breast cancer), and SK-N-SH (neuroblastoma).

Stereoselective total synthesis of (+)-oploxyne A, (-)-oploxyne B, and their C-10 epimers and structure revision of natural oploxyne B.

The first total synthesis of recently isolated diacetylene alcohols oploxyne A, oploxyne B, and their C-10 epimers was accomplished. The structure of natural oploxyne B has been revised. The key steps involved are base-induced double elimination of a carbohydrate-derived ß-alkoxy chloride to generate the chiral acetylenic alcohol and Cadiot-Chodkiewicz cross-coupling reaction. The target compounds displayed potent cytotoxicity against neuroblastoma and prostate cancer cell lines.