Loss of tuberous sclerosis complex 2 sensitizes tumors to nelfinavir−bortezomib therapy to intensify endoplasmic reticulum stress-induced cell death

Abstract

Cancer cells lose homeostatic flexibility because of mutations and dysregulated signaling pathways involved in maintaining homeostasis. Tuberous Sclerosis Complex 1 (TSC1) and TSC2 play a fundamental role in cell homeostasis, where signal transduction through TSC1/TSC2 is often compromised in cancer, leading to aberrant activation of mechanistic target of rapamycin complex 1 (mTORC1). mTORC1 hyperactivation increases the basal level of endoplasmic reticulum (ER) stress via an accumulation of unfolded protein, due to heightened de novo protein translation and repression of autophagy. We exploit this intrinsic vulnerability of tumor cells lacking TSC2, by treating with nelvinavir to further enhance ER stress while inhibiting the proteasome with bortezomib to prevent effective protein removal. We show that TSC2-deficient cells are highly dependent on the proteosomal degradation pathway for survival. Combined treatment with nelfinavir and bortezomib at clinically relevant drug concentrations show synergy in selectively killing TSC2-deficient cells with limited toxicity in control cells. This drug combination inhibited tumor formation in xenograft mouse models and patient-derived cell models of TSC and caused tumor spheroid death in 3D culture. Importantly, 3D culture assays differentiated between the cytostatic effects of the mTORC1 inhibitor, rapamycin, and the cytotoxic effects of the nelfinavir/bortezomib combination. Through RNA sequencing, we determined that nelfinavir and bortezomib tip the balance of ER protein homeostasis of the already ER-stressed TSC2-deficient cells in favor of cell death. These findings have clinical relevance in stratified medicine to treat tumors that have compromised signaling through TSC and are inflexible in their capacity to restore ER homeostasis.

Human pluripotent stem cell modeling of tuberous sclerosis complex reveals lineage-specific therapeutic vulnerabilities

“…However, the same treatments display no genotype specific toxicity in NCCs. These latter findings are in contrast to previous studies demonstrating the potential therapeutic effectiveness of these treatments in vitro; however, we note that the TSC2-deficient cell types used in these studies were murine cell lines with limited tissuespecific relevance to TSC tumors (Babcock et al, 2013;Johnson et al, 2018;Zhou et al, 2009). Indeed, in a preclinical trial using a Tsc2 +/mouse model, bortezomib treatment was ineffective in targeting mesenchymal renal tumors (Auricchio et al, 2012).…”

Connectivity Map Analysis of a Single-Cell RNA-Sequencing -Derived Transcriptional Signature of mTOR Signaling

“…( Figure 6 B, Supplementary Table S4 ). Mutation of TSC2 and its leading activation of MTORC1 upregulates the proteasome [ 30 ], which may facilitate estrogen-enhanced survival of tumor cells [ 31 , 32 ]. MTOR also activates NF-κB [ 33 ], a major regulator of cell survival, pro-inflammatory cytokines such as TNF-α, and cell adhesion molecules which may allow LAM cells to survive [ 34 , 35 ].…”

Finding a cure for tuberous sclerosis complex: From genetics through to targeted drug therapies

“…For instance, rapamycin was very efficient at shrinking the tumor volume, but after removal of rapamycin, cells within the tumor quickly recovered. In contrast, Nelfinavir and Bortezomib were effective at killing the tumors in vitro, where there was not cell recovery after removal of this drug combination (Johnson et al, 2018). These studies demonstrate a potent cytotoxic response to TSC-diseased cells with ER stress inducers, which are tolerated by the normal cells with an intact TSC1/TSC2 signaling pathway (Fig.…”

“…Forward research progress has been made regarding testing ER stress inducers as a potential therapy for TSC. Recent studies showed that combinatory therapy with an ER stress inducer, Nelfinavir, in the presence of an autophagy inhibitor, Chloroquine (Johnson et al, 2015), or a proteosomal inhibitor, Bortezomib (Johnson et al, 2018), showed promise in both in vitro and in vivo models of TSC to reduce tumor size and to selectively kill the TSC-diseased cells. There was a marked difference when comparing rapamycin to these drug combinations.…”