Activating mutations in the rat sarcoma (RAS) genes HRAS, NRAS and KRAS collectively represent the most frequent oncogenic driver in human cancer1. They have previously been considered undruggable, but advances in the past few years have led to the clinical development of agents that target KRAS(G12C) and KRAS(G12D) mutants, yielding promises of therapeutic responses at tolerated doses2. However, clinical agents that selectively target NRAS(Q61*) mutants (* represents 'any'), the second-most-frequent oncogenic driver in melanoma, are still lacking. Here we identify SHOC2, a component of the SHOC2-MRAS-PP1C complex, as a dependency of RAS(Q61*) tumours in a nucleotide-state-dependent and isoform-agnostic manner. Mechanistically, we found that oncogenic NRAS(Q61R) forms a direct interaction with SHOC2, evidenced by X-ray co-crystal structure. In vitro high-throughput screening enabled the discovery of small molecules that bind to SHOC2 and disrupt the interaction with NRAS(Q61*). Structure-based optimization led to a cellularly active tool compound that shows inhibition of mitogen-activated protein kinase (MAPK) signalling and proliferation in RAS-mutant cancer models, most notably in NRAS(Q61*) settings. These findings provide evidence for a neomorph SHOC2-(canonical)RAS protein interaction that is pharmacologically actionable and relevant to cancer sustenance. Overall, this work provides the concept validation and foundation for developing new therapies at the core of the RAS signalling pathway.

Activating mutations in GNAQ/GNA11 occur in over 90% of uveal melanomas (UMs), the most lethal melanoma subtype; however, targeting these oncogenes has proven challenging and inhibiting their downstream effectors show limited clinical efficacy. Here, we performed genome-scale CRISPR screens along with computational analyses of cancer dependency and gene expression datasets to identify the inositol-metabolizing phosphatase INPP5A as a selective dependency in GNAQ/11-mutant UM cells in vitro and in vivo. Mutant cells intrinsically produce high levels of the second messenger inositol 1,4,5 trisphosphate (IP3) that accumulate upon suppression of INPP5A, resulting in hyperactivation of IP3-receptor signaling, increased cytosolic calcium and p53-dependent apoptosis. Finally, we show that GNAQ/11-mutant UM cells and patients’ tumors exhibit elevated levels of IP4, a biomarker of enhanced IP3 production; these high levels are abolished by GNAQ/11 inhibition and correlate with sensitivity to INPP5A depletion. Our findings uncover INPP5A as a synthetic lethal vulnerability and a potential therapeutic target for GNAQ/11-mutant-driven cancers.

Concomitant inhibition of PI3Kβ, IGF1R and MAPK signaling are leading to full long-term pathway blockade. A) +B) Effects of treatment with the indicated inhibitors as single-agents or in combination on WM-266-4 (A) or RVH-421 (B) were evaluated by immunoblotting using phospho-specific or total target protein antibodies. PI3Kβi=rac-KIN-193, PI3Kαi=BYL719, IGF1Ri (A)=AEW541, IGF1Ri (B)=Figitumumab-like antibody, MEKi=MEK162

PDF file, 53KB, A and B. 5x105 A2058 cells were seeded in 10 cm dishes and incubated for 24 h either with increasing amounts of BKM120 (A) or with 5 M of either GDC-0941 (B, top panel) or BEZ235 (B, bottom panel). Cells were then fixed, prepared as described for quantification of the population in the different phases of the cell cycle by fluorescence-activated cell sorting. G1, S and G2/M distribution for control untreated cells are described in the mean text and in Figure 4A. The activities of BKM120 on the cell cycle were plotted along to the inhibitory effects on pAkt levels (A).

Supplementary methods, tables and figures

PDF - 92K, NVP-BYL719 does not inhibit mTOR and PIKKs involved in DNA damage-repair processes. A. TSC1 -/- MEFs cells were grown in a 96-well format and treated for 1 h with increased concentrations of RAD001 or NVP-BYL719 (from 0.5 nmol/L to 10 ?mol/L in 1 third dilution steps) and immediately fixed. S235/236P-RPS6 levels were measured and IC50 determined with the Excel module XLfit. Background (no primary Ab incubated); BL, Baseline. B: TSC1 -/- MEFs cells were treated with increasing concentrations of NVP-BYL719 as indicated or RAD001 at 500 nmol/L or an equivalent DMSO concentration for 30 minutes. Levels of S235/236P-RPS6 and total RPS6 in protein- normalized lysates were detected by Western blot analyzis using an activation-state specific antibody, followed by incubation with species- specific HRP-labeled secondary antibody and signal development by ECL. C: 24 h post seeding, A549 cells were treated at the same time with Actinomycin D (Act D) at a concentration of 5 ?mol/L (an agent used to induce DNA damage), and with increasing concentrations of NVP-BYL719 as indicated or with the vehicle control (DMSO) for 1 h. Levels of S15P-p53 and tubulin in protein-normalized lysates were detected by Western blot analysis using an activation-state specific antibody, followed by incubation with species- specific HRP-labeled secondary antibody and signal development by ECL. D: 24 h post seeding, U2OS cells were pre-treated for 1 h with increased concentrations of NVP-BYL719 or KU55933 a specific small molecular mass inhibitor of ATM (Supplementary reference 1) at a concentration of 10 ?mol/L or with the vehicle control (DMSO). The cells were then irradiated with 15 Gy and re-incubated at 37 degrees C for 1 h and then lysed. Levels of S1981P-ATM in protein- normalized lysates were detected by Western blot analysis using an activation-state specific antibody, followed by incubation with species- specific HRP-labeled secondary antibody and signal development by ECL.

PDF file - 4.6MB, Representative immunofluorescence images of phospho-ATM (Ser1981) foci in H460 and A549 cells at 30 minutes after 10 Gy dose of radiation (x 60 magnification). Green, phospho-ATM foci. Blue, DAPI staining.

Abstract Although KRASG12C inhibitors show clinical activity in patients with KRAS G12C mutated non–small cell lung cancer (NSCLC) and other solid tumor malignancies, response is limited by multiple mechanisms of resistance. The KRASG12C inhibitor JDQ443 shows enhanced preclinical antitumor activity combined with the SHP2 inhibitor TNO155, and the combination is currently under clinical evaluation. To identify rational combination strategies that could help overcome or prevent some types of resistance, we evaluated the duration of tumor responses to JDQ443 ± TNO155, alone or combined with the PI3Kα inhibitor alpelisib and/or the cyclin-dependent kinase 4/6 inhibitor ribociclib, in xenograft models derived from a KRASG12C-mutant NSCLC line and investigated the genetic mechanisms associated with loss of response to combined KRASG12C/SHP2 inhibition. Tumor regression by single-agent JDQ443 at clinically relevant doses lasted on average 2 weeks and was increasingly extended by the double, triple, or quadruple combinations. Growth resumption was accompanied by progressively increased KRAS G12C amplification. Functional genome-wide CRISPR screening in KRASG12C-dependent NSCLC lines with distinct mutational profiles to identify adaptive mechanisms of resistance revealed sensitizing and rescuing genetic interactions with KRASG12C/SHP2 coinhibition; FGFR1 loss was the strongest sensitizer, and PTEN loss the strongest rescuer. Consistently, the antiproliferative activity of KRASG12C/SHP2 inhibition was strongly enhanced by PI3K inhibitors. Overall, KRAS G12C amplification and alterations of the MAPK/PI3K pathway were predominant mechanisms of resistance to combined KRASG12C/SHP2 inhibitors in preclinical settings. The biological nodes identified by CRISPR screening might provide additional starting points for effective combination treatments. Significance: Identification of resistance mechanisms to KRASG12C/SHP2 coinhibition highlights the need for additional combination therapies for lung cancer beyond on-pathway combinations and offers the basis for development of more effective combination approaches. See related commentary by Johnson and Haigis, p. 4005

PDF file - 4.8MB, Representative immunofluorescence images of phospho-DNA-PKcs (Thr2609) foci in H460 cells at 30 minutes after 10 Gy radiation (x 60 magnification). Green, phospho-DNA-PKcs foci. Blue, DAPI staining.

PDF file - 2MB, IL-8, MIF and PAI-1 levels in supernatant from H460 and A549 cells after 96 hours treatment. Supernatant volumes were normalized to cell number and analyzed using cytokine antibody arrays.

PDF file - 43K, BEZ235 inhibits phosphorylation of PI3K/mTOR. Western blot showing phospho-AKT (Ser473) and phospho-S6 (Ser235/236) expression in serum-starved H460 and A549 cells treated with indicated doses of BEZ235 and stimulated with IGF-1 (100 ng/ml).

PDF file, 71KB, The data are scaled by the positive control (1M MG132) and the negative control (DMSO). The percentage of maximum activity (Amax) is represented in function of the crossing point (concentration in M at 50% of MG132 activity). Each data point represents a cell line; the vertical and horizontal lines represent, respectively, the median of the crossing point values (1.33M) and the median of the Amax values (-90.06%), of BKM120 across all cell lines. The populations of cell lines least responding to GDC-0941, among which some are sensitive to BKM120, are highlighted in green.

PDF file, 113KB, A. Effects of Paclitaxel and Nocodazole on Tubulin polymerization. Tubulin was mixed with either Paclitaxel (10 M), Nocodazole (10 M) or the DMSO control in the presence of GTP. The polymerization of monomeric tubulin into microtubule was started by transferring the reaction tubes from 4{degree sign}C to 37{degree sign}C, and monitored by the increase in absorbance (λ=340 nM) over a period of 60 min. B. Competition experiments of NVP-BKM120 with colchicine and podophyllotoxin by NMR spectroscopy. T1ρ relaxation of BKM120 in the presence of tubulin (50-fold excess of compound) remains unchanged after adding podophyllotoxin or colchicine, as emphasized by the drawn arrows. The spectra of the three compounds are shown in three colors at the bottom. C. Structures of GDC0941, BEZ235, Nocodazole and BKM120.

Supplementary Figure 1. Representative images of metastases found in the liver (left panel) and peritoneal wall (right panel) of Pten loxp/loxp Lkb1loxp/loxp female mice bearing Pten Lkb1-deficient endometrial tumors. Supplementary Figure 2. Genotyping of endometrial tumors for the recombined alleles of Pten (A) and Lkb1 (B). Genomic DNAs from Pten Lkb1-deficient endometrial tumors (lanes 1–4) or mouse tail (lanes 5) were extracted. PCR analysis was done as previously described (1, 2). Supplementary Figure 3. Representative images of immunohistochemical staining of Pten (A) and Lkb1 (B) proteins in Pten Lkb1-deficient endometrial tumors counterstained with hematoxylin. Bars, 25μM. Normal uterus was shown as a control. Supplementary Figure 4. Representative histopathology images of normal uterus (A) and precursor endometrial lesions (B) from Pten loxp/loxp Lkb1 loxp/loxp female mice with endometrium-specific co-deletion of Pten and Lkb1 (2 weeks post intrauterine injection of Ade-Cre). Scale bars, 25μM. Supplementary Figure 5. Representative images of histopathology of endometrial lesions from Pten loxp/loxp female mice with endometrium-specific deletion of Pten. Mice were sacrificed 10 months post intrauterine injection of Ade-Cre. Scale bars, 50μM. Supplementary Figure 6. Representative histopathology images of endometrial lesions from Lkb1loxp/loxp female mice with endometrium-specific deletion of Lkb1. Mice were sacrificed 10 months post intrauterine injection of Ade-Cre. Scale bars, 100μM. Supplementary Figure 7. Representative images of immunohistochemical staining of CK8 in Pten Lkb1-deficient endometrial tumors. Scale bars, 50μM (left panel); 25μM (right panel). Supplementary Figure 8. Representative histopathology images of metastases found in the lungs of Ptenloxp/loxp Lkb1loxp/loxp female mice bearing Pten Lkb1-deficient endometrial tumors. Bars, 50μM. Supplementary Figure 9. Representative histopathology image of Pten Lkb1-deficient endometrial cancer lesions with desmoplastic stromal response. Scale bar, 25μM. Supplementary Figure 10. Representative images of immunohistochemical staining of proteins as indicated in normal uterus (upper panels, A and B) and Pten Lkb1-deficient endometrial tumors (lower panels, A and B). Scale bars, 25μM.Supplementary Figure 11. Western blot analysis of LKB1 expression in ETN-1 and HEC108 cells with stable expression of vector or flag-tagged LKB1 wild-type. Vinculin was used a loading control.