Genetic and pharmacological interrogation of cancer vulnerability using a multiplexed cell line screening platform.

The multiplexed cancer cell line screening platform PRISM demonstrated its utility in testing hundreds of cell lines in a single run, possessing the potential to speed up anti-cancer drug discovery, validation and optimization. Here we described the development and implementation of a next-generation PRISM platform combining Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9-mediated gene editing, cell line DNA barcoding and next-generation sequencing to enable genetic and/or pharmacological assessment of target addiction in hundreds of cell lines simultaneously. Both compound and CRISPR-knockout PRISM screens well recapitulated the results from individual assays and showed high consistency with a public database.

Avadomide induces degradation of ZMYM2 fusion oncoproteins in hematologic malignancies.

Thalidomide analogs exert their therapeutic effects by binding to the CRL4CRBN E3 ubiquitin ligase, promoting ubiquitination and subsequent proteasomal degradation of specific protein substrates. Drug-induced degradation of IKZF1 and IKZF3 in B-cell malignancies demonstrates the clinical utility of targeting disease-relevant transcription factors for degradation. Here, we found that avadomide (CC-122) induces CRBN-dependent ubiquitination and proteasomal degradation of ZMYM2 (ZNF198), a transcription factor involved in balanced chromosomal rearrangements with FGFR1 and FLT3 in aggressive forms of hematologic malignancies. The minimal drug-responsive element of ZMYM2 is a zinc-chelating MYM domain and is contained in the N-terminal portion of ZMYM2 that is universally included in the derived fusion proteins. We demonstrate that avadomide has the ability to induce proteasomal degradation of ZMYM2-FGFR1 and ZMYM2-FLT3 chimeric oncoproteins, both in vitro and in vivo. Our findings suggest that patients with hematologic malignancies harboring these ZMYM2 fusion proteins may benefit from avadomide treatment.

Deciphering the mechanisms of CC-122 resistance in DLBCL via a genome-wide CRISPR screen.

CC-122 is a next-generation cereblon E3 ligase-modulating agent that has demonstrated promising clinical efficacy in patients with relapsed or refractory diffuse large B-cell lymphoma (R/R DLBCL). Mechanistically, CC-122 induces the degradation of IKZF1/3, leading to T-cell activation and robust cell-autonomous killing in DLBCL. We report a genome-wide CRISPR/Cas9 screening for CC-122 in a DLBCL cell line SU-DHL-4 with follow-up mechanistic characterization in 6 DLBCL cell lines to identify genes regulating the response to CC-122. Top-ranked CC-122 resistance genes encode, not only well-defined members or regulators of the CUL4/DDB1/RBX1/CRBN E3 ubiquitin ligase complex, but also key components of signaling and transcriptional networks that have not been shown to modulate the response to cereblon modulators. Ablation of CYLD, NFKBIA, TRAF2, or TRAF3 induces hyperactivation of the canonical and/or noncanonical NF-κB pathways and subsequently diminishes CC-122-induced apoptosis in 5 of 6 DLBCL cell lines. Depletion of KCTD5, the substrate adaptor of the CUL3/RBX1/KCTD5 ubiquitin ligase complex, promotes the stabilization of its cognate substrate, GNG5, resulting in CC-122 resistance in HT, SU-DHL-4, and WSU-DLCL2. Furthermore, knockout of AMBRA1 renders resistance to CC-122 in SU-DHL-4 and U-2932, whereas knockout of RFX7 leads to resistance specifically in SU-DHL-4. The ubiquitous and cell line-specific mechanisms of CC-122 resistance in DLBCL cell lines revealed in this work pinpoint genetic alternations that are potentially associated with clinical resistance in patients and facilitate the development of biomarker strategies for patient stratification, which may improve clinical outcomes of patients with R/R DLBCL.

CC-90009, a novel cereblon E3 ligase modulator, targets acute myeloid leukemia blasts and leukemia stem cells.

A number of clinically validated drugs have been developed by repurposing the CUL4-DDB1-CRBN-RBX1 (CRL4CRBN) E3 ubiquitin ligase complex with molecular glue degraders to eliminate disease-driving proteins. Here, we present the identification of a first-in-class GSPT1-selective cereblon E3 ligase modulator, CC-90009. Biochemical, structural, and molecular characterization demonstrates that CC-90009 coopts the CRL4CRBN to selectively target GSPT1 for ubiquitination and proteasomal degradation. Depletion of GSPT1 by CC-90009 rapidly induces acute myeloid leukemia (AML) apoptosis, reducing leukemia engraftment and leukemia stem cells (LSCs) in large-scale primary patient xenografting of 35 independent AML samples, including those with adverse risk features. Using a genome-wide CRISPR-Cas9 screen for effectors of CC-90009 response, we uncovered the ILF2 and ILF3 heterodimeric complex as a novel regulator of cereblon expression. Knockout of ILF2/ILF3 decreases the production of full-length cereblon protein via modulating CRBN messenger RNA alternative splicing, leading to diminished response to CC-90009. The screen also revealed that the mTOR signaling and the integrated stress response specifically regulate the response to CC-90009 in contrast to other cereblon modulators. Hyperactivation of the mTOR pathway by inactivation of TSC1 and TSC2 protected against the growth inhibitory effect of CC-90009 by reducing CC-90009-induced binding of GSPT1 to cereblon and subsequent GSPT1 degradation. On the other hand, GSPT1 degradation promoted the activation of the GCN1/GCN2/ATF4 pathway and subsequent apoptosis in AML cells. Collectively, CC-90009 activity is mediated by multiple layers of signaling networks and pathways within AML blasts and LSCs, whose elucidation gives insight into further assessment of CC-90009s clinical utility. These trials were registered at www.clinicaltrials.gov as #NCT02848001 and #NCT04336982).

Scoring system to facilitate diagnosis of Gaucher disease.

BACKGROUND: Gaucher disease (GD) manifests heterogeneously and other conditions are often misdiagnosed in its place, leading to diagnostic delays. The Gaucher Earlier Diagnosis Consensus (GED-C) initiative proposed a point-scoring system (PSS) based on the signs and covariables that are most indicative of GD to help clinicians identify which individuals to test for GD. AIMS: To validate the PSS retrospectively in a test population including patients with GD and other conditions with overlapping manifestations. METHODS: Four cohorts of adults with GD, liver disease, haematological malignancy or immune thrombocytopenia were identified from hospital records. Clinical data were audited for GED-C factors identified as potentially indicative of GD and aggregate scores calculated (sum of scores/number of factors) based on published PSS weightings. Threshold discriminatory PSS scores, sensitivity and specificity were determined by receiver-operating characteristic analysis. RESULTS: Among 100 patients (GD, n = 25; non-GD, n = 75), analyses based on 11 possible factors estimated group mean (standard deviation) PSS scores of: GD (n = 14), 1.08 (0.25); non-GD (n = 38), 0.58 (0.31). Mean between-group difference (95% confidence interval) was -0.49 (-0.68, -0.31) and area under the receiver-operating characteristic analysis curve (95% confidence interval) was 0.88 (0.78, 0.97). A threshold PSS score of 0.82 identified all 14 patients with GD in the analysis set (100% sensitivity) and 27 of 38 patients in the non-GD group (71% specificity). Patients with liver disease and haematological malignancy were most likely to have manifestations overlapping GD. CONCLUSIONS: Preliminary validation of the GED-C PSS discriminated effectively between patients with GD and those with overlapping signs.

Early indicators of disease progression in Fabry disease that may indicate the need for disease-specific treatment initiation: findings from the opinion-based PREDICT-FD modified Delphi consensus initiative.

OBJECTIVES: The PRoposing Early Disease Indicators for Clinical Tracking in Fabry Disease (PREDICT-FD) initiative aimed to reach consensus among a panel of global experts on early indicators of disease progression that may justify FD-specific treatment initiation. DESIGN AND SETTING: Anonymous feedback from panellists via online questionnaires was analysed using a modified Delphi consensus technique. Questionnaires and data were managed by an independent administrator directed by two non-voting cochairs. First, possible early indicators of renal, cardiac and central/peripheral nervous system (CNS/PNS) damage, and other disease and patient-reported indicators assessable in routine clinical practice were compiled by the cochairs and administrator from panellists' free-text responses. Second, the panel scored indicators for importance (5-point scale: 1=not important; 5=extremely important); indicators scoring ≥3 among >75% of panellists were then rated for agreement (5-point scale: 1=strongly disagree; 5=strongly agree). Indicators awarded an agreement score ≥4 by >67% of panellists achieved consensus. Finally, any panel-proposed refinements to consensus indicator definitions were adopted if >75% of panellists agreed. RESULTS: A panel of 21 expert clinicians from 15 countries provided information from which 83 possible current indicators of damage (kidney, 15; cardiac, 15; CNS/PNS, 13; other, 16; patient reported, 24) were compiled. Of 45 indicators meeting the importance criteria, consensus was reached for 29 and consolidated as 27 indicators (kidney, 6; cardiac, 10; CNS/PNS, 2; other, 6; patient reported, 3) including: (kidney) elevated albumin:creatinine ratio, histological damage, microalbuminuria; (cardiac) markers of early systolic/diastolic dysfunction, elevated serum cardiac troponin; (CNS/PNS) neuropathic pain, gastrointestinal symptoms suggestive of gastrointestinal neuropathy; (other) pain in extremities/neuropathy, angiokeratoma; (patient-reported) febrile crises, progression of symptoms/signs. Panellists revised and approved proposed chronologies of when the consensus indicators manifest. The panel response rate was >95% at all stages. CONCLUSIONS: PREDICT-FD captured global opinion regarding current clinical indicators that could prompt FD-specific treatment initiation earlier than is currently practised.

Crystal structure of the SALL4-pomalidomide-cereblon-DDB1 complex.

Thalidomide-dependent degradation of the embryonic transcription factor SALL4 by the CRL4CRBN E3 ubiquitin ligase is a plausible major driver of thalidomide teratogenicity. The structure of the second zinc finger of SALL4 in complex with pomalidomide, cereblon and DDB1 reveals the molecular details of recruitment. Sequence differences and a shifted binding position relative to Ikaros offer a path to the rational design of cereblon-binding drugs with reduced teratogenic risk.

Attributes of analgesics for emergency pain relief: results of the Consensus on Management of Pain Caused by Trauma Delphi initiative.

OBJECTIVES: Management of pain is suboptimal in many prehospital and emergency department settings, and European guidelines are lacking. We carried out the Consensus On Management of PAin Caused by Trauma (COMPACT) Delphi initiative to gain insights into the factors physicians consider important when selecting analgesics for trauma pain. PATIENTS AND METHODS: A pan-European panel of experts in emergency medicine or pain (N = 31) was recruited to participate in the COMPACT Delphi initiative. In round 1, panelists supplied free-text responses to an open question about the attributes of analgesics for emergency pain relief favored by physicians. Common themes were consolidated into factors. In round 2, factors rated important by more than 75% of the panel were taken forward into round 3. In round 3, the point at which the consensus was achieved was defined a priori as at least 75% of panelists agreeing or strongly agreeing that a factor was important. RESULTS: Twenty-nine experts participated, representing 12 European countries and with a mean (SD) of 20 (8.6) years of clinical experience. Most worked in an emergency department (79.3%). The consensus was achieved for 10 factors that were important to consider when selecting analgesics for trauma pain relief. The highest level of consensus was achieved for 'efficacy' (100%), followed by 'safety and tolerability' (96.6%), and 'ease of use' (93.1%). CONCLUSION: These findings may facilitate the development of evidence-based guidelines supporting the provision of pain management in prehospital, emergency department, and critical care settings.

CB-6644 Is a Selective Inhibitor of the RUVBL1/2 Complex with Anticancer Activity.

RUVBL1 and RUVBL2 are ATPases associated with diverse cellular activities (AAAs) that form a complex involved in a variety of cellular processes, including chromatin remodeling and regulation of gene expression. RUVBLs have a strong link to oncogenesis, where overexpression is correlated with tumor growth and poor prognosis in several cancer types. CB-6644, an allosteric small-molecule inhibitor of the ATPase activity of the RUVBL1/2 complex, interacts specifically with RUVBL1/2 in cancer cells, leading to cell death. Importantly, drug-acquired-resistant cell clones have amino acid mutations in either RUVBL1 or RUVBL2, suggesting that cell killing is an on-target consequence of RUVBL1/2 engagement. In xenograft models of acute myeloid leukemia and multiple myeloma, CB-6644 significantly reduced tumor growth without obvious toxicity. This work demonstrates the therapeutic potential of targeting RUVBLs in the treatment of cancer and establishes a chemical entity for probing the many facets of RUVBL biology.

UBE2G1 governs the destruction of cereblon neomorphic substrates.

The cereblon modulating agents (CMs) including lenalidomide, pomalidomide and CC-220 repurpose the Cul4-RBX1-DDB1-CRBN (CRL4CRBN) E3 ubiquitin ligase complex to induce the degradation of specific neomorphic substrates via polyubiquitination in conjunction with E2 ubiquitin-conjugating enzymes, which have until now remained elusive. Here we show that the ubiquitin-conjugating enzymes UBE2G1 and UBE2D3 cooperatively promote the K48-linked polyubiquitination of CRL4CRBN neomorphic substrates via a sequential ubiquitination mechanism. Blockade of UBE2G1 diminishes the ubiquitination and degradation of neomorphic substrates, and consequent antitumor activities elicited by all tested CMs. For example, UBE2G1 inactivation significantly attenuated the degradation of myeloma survival factors IKZF1 and IKZF3 induced by lenalidomide and pomalidomide, hence conferring drug resistance. UBE2G1-deficient myeloma cells, however, remained sensitive to a more potent IKZF1/3 degrader CC-220. Collectively, it will be of fundamental interest to explore if loss of UBE2G1 activity is linked to clinical resistance to drugs that hijack the CRL4CRBN to eliminate disease-driving proteins.

SALL4 mediates teratogenicity as a thalidomide-dependent cereblon substrate.

Targeted protein degradation via small-molecule modulation of cereblon offers vast potential for the development of new therapeutics. Cereblon-binding therapeutics carry the safety risks of thalidomide, which caused an epidemic of severe birth defects characterized by forelimb shortening or phocomelia. Here we show that thalidomide is not teratogenic in transgenic mice expressing human cereblon, indicating that binding to cereblon is not sufficient to cause birth defects. Instead, we identify SALL4 as a thalidomide-dependent cereblon neosubstrate. Human mutations in SALL4 cause Duane-radial ray, IVIC, and acro-renal-ocular syndromes with overlapping clinical presentations to thalidomide embryopathy, including phocomelia. SALL4 is degraded in rabbits but not in resistant organisms such as mice because of SALL4 sequence variations. This work expands the scope of cereblon neosubstrate activity within the formerly 'undruggable' C2H2 zinc finger family and offers a path toward safer therapeutics through an improved understanding of the molecular basis of thalidomide-induced teratogenicity.

The p97 Inhibitor CB-5083 Is a Unique Disrupter of Protein Homeostasis in Models of Multiple Myeloma.

Inhibition of the AAA ATPase, p97, was recently shown to be a novel method for targeting the ubiquitin proteasome system, and CB-5083, a first-in-class inhibitor of p97, has demonstrated broad antitumor activity in a range of both hematologic and solid tumor models. Here, we show that CB-5083 has robust activity against multiple myeloma cell lines and a number of in vivo multiple myeloma models. Treatment with CB-5083 is associated with accumulation of ubiquitinated proteins, induction of the unfolded protein response, and apoptosis. CB-5083 decreases viability in multiple myeloma cell lines and patient-derived multiple myeloma cells, including those with background proteasome inhibitor (PI) resistance. CB-5083 has a unique mechanism of action that combines well with PIs, which is likely owing to the p97-dependent retro-translocation of the transcription factor, Nrf1, which transcribes proteasome subunit genes following exposure to a PI. In vivo studies using clinically relevant multiple myeloma models demonstrate that single-agent CB-5083 inhibits tumor growth and combines well with multiple myeloma standard-of-care agents. Our preclinical data demonstrate the efficacy of CB-5083 in several multiple myeloma disease models and provide the rationale for clinical evaluation as monotherapy and in combination in multiple myeloma. Mol Cancer Ther; 16(11); 2375-86. ©2017 AACR.

Valosin-Containing Protein/p97 as a Novel Therapeutic Target in Acute Lymphoblastic Leukemia.

B acute lymphoblastic leukemia (B-ALL) cells are distinctively vulnerable to endoplasmic reticulum (ER) stress. Recently, inhibition of p97 was shown to induce ER stress and subsequently cell death in solid tumors and in multiple myeloma. We investigated the role of a novel, orally available, p97 inhibitor (CB-5083; Cleave Biosciences) in B-ALL. CB-5083 induced a significant reduction in viability in 10 human B-ALL cell lines, harboring the most common fusion-genes involved in pediatric and adult B-ALL, with IC50s ranging from 0.34 to 0.76 µM. Moreover, CB-5083 significantly reduced the colony formation of OP1 and NALM6 cells. Early and strong induction of apoptosis was demonstrated in BALL1 and OP1 cells, together with a robust cleavage of PARP. CB-5083 induced ER stress, as documented through: 1) prominent expression of chaperones (GRP78, GRP94, PDI, DNAJC3, and DNAJB9); 2) increased activation of IRE1-alpha, as demonstrated by the splicing of XBP1; and 3) activation of PERK, which resulted in a significant overexpression of CHOP, and its downstream genes. CB-5083 reduced the viability also in GRP78-/-, GRP94-/-, and XBP1-/- cells, suggesting that none of these proteins alone was strictly required for CB-5083 activity. Moreover, we showed that the absence of XBP1 (XBP1-/-) increased the sensitivity to CB-5083, leading to the hypothesis that XBP1 splicing counteracts the activity of CB-5083, probably mitigating ER stress. Finally, vincristine was synergistic with CB-5083 in both BALL1 and OP1 cells. In summary, the targeting of p97 with CB-5083 is a novel promising therapeutic approach that should be further evaluated in B-ALL.

The Holy Grail: Solid Tumor Efficacy by Proteasome Inhibition.

Proteasome inhibitors have revolutionized the treatment of multiple myeloma but have had disappointing results when treating solid tumors. The work of Weyburne et al. (2017), published in this issue of Cell Chemical Biology, sheds some light on this conundrum and suggests a novel approach to achieve solid tumor efficacy.

Middle Lobe Torsion after Unilateral Lung Transplant.

Lobar torsion is well documented after pneumonectomy, but is very rare after lung transplant. To the best of our knowledge, this is the twelfth reported case of lobar torsion after lung transplant. In our case, bronchoscopies and chest radiographs were inconclusive; however, CT scan clearly demonstrated findings consistent with right middle lobe torsion. We review the literature and discuss the epidemiology, clinical presentation, imaging features, and treatment considerations for this condition. We also propose that if a clinical picture could be secondary to torsion and bronchoscopies and chest x ray are inconclusive that a CT scan should be obtained as soon as possible since early recognition increases the likelihood of being able to successfully detorse the lung and avoid lobectomy.

Discovery of a First-in-Class, Potent, Selective, and Orally Bioavailable Inhibitor of the p97 AAA ATPase (CB-5083).

The AAA-ATPase p97 plays vital roles in mechanisms of protein homeostasis, including ubiquitin-proteasome system (UPS) mediated protein degradation, endoplasmic reticulum-associated degradation (ERAD), and autophagy. Herein we describe our lead optimization efforts focused on in vitro potency, ADME, and pharmaceutical properties that led to the discovery of a potent, ATP-competitive, D2-selective, and orally bioavailable p97 inhibitor 71, CB-5083. Treatment of tumor cells with 71 leads to significant accumulation of markers associated with inhibition of UPS and ERAD functions, which induces irresolvable proteotoxic stress and cell death. In tumor bearing mice, oral administration of 71 causes rapid accumulation of markers of the unfolded protein response (UPR) and subsequently induces apoptosis leading to sustained antitumor activity in in vivo xenograft models of both solid and hematological tumors. 71 has been taken into phase 1 clinical trials in patients with multiple myeloma and solid tumors.

Targeting the AAA ATPase p97 as an Approach to Treat Cancer through Disruption of Protein Homeostasis.

p97 is a AAA-ATPase with multiple cellular functions, one of which is critical regulation of protein homeostasis pathways. We describe the characterization of CB-5083, a potent, selective, and orally bioavailable inhibitor of p97. Treatment of tumor cells with CB-5083 leads to accumulation of poly-ubiquitinated proteins, retention of endoplasmic reticulum-associated degradation (ERAD) substrates, and generation of irresolvable proteotoxic stress, leading to activation of the apoptotic arm of the unfolded protein response. In xenograft models, CB-5083 causes modulation of key p97-related pathways, induces apoptosis, and has antitumor activity in a broad range of both hematological and solid tumor models. Molecular determinants of CB-5083 activity include expression of genes in the ERAD pathway, providing a potential strategy for patient selection.

Reconstruction of pulmonary venous conduit with CorMatrix in lung transplant.

Anomalous pulmonary vein anatomy is infrequently encountered during reconstructive lung surgery, especially lung transplantation. Complications of pulmonary venous anastomosis carry high morbidity and mortality. We report a case of anomalous pulmonary vein reconstruction with a decellularized porcine small intestinal submucosa-derived extracellular matrix during bilateral lung transplantation in an 18-year-old woman with cystic fibrosis.

Fosfomycin/tobramycin for inhalation in patients with cystic fibrosis with pseudomonas airway infection.

RATIONALE: Fosfomycin/tobramycin for inhalation (FTI), a unique, broad-spectrum antibiotic combination, may have therapeutic potential for patients with cystic fibrosis (CF). OBJECTIVES: To evaluate safety and efficacy of FTI (160/40 mg or 80/20 mg), administered twice daily for 28 days versus placebo, in patients greater than or equal to 18 years of age, with CF, chronic Pseudomonas aeruginosa (PA) airway infection, and FEV(1) greater than or equal to 25% and less than or equal to 75% predicted. METHODS: This double-blind, placebo-controlled, multicenter study assessed whether FTI/placebo maintained FEV(1) % predicted improvements achieved following a 28-day, open-label, run-in course of aztreonam for inhalation solution (AZLI). MEASUREMENTS AND MAIN RESULTS: A total of 119 patients were randomized to FTI (160/40 mg: n = 41; 80/20 mg: n = 38) or placebo (n = 40). Mean age was 32 years and mean FEV(1) was 49% predicted at screening. Relative improvements in FEV(1) % predicted achieved by the AZLI run-in were maintained in FTI groups compared with placebo (160/40 mg vs. placebo: 6.2% treatment difference favoring FTI, P = 0.002 [primary endpoint]; 80/20 mg vs. placebo: 7.5% treatment difference favoring FTI, P < 0.001). The treatment effect on mean PA sputum density was statistically significant for the FTI 80/20 mg group versus placebo (-1.04 log(10) PA colony-forming units/g sputum difference, favoring FTI; P = 0.01). Adverse events, primarily cough, were consistent with CF disease. Respiratory events, including dyspnea and wheezing, were less common with FTI 80/20 mg than FTI 160/40 mg. No clinically significant differences between groups were reported for laboratory values. CONCLUSIONS: FTI maintained the substantial improvements in FEV(1) % predicted achieved during the AZLI run-in and was well tolerated. FTI is a promising antipseudomonal therapy for patients with CF.

Pharmacological and genetic evaluation of proposed roles of mitogen-activated protein kinase/extracellular signal-regulated kinase kinase (MEK), extracellular signal-regulated kinase (ERK), and p90(RSK) in the control of mTORC1 protein signaling by phorbol esters.

The mammalian target of rapamycin complex 1 (mTORC1) links the control of mRNA translation, cell growth, and metabolism to diverse stimuli. Inappropriate activation of mTORC1 can lead to cancer. Phorbol esters are naturally occurring products that act as potent tumor promoters. They activate isoforms of protein kinase C (PKCs) and stimulate the oncogenic MEK/ERK signaling cascade. They also activate mTORC1 signaling. Previous work indicated that mTORC1 activation by the phorbol ester PMA (phorbol 12-myristate 13-acetate) depends upon PKCs and may involve MEK. However, the precise mechanism(s) through which they activate mTORC1 remains unclear. Recent studies have implicated both the ERKs and the ERK-activated 90-kDa ribosomal S6 kinases (p90(RSK)) in activating mTORC1 signaling via phosphorylation of TSC2 (a regulator of mTORC1) and/or the mTORC1 component raptor. However, the relative importance of each of these kinases and phosphorylation events for the activation of mTORC1 signaling is unknown. The recent availability of MEK (PD184352) and p90(RSK) (BI-D1870) inhibitors of improved specificity allowed us to address the roles of these protein kinases in controlling mTORC1 in a variety of human and rodent cell types. In parallel, we used specific shRNAs against p90(RSK1) and p90(RSK2) to further test their roles in regulating mTORC1 signaling. Our data indicate that p90(RSKs) are dispensable for the activation of mTORC1 signaling by phorbol esters in all cell types tested. Our data also reveal striking diversity in the requirements for MEK/ERK in the control of mTORC1 between different cell types, pointing to additional signaling connections between phorbol esters and mTORC1, which do not involve MEK/ERK. This study provides important information for the design of efficient strategies to combat the hyperactivation of mTORC1 signaling by oncogenic pathways.