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

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.


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Financial Support: 24 This work was supported by Health and Care Research Wales (the Wales Cancer Research 25 Centre) (to ED, AT, PG and TH), the Tuberous Sclerosis Association (to ED, AT and MS), 26 Cancer Research Wales (to CJ, RJE and AT), the Tuberous Sclerosis Alliance (to ED and AT) 27 and BBSRC (to RJE). inflexible in their capacity to restore ER homeostasis.

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Cancer cells often exhibit enhanced endoplasmic reticulum (ER) stress, due to a combination 82 of inappropriately activated protein synthesis, high mutational load, oxidative stress and 83 relative nutrient depletion that leads to the accumulation of misfolded protein [1]. The 84 unfolded protein response (UPR) pathway restores ER homeostasis by three main 85 mechanisms; by slowing the rates of global protein translation, by targeting unfolded 86 protein to proteolytic degradation pathways (such as through autophagy and the 87 proteasome), and through enhancement of protein folding orchestrated by molecular 88 protein chaperones within the ER. If the UPR fails to restore the ER protein folding 89 environment in a timely manner, cell death will ensue. 90 Mechanistic target of rapamycin (mTOR) (also referred to as mammalian target of 91 rapamycin) orchestrates cell growth control by functioning as a key regulator of protein 92 translation. Hyperactivation of mTOR complex 1 (mTORC1) is known to elevate the basal 93 levels of ER stress through inappropriately high levels of protein synthesis and an 94 accumulation of unfolded protein [2]. Aberrant signal transduction through mTORC1 can 95 also potently repress autophagy (reviewed in [3]). When autophagy is compromised, the 96 proteasome becomes the primary proteolytic pathway to clear unfolded protein aggregates 97 from the cell, thereby restoring ER homeostasis and preventing cell death [4]. 98 Inactivating mutations in either Tuberous Sclerosis Complex 1 (TSC1) or TSC2 give rise to 99 Tuberous Sclerosis (TS), a genetic disorder where patients are predisposed to mTORC1-100 dependent tumor growth in various organs including the brain, kidney, eyes, lung, heart, 101 and skin (for review see [5]). Functional loss of TSC2 and resulting activation of mTORC1 was 102 shown to upregulate the proteasome [6]. We hypothesise that mTORC1-driven cancers have 103 an increased dependency on the proteasome for survival in response to ER stress. 104 Therefore, a feasible therapeutic strategy might be to inhibit the proteasome with the aim 105 to increase ER stress beyond a tolerated survival threshold. In support of this concept, 106 selective cytotoxicity of proteasome inhibitors has been shown in cancer cell models with 107 heightened mTORC1 signaling [7,8]. However, the proteasome inhibitor, bortezomib, had 108 little efficacy as a single agent in preventing renal cystadenoma development in vivo in a 109 Tsc2+/-mouse model [9]. This study suggested that targeting the proteasome alone is 110 unlikely to cause cytotoxicity in mTORC1-active tumors. We therefore examined the effects 111 of targeting the proteasome with bortezomib in combination with nelfinavir. Nelfinavir was 112 originally used as a HIV protease inhibitor but has shown activity against a broad range of 113 cancer models. One of its proposed mechanisms of anti-cancer action is via induction of ER

ER stress is elevated upon combined treatment with nelfinavir and bortezomib in Tsc2-
132 /-MEFs. 133 To assess ER stress induction after combined nelfinavir and bortezomib treatment, we 134 analysed downstream ER stress markers by western blotting. As a control we employed 135 MG132 to inhibit the proteasome. Nelfinavir and bortezomib individually enhanced the level 136 of ER stress in Tsc2-/-MEFs, as shown by increases in ATF4, CHOP and GADD34 protein 137 levels, while induction of ATF4, CHOP and GADD34 in Tsc2+/+ MEFs was less evident ( Figure   138 1A). Combined nelfinavir and bortezomib treatment further elevated the protein levels of 139 ATF4, CHOP and GADD34 compared to single drug treatment, particularly in Tsc2-/-MEFs. 140 We next analysed Xbp1 mRNA splicing, which is a functional readout of ER stress and IRE1α 141 activation. Tharpsigargin was employed as a control drug to induce ER stress. We observed 142 more Xbp1 mRNA splicing upon nelfinavir treatment in both the Tsc2+/+ and Tsc2-/-MEFs. 143 Bortezomib treatment did not result in Xbp1 mRNA splicing as a single agent and did not 144 further enhance the splicing of Xbp1 mRNA when combined with nelfinavir ( Figure 1B). To 145 confirm this differential ER stress induction between cells with and without Tsc2, we  To examine whether drug treatment was inducing CHOP via the PERK pathway, we 156 employed a PERK inhibitor, GSK2606414 ( Figure 1D). CHOP expression induced by nelfinavir 157 and bortezomib was markedly repressed with GSK2606414, revealing that these drugs are 158 inducing an ER stress response through PERK. 159 Given that bortezomib promotes ER stress via proteasomal inhibition and that nelfinavir has  Figure 1F). We observed that 170 the Tsc2-/-cells had almost 4-fold elevation of protein synthesis compared to wild-type, 171 showing that basally ER stressed Tsc2-/-cells maintain a high level of protein synthesis. After 172 6 h of nelfinavir and bortezomib dual treatment, protein translation was markedly reduced.  174 nelfinavir. 175 We speculated that combined nelfinavir and bortezomib treatment might selectively induce 176 cell death in Tsc2-/-MEFs compared to Tsc2+/+ MEFs. We quantified cell death by flow 177 cytometry with DRAQ7 labelling (Figures 2A and 2B). DRAQ7 is a membrane impermeable   210 We next assessed evidence for synergy between nelfinavir and bortezomib in inducing cell

Nelfinavir and bortezomib treatment downregulates pro-survival and upregulates
262 pro-apoptosis genes, likely mediated through ER stress 263 To gain a better understanding of the early changes that nelfinavir and bortezomib cause to 264 gene expression within cells, RNA sequencing was performed in Tsc2+/+ and Tsc2-/-MEFs 265 after 6 h of combined treatment or DMSO vehicle control. Figure 6A shows a panel of genes 266 associated with ER stress, a selection of which is highlighted graphically in Figure 6B 286 287 To determine the anti-tumor efficacy of nelfinavir and bortezomib in vivo, mice bearing

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In this study, using clinically relevant drugs that could be repositioned to treat tumors 305 displaying high ER stress profiles, we reveal that mTORC1-overactive cells are sensitive to 306 combined nelfinavir and bortezomib treatment. We show that nelfinavir and bortezomib act 307 to amplify ER stress levels, and combine synergistically to promotecell death. Whilst wild-308 type cells tolerate this drug combination with minimal cell death, cytotoxicity in Tsc2-309 deficient cells is evident even at low drug concentrations and is likely attributable to their 310 inability to manage the ER stress burden. Indeed, we see that ER stress is not fully restored 311 in the Tsc2-deficient cells after 24 h of combined drug treatment, as observed by a 312 reoccurrence of ATF4 and CHOP protein expression ( Figure 6E) repressing mTORC1 signal transduction. We observed high protein expression levels of 320 GADD34 after ER stress induction in all our cell lines, more so in Tsc2-/-MEFs. 321 Normally, protein synthesis is down-regulated upon ER stress as an efficient strategy to 322 prevent further build-up of unfolded protein within the ER. We observed that Tsc2-deficient 323 cells have elevated protein synthesis despite the higher background levels of ER stress, with a 3 to 4-fold increase in protein synthesis in Tsc2-/-MEFs compared to wild-type ( Figure 1F). 325 The elevated levels of protein synthesis would likely enhance ER stress within the Tsc2-   383 Samples were collected and analysed as previously described [15]. Bip was analysed using 384 Quantitect primers (QT00172361, Qiagen).   402 Cell death was quantified as previously described [15]. To determine synergy, cells were 403 treated with a range of drug concentrations and the affected fraction was used to 404 determine combination index (CI) values using CompuSyn software (ComboSyn, Inc.) using a 405 non-constant ratio approach.