- Email: [email protected]
LKB1 Deficiency Renders NSCLC Cells Sensitive to ERK Inhibitors
Journal Pre-proof LKB1 deficiency renders non-small-cell lung cancer cells sensitive to ERK inhibitors. Elisa Caiola, PhD, Alice Iezzi, MSc, Michele Tomanelli, MSc, Elisa Bonaldi, MSc, Arianna Scagliotti, MSc, Marika Colombo, MSc, Federica Guffanti, MSc, Edoardo Micotti, PhD, Marina Chiara Garassino, MD, Lucia Minoli, DVM, Eugenio Scanziani, DVM, Massimo Broggini, PhD, Mirko Marabese, PhD PII:
S1556-0864(19)33568-3
DOI:
https://doi.org/10.1016/j.jtho.2019.10.009
Reference:
JTHO 1617
To appear in:
Journal of Thoracic Oncology
Received Date: 27 March 2019 Revised Date:
7 October 2019
Accepted Date: 12 October 2019
Please cite this article as: Caiola E, Iezzi A, Tomanelli M, Bonaldi E, Scagliotti A, Colombo M, Guffanti F, Micotti E, Garassino MC, Minoli L, Scanziani E, Broggini M, Marabese M, LKB1 deficiency renders nonsmall-cell lung cancer cells sensitive to ERK inhibitors., Journal of Thoracic Oncology (2019), doi: https:// doi.org/10.1016/j.jtho.2019.10.009. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 International Association for the Study of Lung Cancer. Published by Elsevier Inc. All rights reserved.
LKB1 deficiency renders non-small-cell lung cancer cells sensitive to ERK inhibitors.
Elisa Caiola§a, PhD Alice Iezzi§a, MSc Michele Tomanellia, MSc Elisa Bonaldia, MSc Arianna Scagliottia, MSc Marika Colomboa, MSc Federica Guffanti a, MSc Edoardo Micottib, PhD Marina Chiara Garassinoc, MD Lucia Minolid,e, DVM Eugenio Scanzianid,e, DVM Massimo Broggini#a, PhD Mirko Marabese#a, PhD
a
Laboratory of Molecular Pharmacology, Department of Oncology, Istituto di Ricerche
Farmacologiche Mario Negri IRCCS, Milan, Italy. bDepartment of Neuroscience, Istituto di Ricerche Farmacologiche Mario Negri IRCCS, Milan, Italy. cUnit of Thoracic Oncology, Fondazione IRCCS Istituto Nazionale dei Tumori, Milan, Italy.
Mouse & Animal
d
Pathology Lab, Fondazione Filarete, Milan, Italy. eDepartment of Veterinary Medicine, University of Milan, Milan, Italy 1
§
Elisa Caiola and Alice Iezzi are co-first authors
# Mirko Marabese and Massimo Broggini are co-last authors
Corresponding author: Mirko Marabese, Laboratory of Molecular Pharmacology, Department of Oncology, Istituto di Ricerche Farmacologiche Mario Negri IRCCS, [email protected]
Funding information The research leading to these results has received funding from AIRC under MFAG 2016 ID. 18386 project – P.I. Marabese; IG 2015 – ID. 16792 project – P.I. Broggini and IG 2017 – ID. 20085 project – P.I. Garassino
Dr. Garassino reports grants from AstraZeneca, Bristol-Myers Squibb, MSD Oncology, Roche, Takeda, Incyte, Lilly, Merck, Pfizer, Clovis, Merck Senoro, Bayer, Spectrum Pharmaceuticals, F. Hoffmann-La Roche, Blueprint Medicine; personal fees from AstraZeneca, Bristol-Myers Squibb, MSD Oncology, Novartis, Roche, Takeda, Tiziana Life Sciences, Celgene, Incyte, Boehringer Ingelheim, Otsuka Pharma, Bayer, Inivata, SanofiAventis, SeaGen International GmbH; non-financial support from AstraZeneca, Roche, Pfizer, F. Hoffmann-La Roche. The remaining authors have nothing to disclose.
2
Abstract Background Liver kinase B1 (LKB1/STK11) is one of the most mutated genes in non-small-cell lung cancer (NSCLC) accounting for about one third of cases and its activity is impaired in about half of KRAS mutated NSCLC. At present, these patients cannot benefit from any specific therapy. Methods Through CRISPR/Cas9 technology, we systematically deleted LKB1 in both wild-type and KRAS-mutated human NSCLC cells. By using these isogenic systems together with genetically engineered mouse models we investigated the cell response to ERK inhibitors both in vitro and in vivo. Results In all the systems used here, the loss of LKB1 creates a vulnerability and renders these cells particularly sensitive to ERK inhibitors both in vitro and in vivo. The same cells expressing a wt LKB1 poorly respond to these drugs. At molecular level, ERK inhibitors induced, in the absence of LKB1, a marked inhibition of p90 ribosomal S6 kinase activation, which in turn abolished S6 protein activation, promoting the cytotoxic effect. Conclusions This work shows that ERK inhibitors are effective in LKB1 and LKB1/KRAS mutated tumors, thus offering a therapeutic strategy for this prognostically unfavorable subgroup of patients. Since ERK inhibitors are already in clinical development, our findings could be easily translatable to the clinic. Importantly, the lack of effect in cells expressing wild-type 3
LKB1, predicts that treatment of LKB1 mutated tumors with ERK inhibitors should have a favorable toxicity profile.
Short title ERK inhibitors in LKB1 mutated NSCLC
Keywords LKB1/STK11, ERK inhibitor, SCH772984, ulixertinib, NSCLC
4
Introduction Liver kinase B1 (LKB1/STK11) is one of the most commonly mutated genes in non-small cell lung cancer (NSCLC) accounting for about one third of cases1. In addition, its activity is impaired in about 50% of KRAS mutated NSCLC2. This large number of patients has an aggressive, chemoresistant subtype of cancer and, at present, has no specific therapy. LKB1 is a serine/threonine kinase that serves as a master regulator of the cell metabolism3. The best characterized LKB1 substrate is the AMP-activated protein kinase (AMPK) and together they control cell metabolism in response to the availability of nutrients4. Besides metabolism, both AMPK and LKB1 were reported to have roles in the control of cell growth5. LKB1/AMPK pathway activation results in negative regulation of one of the most deregulated
signaling
systems
in
cancer,
the
PhosphatidylInositol
3-Kinase
(PI3K)/AKT/mammalian target of the rapamycin (mTOR) pathway6. mTOR is a pivotal protein in mTORC1 and mTORC2 complexes and acts as an integrator of nutrients and growth factor signals, resulting in the control of cell growth in all eukaryotes. These signals act on ribosome biogenesis and the translation of proteins that promote cell growth and division7. The PI3K pathway is not the only regulator of mTOR. Extracellular signalregulated kinase (ERK) and its target, p90 ribosomal S6 kinase (p90RSK), both target mTORC1 activity8, 9. SCH772984, an analog of the orally available clinical candidate SCH900353/MK-8353, has a dual mechanism of action: allosteric inhibition of MEK1/2 binding to ERK, resulting in the lack of ERK phosphorylation and ATP-competitive ERK inhibition, resulting in the absence of phosphorylation of its substrates10. This compound was described as an 5
extremely selective ERK1/2 inhibitor able to suppress the mitogen-activated protein kinase (MAPK) pathway signaling and cell proliferation in many tumor models. In particular, it has a strong activity in cell lines positive for KRAS and BRAF mutations11. Ulixertinib (BVD-523, VRT752271) is a small molecule under phase I clinical evaluation12. It potently and selectively inhibits ERK1/2 kinases in a reversible, ATP-competitive way. Signal transduction, cell survival and cell proliferation are negatively regulated particularly in cell lines harboring MAPK pathway activating mutations. Like SCH772984, ulixertinib inhibits cells growth in BRAF- and in KRAS-mutant tumors13. In the present work, we investigated the activity of SCH772984 and ulixertinib in different NSCLC models with different LKB1 and KRAS status.
6
Methods Cell cultures, drugs, growth assay, siRNA and plasmids NSCLC cell lines (H1299, LU99, H460, A549 and H358) were grown in RPMI-1640 supplemented with 10% FBS. H1299 derived clones (wt and G12C KRAS) were grown in normal medium including 500 µg/mL of G418 (Gibco). LKB1 KO clones in H1299, LU99 and H358 were generated using LKB1 CRISPR/Cas9 KO Plasmid and HDR Plasmid (Santa Cruz Biotechnology) according to the manufacturer's instructions. Cell lines and clones are routinely tested by PCR for mycoplasma contamination and authenticated with the PowerPlex 16 HS System (Promega) every six months by comparing the STR profiles with those deposited in ATCC and/or DSMZ databases. SCH772984 (TargetMol), ulixertinib (TargetMol), ARQ 751 (ArQule) and PIK-75 (Selleckchem) DMSO stock solutions were dissolved in medium just before use. In all the cytotoxicity experiments, cells were continuously treated for 72h. Drug effects were estimated by the CellTiter AQueous MTS as reported14. The mean and SD of at least three independent experiments are presented. EsiRNA were purchased from Sigma Aldrich and transfected by Lipofectamine 2000 (Invitrogen). pcDNA3-FLAG-LKB14 was a gift from Lewis Cantley (Addgene plasmid # 8590). The colony assay was performed as reported15.
Western blotting analyses Proteins were extracted and visualized as reported15. Immunoblotting was carried out with the antibodies listed in the supplementary material.
7
Xenograft studies The IRFMN adheres to the principles declared in the supplementary material. For the xenograft experiments, six-week-old female nude athymic mice (Envigo) were housed at constant temperature and humidity, according to institutional guidelines. SCH772984 (ChemieTek) was dissolved in 50% DMSO, 20% 2-Hydroxypropyl)-beta-cyclodextrin (Carlo Erba Reagents S.r.l) and given i.p. twice a day for 14 days consecutively. ARQ 751 (ArQule) was dissolved in 0.5% methylcellulose (Carlo Erba Reagents S.r.l) and given p.o. for three days a week for 3 times. Additional details are reported in supplementary material.
Genetically engineered murine models of NSCLC and derived cell lines Conditional transgenic models (KRAStm4Tyj/J and LKB1/STK11tm1.1Sjm/J) were purchased from Jackson Laboratories. KRAStm4Tyj/J (KRASG12D/WT) and LKB1/STK11tm1.1Sjm/J (LKB1KO/KO) mice were crossed to generate conditional double KO mice (KRASG12D/WT/LKB1KO/KO) which were genotyped using PCR protocols obtained from the Jackson Laboratories. KRASG12D/WT and KRASG12D/WT/LKB1KO/KO mice were treated intranasally with Adeno-Cre (SignaGen Laboratories) at 6 weeks of age as previously reported16. Four weeks after Adeno-Cre treatment, the animals were randomized to receive SCH772984 or vehicle as for xenograft experiments. At the end of treatment, 6 animals per group were sacrificed and lungs fixed in formalin for histopathological evaluation.
8
Details for isolation of cell lines from lung nodules are reported in supplementary material. Two independent clones from KRASG12D/WT/LKB1KO/KO lung tumors (KL 7 and KL 95) were isolated and their behavior compared to that of a KRASG12D/WT cell line (KP1.9).
MRI scan Before treatment and after 7 days post-treatment, the group of animals for nodule count was analyzed by MRI using a small rodents 7 Tesla BRUKER scanner (Bruker Biospec 70/30), equipped with a 72 volume coil in transmission and a saddle shaped 2x2 surface array for receiving the signal. Details are reported in the supplementary material.
Histopathology Lungs were formalin fixed, paraffin embedded and 4 um thick sections were routinely stained with Hematoxylin and Eosin. Pulmonary proliferative lesions were classified according to international guidelines17. The tumoral burden was assessed by counting the number of pulmonary tumors on an approximatly 50 mm2 of parenchymal area.
Statistical analyses Statistical analyses were done using GraphpadPrism version 7. Differences between groups were considered significant when the p-values were <0.05.
9
Results While exploring drug responses in NSCLC cell lines in vitro, we found striking different responses to the ERK inhibitor SCH772984 in two cell lines, H727 and H1299. In H1299 the drug was completely inactive (IC50 >7 µM) while in H727 there was a very good response, with an IC50 of approximately 0.13 µM (Confidence Interval (CI): 0.07-0.24 µM) (Supplementary Figure 1a). Among the mutations harbored in the two cell lines, our attention was caught on KRAS and LKB1 given H727 cell line is mutated in both KRAS and LKB1 while these proteins were wild-type (wt) in H1299 (COSMIC database, https://cancer.sanger.ac.uk/cosmic). Being H727 cells derived from carcinoid, we no more used this model for subsequent experiments and concentrated our experiments on carcinomas.
Lack of LKB1 expression increases sensitivity to the ERK inhibitor SCH772984 in KRAS wt NSCLC cells by impairing S6 Ribosomal Protein activation through p90RSK protein The H1299 cell line (KRAS wt/LKB1 wt) was genetically manipulated with CRISPR/Cas9 technique to generate a deletion in the LKB1 gene (resulting in lack of protein expression), and we selected two independent LKB1 knock-out (KO) clones (H1299-LKB1 KO 1 and 2) (Figure 1a). SCH772984 treatment gave a marked, statistically significant response in LKB1 deficient cells compared to the parental cell line (Figure 1b). Whereas for the latter it was not possible to calculate an IC50 with the drug concentrations tested, the IC50 of the two LKB1 deficient clones were respectively 2.4 µM (CI: 1.9-3.0 µM) and 2.8 µM (CI: 2.3-3.5
10
µM). These results were confirmed by using the colony formation assay (Figure 1c and 1d). To corroborate these data with a different approach, LKB1 was silenced with specific LKB1 endoribonuclease-prepared siRNA (esiRNA) transfection, which strongly downregulated LKB1 protein expression, while universal negative esiRNA had no effect (Figure 1e). As in H1299-LKB1 KO clones, transient silencing of LKB1 in H1299 cells induced significant sensitization to SCH772984 compared to cells transfected with the negative esiRNA (Figure 1f). In agreement with these data, the re-introduction of LKB1 in the H1299 LKB1 KO clone (Figure 1g) strongly reduced the activity of SCH772984 to the level of parental H1299 cells, while LKB1 overexpression did not alter the behavior of H1299 after drug treatment (Figure 1h). To check whether the absence of LKB1 increased the sensitivity to SCH772984 in vivo, both LKB1 wt and KO cells were subcutaneously implanted into nude mice. Although the LKB1 KO clone took much longer (40 days) to reach randomization weight than the LKB1 wt cell line (19 days), their growth rates from this point were comparable. SCH772984 treatment induced a tumor growth reduction in LKB1 KO clone reaching statistical significance on day 19 (p<0.05) and 21 (p<0.01) after treatment start, with a best treated/control ratio (T/C) of 33% on day 16 (Figure 1i). The same treatment schedule in LKB1 wt expressing tumors had a best T/C of only 77% on day 9 and did not reach a significant reduction at any time during the experiment (Figure 1j). SCH772984 was well tolerated and there was no significant reduction in the animals’ body weights (Supplementary Figure 1b). The drug
11
reached and inhibited its target in both clones, as shown by the similar reduction of ERK phosphorylation 6 hours after drug administration (Figure 1k). To clarify the molecular mechanism responsible for the selective activity of SCH772984 in LKB1 deleted cells, we analyzed the MAPK pathway and mTOR downstream effector activation in cells growing in culture (Figure 2a). ERK phosphorylation was abolished at 6h and reappeared 24h after treatment in both wt and LKB1 deleted cell lines. In both cells the drug induced phosphorylation of AKT. The phosphorylation status of p70S6K, the direct target of mTOR, was qualitatively comparable for both cells. However, the activation of the S6 Ribosomal Protein (S6) was differently affected in these cells. The phosphorylation levels were in fact strongly reduced by SCH772984 in LKB1 KO cells but only slightly perturbed in the LKB1 wt counterpart. Since the activation of p70S6K was comparable, we investigated the role of p90 ribosomal S6 kinase (p90RSK) given it is a ERK target and regulates S6. The phosphorylation at both investigated sites was almost abolished only in cells deleted in LKB1. To further address the role of p90RSK as a mediator of the SCH772984 response, we used CRISPR/Cas9 technology to generate H1299 clones lacking the p90RSK1 protein expression. Figure 2b shows the expression of p90RSK1 in the heterozygous (H1299-RSK KO16) and the homozygous (H1299-RSK KO9 and H1299-RSK KO31) deleted clones. The lack of p90RSK1 resulted in cell responses to the ERK inhibitor (IC50 H1299-RSK KO9 5.70 µM CI: 5.04-6.55 µM and IC50 H1299-RSK KO31 6.03 µM CI: 5.60-6.50 µM) comparable to those obtained by the loss of LKB1 (IC50 4.04 µM, CI: 3.57-4.56 µM) (Figure 2c). The p90RSK1 heterozygous clone’s response was comparable to that observed in the parental cell line (IC50 >5 µM for both). Molecular 12
analyses indicated that homozygous loss of p90RSK1 impaired the phosphorylation of S6 even when the wt LKB1 was expressed (Figure 2d). In vivo too, the inhibition of S6 was noteworthy in the LKB1 KO-derived tumor but not in the parental-derived tumor (Figure 2e).
KRAS mutated NSCLCs deficient in LKB1 are sensitive to the ERK inhibitor SCH772984 To investigate the activity of SCH772984 in cells harboring both a KRAS mutation and an impairment of LKB1, we generated further ad hoc isogenic cell systems. We selected the already available H1299-derived isogenic system expressing wt or G12C KRAS18-21 and applied the CRISPR/Cas9 system, targeting the LKB1 gene. We selected the KRAS(wt)/LKB1 KO and KRAS(G12C)/LKB1 KO clones 22. The presence of a mutant KRAS did not change the response to SCH772984 which remained negligible (IC50 >3 µM). In contrast, treatment of LKB1-deleted clone resulted in a marked response (IC50 KRAS WT/LKB1 KO 0.31 µM CI: 0.24-0.41 µM and IC50 KRAS G12C/LKB1 KO 0.87 µM CI: 0.711.07 µM) (Figure 3a). We then extended our findings in a cell model derived from lung tumors developed in GEMMs expressing mutated KRAS (KP1.9) or mutated KRAS and LKB1 loss (KL7 and KL95). As reported in Figure 3b, the treatment of KL cells resulted in a strong response (IC50 KL7 cells 1.20 µM CI: 1.14-1.26 µM; IC50 KL95 cells 0.49 µM CI: 0.45-0.53 µM) while the same treatment was ineffective in KP cells (IC50 >10 µM). The transient re-introduction of LKB1 in the KL95 clone (Figure 3c) reduced the activity of SCH772984 (Figure 3d).
13
The same cells were implanted s.c. in the syngenic C57Bl/6J mice. The KL tumors treated with SCH772984 showed a tumor weight reduction compared to the control groups reaching statistical significance on days 9, 12 and 15 (p<0.0001) after treatment start, with a best T/C of 39% on day 9 (Figure 3e). The KP tumors had a best T/C of only 77% on day 9 and did not reach a statistically significant reduction at any time in the experiment (Figure 3f). SCH772984 was well tolerated and there was no significant reduction in the animals’ body weights (Supplementary Figure 1c). Also in this in vivo model there was a similar inhibition of ERK phosphorylation in tumor samples from both KP and KL mice, while the reduction in S6 phosphorylation could be appreciated only in KL tumors, thus confirming previous data (Figure 3g). To corroborate these data we moved to the well-characterized Cre-inducible transgenic NSCLC model KRASG12D/WT/LKB1KO/KO. Four weeks after viral induction, mice were randomized to receive SCH772984 or vehicle. By MRI scan we were able to detect a clear effect of the drug (Supplementary Figure 2) and 7 days after treatment start 6 mice per group were sacrificed and lungs histologically examined to determine the tumoral burden. Histological proliferative lesions were represented by multifocal areas of alveolar hyperplasia and multiple adenomas. The mean number of adenomas counted in SCH772984 treated group was 2.5 ± 0.34 compared to 7 ± 0.63 in the vehicle group (p<0.0001) (Figure 3h). To further extend these data in physiologically KRAS mutated cells, LKB1 was selectively knocked down in mutated KRAS NSCLC cell lines proficient in LKB1. Considering the previous data, which strongly suggest that the mTOR pathway is involved in the action of 14
SCH772984, we searched for cells with wt LKB1 and not mutated PI3K/AKT/mTOR pathway. However, all the analyzed NSCLC cell lines harbored a mutation which leads to a constitutive activation of the PI3K/AKT/mTOR pathway which, according to our prediction, could prevent the activity of SCH772984. The LU99 cell line harbors an activating mutation (p.T1025A) in the PIK3CA gene encoding the PI3K catalytic isoform p110α (COSMIC database). This cell line (KRAS mut/LKB1 wt) was used to generate isogenic clones deleted in LKB1 (Figure 4a). As predicted, SCH772984 resulted in an equal lack of response in clones and in the parental cell line (Supplementary Figure 3a). Co-treatment of the LU99 system with the PI3K p110α specific inhibitor PIK-75, using a concentration able to kill less than 15% of cells when used as single agent (Supplementary Figure 3b), restored the sensitivity of LKB1 deficient cells to the ERK inhibitor (IC50 0.28 µM, CI: 0.16-0.42 µM) while no effect was observed for the parental line (IC50 >5 µM) (Figure 4b). To corroborate these results, the PI3K downstream effector AKT was inhibited with ARQ 751 using a concentration of ARQ 751 able to kill less than 15% of cells when used as single agent (Supplementary Figure 3c) but able to inhibit the target as shown by the phosphorylation reduction of the AKT effector PRAS40 (Figure 4c). Again, we restored the sensitivity of LKB1 KO cells to SCH772984 (IC50 0.93 µM, CI: 0.35-1.57 µM) (Figure 4b). From a molecular point of view, as expected, SCH772984 was able to inhibit ERK phosphorylation in both cell lines and a slight S6 inactivation in LKB1 KO cells. The combination SCH772984/ARQ 751 almost completely abolished the S6 activation in the responsive cells only (Figure 4c).
15
We performed the same experiments in the H358 (KRAS mut/LKB1 wt) isogenic system, which is mutated in the PHLPP2 gene (p.D724Y) (COSMIC database). This gene encodes for a phosphatase involved in the regulation of PI3K signalling, in particular acting on ser473 of AKT23. Knocking down LKB1 in H358 (Supplementary Figure 3d) did not significantly alter the sensitivity to SCH772984 (Supplementary Figure 3e). As predicted, co-treatment with the AKT inhibitor ARQ 751, using a dose able to kill less than 15% of cells when used as single agent but able to inhibit the target (Supplementary Figure 3f and 3g) strongly sensitized LKB1 KO (IC50 0.40 µM, CI: 0.23-0.71 µM) but not LKB1 wt cells (IC50 >3 µM) (Figure 3h) to SCH772984. The transient re-introduction of LKB1 in the H358 LKB1 KO clone (Supplementary Figure 3i) strongly reduced the activity of SCH772984 (Supplementary Figure 3j). These findings were extended in vivo using LU99 model where the combination ARQ 751/SCH772984 induced a tumor growth reduction in LKB1 KO clone reaching statistical significance on day 14 (p<0.05) and 16 (p<0.001) after treatment start, with a best T/C of 33% on day 10 (Figure 4d). The LKB1 wt expressing cells had a best T/C of 58% on day 5 and did not reach a significant reduction at any time in the experiment (Figure 4e). It is to note that mice bearing wt cells were sacrificed before the end of the scheduled treatment because the maximum average tumor volume allowed (1500 mg) was reached. All treatments were well tolerated and there was no significant reduction in the animals’ body weights (Supplementary Figure 3k and 3l). Finally, we verified the activity of the ERK inhibitor in two non-manipulated cell lines, A549 and H460, where the expression of LKB1 is naturally lost. Both cell lines, in addition 16
to the LKB1 nonsense mutation (p.Q37*), harbor activating mutations in the KRAS gene (A549: p.G12S; H460: p.Q61H) and mutations activating the PI3K pathway (A549: CPPED1 p.Q188H involved in the regulation of AKT, in particular acting on ser47324; H460: PIK3CA p.E545K; all mutations are described in COSMIC database). As expected, SCH772984 alone was not effective in either cell lines (IC50 >7.5 µM) but the combination of the ERK inhibitor with the AKT inhibitor ARQ 751 induced a cell response (IC50 A549 1.20 µM CI: 0.97-1.47 µM and IC50 H460 1.85 µM CI: 1.38-2.52 µM) (Figure 4f). SCH772984 alone was able to inhibit ERK phosphorylation in both cell lines but had no effect on S6 regulation. The combination SCH772984/ARQ 751 almost completely abolished the activation of S6 (Figure 4g).
Ablation of LKB1 sensitizes NSCLC cells to the ERK inhibitor ulixertinib To corroborate and generalize our findings, avoiding a peculiar mechanism of SCH772984, we treated cells with ulixertinib, a different ERK inhibitor. All the isogenic systems described above were treated with the second ERK inhibitor alone or with the drug combinations that were previously shown to be active. H1299 and the LKB1 KO derived clone were treated with ulixertinib and the lack of LKB1 resulted in a marked response to the drug while cells harboring wt LKB1 were resistant to the treatment (Supplementary Figure 4a). Again, ulixertinib treatment of the H1299 system expressing different mutant isoforms of KRAS and the derived LKB1 KO clones resulted in responses of cells deficient for LKB1 expression, independently from KRAS
17
status, while wt LKB1 cells did not respond to the ERK inhibitor (Supplementary Figure 4b). Similarly, only the KRAS mutated LU99-derived clone deleted in LKB1, but harboring a PIK3CA gene mutation responded to ulixertinib when combined with PIK-75, while ulixertinib alone was not active (Supplementary Figure 4c), in accordance with what was found for SCH772984. The results were the same when AKT was inhibited with ARQ 751 (Supplementary Figure 4d). In the H358 cell line, the combination of ulixertinib with the AKT inhibitor ARQ 751 resulted in a response of the LKB1 KO clone, while the parental cell line was not affected (Supplementary Figure 4e), again similarly to what was seen when SCH772984 was used to inhibit ERK.
18
Discussion This study provides, in a wide panel of experimental systems, evidence that NSCLC tumors lacking LKB1 activity are susceptible to ERK inhibitors activity. This is potentially clinically relevant since the LKB1 gene is mutated, and consequently inactivated, in roughly 30% of sporadic human NSCLCs1. LKB1 phosphorylates and activates AMPK which in turn regulates cellular energy metabolism when the ATP concentration is low25. One of the main activities of AMPK is the inactivation of mTOR when ATP levels are too low to sustain protein synthesis and cell growth6. In this scenario, the lack of LKB1 leads to dysregulation of cellular processes such as cell growth and metabolism under energy stress conditions, resulting in particular sensitivity to treatments that target energy pathways. Our idea, therefore, was to target the MAPK pathway, another signaling pathway involved in cell proliferation, metastatization and metabolism, closely connected to the LKB1/AMPK/mTOR pathway26. Consistent with our hypothesis, LKB1 deficient cells were particularly sensitive to the inhibition of ERK, a protein belonging to the MAPK pathway. Approximately 25-30% of NSCLC patients present with KRAS activating mutations. About 50% of the NSCLC tumors with activating KRAS mutations also harbor LKB1 inactivating mutations2. Current estimates suggest that at least 10% of all NSCLCs are co-mutated for both KRAS and LKB127. LKB1 deficiency in combination with mutant KRAS leads to an aggressive tumor phenotype with high prevalence in mouse models. It has been reported that the KRAS/LKB1 double mutation in murine lung tumors promotes tumor burden, invasion and metastasis28, 29. Our data suggest that ERK inhibition could also be effective in 19
tumors harboring KRAS/LKB1 double mutation, thus increasing the therapeutic strategies for this prognostically unfavorable subgroup of patients for whom no targeted therapy is currently available. We found that ERK inhibition was likely to act through inhibition of the mTOR downstream pathway, as S6 phosphorylation was selectively reduced in cells sensitive to the ERK inhibitors. This is not surprising since there is evidence of connections between the MAPK, PI3K/mTOR and LKB1 pathways3. From this, we hypothesized that constitutive activation of the parallel PI3K/mTOR pathway would abolish the selective activity of ERK inhibitors observed in LKB1 KO cells. The experiments in cell lines with activated PI3K or AKT clearly showed that this was the case. Using specific inhibitors targeting PI3K (PIK-75) and AKT (ARQ 751) at concentrations able to inhibit the targets without inducing cytotoxicity when used as single agent, we showed that LKB1 KO cells, but not LKB1 wt cells, were selectively sensitized to ERK inhibitors. A recently published paper demonstrates that the dual inhibition of mTOR and PI3K can be a promising therapeutic strategy for LKB1-deficient tumors30. These data are in line with our results where we inhibited the PI3K and mTOR target S6 through the inhibition of ERK/p90RSK/S6 axis. These results are potentially and easily translatable to the clinic where the combination of PI3K inhibitors and ERK inhibitors would selectively kill LKB1 deficient (tumor) cells but not LKB1 wt (normal) cells. On the other hand, we were not able to find a panel of PI3K pathway wild-type NSCLC cell lines to further proof that loss of LKB1 is sufficient to sensitize cells with different mutational background to ERK inhibitors. 20
Although SCH772984 is not currently under clinical evaluation, we showed that the results obtained with this inhibitor were also true for another molecule, ulixertinib, an ERK inhibitor already in clinical development12 together with other ERK inhibitors31. In conclusion, our data suggest that the lack of a functional LKB1, now associated with a poor prognosis for patients with NSCLC32, could offer a strong therapeutic advantage. The lack of effect in cells expressing wt LKB1 suggests that treatment of LKB1 mutated tumors with ERK inhibitors should have a low toxicity profile. In this context, we must always consider the status of PI3K/AKT/mTOR pathway, given that the activation of the latter renders unsuccessful the ERK inhibitor treatment. This limitation can be easily overcome considering a combination treatment with, at least, PI3K and AKT inhibitors. Although PI3K inhibitors were described to have several limitations due to the emergence of doselimiting for the presence of adverse effects33, our data open the possibility to reconsider these drugs for the use in combinations with ERK inhibitors, given that these inhibitors should be used at low doses (sufficient to inhibit the target) and hence likely not to induce severe toxicity. On the other hand, the analysis of data available in the cBioPortal
34
revealed that, while the NSCLC LKB1 loss cell lines are 100% mutated in the PI3K/AKT/mTOR pathway, the 60-85% of NSCLC patients mutated in LKB1 do not present additional mutations in genes belonging to the PI3K/AKT/mTOR pathway, rendering the majority of LKB1 mutated patients suitable for a monotherapy treatment. LKB1 mutation in NSCLC is associated with loss of protein. Wt LKB1 expression is regulated both at transcriptional and pos-transcriptional levels35, 36. Any treatment that downregulates LKB1 expression (as we showed using siRNAs in cell cultures) should 21
result in sensitization to ERK inhibitors. Transient and specific (in tumor) LKB1 downregulation in NSCLC is potentially feasible, for example by targeted delivery of siRNAs or miRNAs in the lung, thus increasing the number of the patients who could potentially benefit from this treatment. The selective advantage for NSCLC patients with LKB1 mutated tumors might therefore possibly be extended to other NSCLC patients with wt LKB1.
22
Acknowledgements Prof. Zippelius University Hospital and University of Basel (Switzerland) kindly provided the KRASG12D/WT cell line (KP1.9). ARQ 751 was kindly provided by ArQule.
23
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28
Figures legend Figure 1: Lack of LKB1 expression causes increased sensitivity to the ERK inhibitor SCH772984 in KRAS wt NSCLC cells. a) expression of LKB1 in H1299 and isogenic H1299-LKB1 KO clones. Ran was used as loading control. b) activity of SCH772984 in H1299 and H1299-LKB1 KO clones. c) activity of SCH772984 in H1299 and H1299-LKB1 KO clone assessed by colony assay. d) representative images of colony assay: H1299 in the left and H1299-LKB1 KO in the right panel. Drug doses are expressed in nM. e) expression of LKB1 in H1299 and isogenic H1299-LKB1 KO clones treated with esiRNA targeting LKB1 (H1299-esiRNA) or scramble (H1299-Neg). Ran was used as loading control. f) activity of esiRNA targeting LKB1 or scramble in H1299. g) expression of LKB1 in H1299 and isogenic H1299-LKB1 KO clone transfected with pCDNA3/LKB1 or pCDNA3 empty vector. Ran was used as loading control. h) activity of SCH772984 in H1299 and H1299-LKB1 KO clone transfected with pCDNA3/LKB1 (L) or pCDNA3 empty vector (p). i) tumor growth inhibition in H1299LKB1 KO clone injected mice (N=8) treated with SCH772984 25 mg/kg or vehicle. *p<0.05 **p<0.01 SCH772984 vs. vehicle j) tumor growth inhibition activity on H1299 injected mice (N=8) treated with SCH772984 25 mg/kg or vehicle. k) activation of ERK in tumors removed from mice 6h after the last treatment. Actin was used as loading control. All in vitro data are depicted as mean ± SD (6 replicates for 3 independent experiments). Statistical analyses are reported in supplementary material.
Figure 2: Molecular analysis after SCH772984 treatment. 29
a) activation of MAPK pathway and mTOR downstream effectors in H1299 and H1299LKB1 KO cells, either untreated or treated with SCH772984 0.5 µM, at the indicated time points. b) expression of p90RSK and LKB1 in H1299, H1299-LKB1 KO and H1299-RSK KO cells. Actin was used as loading control. c) activity of SCH772984 in H1299, H1299-LKB1 KO and H1299-RSK KO clones. Data are depicted as mean ± SD (6 replicates for 3 independent experiments). Statistical analyses are reported in supplementary material. d) activation of ERK, S6 and expression of p90RSK and in H1299, H1299-LKB1 KO and H1299-RSK KO cells, either untreated or treated with SCH772984 0.5 µM, estimated at the indicated time points. e) activation of S6 in tumors removed from mice 24h after the last treatment. Actin was used as loading control.
Figure 3: KRAS mutated NSCLC murine models deficient in LKB1 are sensitive to the ERK inhibitor SCH772984. a) activity of SCH772984 in KRAS(wt) or KRAS(G12C) H1299 and their isogenic LKB1 KO clones. b) activity of SCH772984 in KP or KL cells. c) expression of LKB1 in KL95 cells transiently transfected with pCDNA3/LKB1 or pCDNA3 empty vector. Ran was used as loading control. d) activity of SCH772984 in KL95 cells transfected with pCDNA3 empty vector (upper) or pCDNA3/LKB1 (lower). e) tumor growth inhibition on KL cells injected mice (N=8) treated with SCH772984 25 mg/kg or vehicle. ****p<0.0001 SCH772984 vs. vehicle f) tumor growth inhibition on KP cells injected mice (N=8) treated with SCH772984 25 mg/kg or vehicle. g) activation of ERK and S6 in KP and KL tumors removed from mice
30
6h after the last treatment. Actin was used as loading control. h) mean number of counted masses in lungs of KL mice treated with SCH772984 (N=6) or vehicle (N=6).
Figure 4: KRAS mutated NSCLC human models deficient in LKB1 are sensitive to the ERK inhibitor SCH772984. a) expression of LKB1 in LU99 and in isogenic LU99-LKB1 KO clones. Ran was used as loading control. b) activity of SCH772984 alone or in combination with PIK-75 10 nM or ARQ 751 50 nM in LU99 and LU99-LKB1 KO clone. c) activation of ERK, PRAS40 and S6 in LU99 and LU99-LKB1 KO cells after 24h of treatment with SCH772984 0.5 µM and ARQ 751 50 nM. Ran was used as loading control. d) tumor growth inhibition in LU99-LKB1 KO clone injected mice (N=8) treated with SCH772984 25 mg/kg, ARQ 751 50 mg/kg, SCH772984/ARQ 751 or vehicle. *p<0.05; ***p<0.001 SCH772984/ARQ 751 vs. vehicle. e) tumor growth inhibition activity on LU99 injected mice (N=8) treated with SCH772984 25 mg/kg, ARQ 751 50 mg/kg, SCH772984/ARQ 751 or vehicle. f) activity of SCH772984 alone or in combination with ARQ 751 50 nM in A549 and H460 cells. g) activation of ERK, PRAS40 and S6 in A549 and H460 cells after 24h of treatment with SCH772984 0.5 µM and ARQ 751 50 nM. Ran was used as loading control. All in vitro data are depicted as mean ± SD (6 replicates for 3 independent experiments). Statistical analyses are reported in supplementary material.
31
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S1556-0864(19)33568-3
DOI:
https://doi.org/10.1016/j.jtho.2019.10.009
Reference:
JTHO 1617
To appear in:
Journal of Thoracic Oncology
Received Date: 27 March 2019 Revised Date:
7 October 2019
Accepted Date: 12 October 2019
Please cite this article as: Caiola E, Iezzi A, Tomanelli M, Bonaldi E, Scagliotti A, Colombo M, Guffanti F, Micotti E, Garassino MC, Minoli L, Scanziani E, Broggini M, Marabese M, LKB1 deficiency renders nonsmall-cell lung cancer cells sensitive to ERK inhibitors., Journal of Thoracic Oncology (2019), doi: https:// doi.org/10.1016/j.jtho.2019.10.009. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 International Association for the Study of Lung Cancer. Published by Elsevier Inc. All rights reserved.
LKB1 deficiency renders non-small-cell lung cancer cells sensitive to ERK inhibitors.
Elisa Caiola§a, PhD Alice Iezzi§a, MSc Michele Tomanellia, MSc Elisa Bonaldia, MSc Arianna Scagliottia, MSc Marika Colomboa, MSc Federica Guffanti a, MSc Edoardo Micottib, PhD Marina Chiara Garassinoc, MD Lucia Minolid,e, DVM Eugenio Scanzianid,e, DVM Massimo Broggini#a, PhD Mirko Marabese#a, PhD
a
Laboratory of Molecular Pharmacology, Department of Oncology, Istituto di Ricerche
Farmacologiche Mario Negri IRCCS, Milan, Italy. bDepartment of Neuroscience, Istituto di Ricerche Farmacologiche Mario Negri IRCCS, Milan, Italy. cUnit of Thoracic Oncology, Fondazione IRCCS Istituto Nazionale dei Tumori, Milan, Italy.
Mouse & Animal
d
Pathology Lab, Fondazione Filarete, Milan, Italy. eDepartment of Veterinary Medicine, University of Milan, Milan, Italy 1
§
Elisa Caiola and Alice Iezzi are co-first authors
# Mirko Marabese and Massimo Broggini are co-last authors
Corresponding author: Mirko Marabese, Laboratory of Molecular Pharmacology, Department of Oncology, Istituto di Ricerche Farmacologiche Mario Negri IRCCS, [email protected]
Funding information The research leading to these results has received funding from AIRC under MFAG 2016 ID. 18386 project – P.I. Marabese; IG 2015 – ID. 16792 project – P.I. Broggini and IG 2017 – ID. 20085 project – P.I. Garassino
Dr. Garassino reports grants from AstraZeneca, Bristol-Myers Squibb, MSD Oncology, Roche, Takeda, Incyte, Lilly, Merck, Pfizer, Clovis, Merck Senoro, Bayer, Spectrum Pharmaceuticals, F. Hoffmann-La Roche, Blueprint Medicine; personal fees from AstraZeneca, Bristol-Myers Squibb, MSD Oncology, Novartis, Roche, Takeda, Tiziana Life Sciences, Celgene, Incyte, Boehringer Ingelheim, Otsuka Pharma, Bayer, Inivata, SanofiAventis, SeaGen International GmbH; non-financial support from AstraZeneca, Roche, Pfizer, F. Hoffmann-La Roche. The remaining authors have nothing to disclose.
2
Abstract Background Liver kinase B1 (LKB1/STK11) is one of the most mutated genes in non-small-cell lung cancer (NSCLC) accounting for about one third of cases and its activity is impaired in about half of KRAS mutated NSCLC. At present, these patients cannot benefit from any specific therapy. Methods Through CRISPR/Cas9 technology, we systematically deleted LKB1 in both wild-type and KRAS-mutated human NSCLC cells. By using these isogenic systems together with genetically engineered mouse models we investigated the cell response to ERK inhibitors both in vitro and in vivo. Results In all the systems used here, the loss of LKB1 creates a vulnerability and renders these cells particularly sensitive to ERK inhibitors both in vitro and in vivo. The same cells expressing a wt LKB1 poorly respond to these drugs. At molecular level, ERK inhibitors induced, in the absence of LKB1, a marked inhibition of p90 ribosomal S6 kinase activation, which in turn abolished S6 protein activation, promoting the cytotoxic effect. Conclusions This work shows that ERK inhibitors are effective in LKB1 and LKB1/KRAS mutated tumors, thus offering a therapeutic strategy for this prognostically unfavorable subgroup of patients. Since ERK inhibitors are already in clinical development, our findings could be easily translatable to the clinic. Importantly, the lack of effect in cells expressing wild-type 3
LKB1, predicts that treatment of LKB1 mutated tumors with ERK inhibitors should have a favorable toxicity profile.
Short title ERK inhibitors in LKB1 mutated NSCLC
Keywords LKB1/STK11, ERK inhibitor, SCH772984, ulixertinib, NSCLC
4
Introduction Liver kinase B1 (LKB1/STK11) is one of the most commonly mutated genes in non-small cell lung cancer (NSCLC) accounting for about one third of cases1. In addition, its activity is impaired in about 50% of KRAS mutated NSCLC2. This large number of patients has an aggressive, chemoresistant subtype of cancer and, at present, has no specific therapy. LKB1 is a serine/threonine kinase that serves as a master regulator of the cell metabolism3. The best characterized LKB1 substrate is the AMP-activated protein kinase (AMPK) and together they control cell metabolism in response to the availability of nutrients4. Besides metabolism, both AMPK and LKB1 were reported to have roles in the control of cell growth5. LKB1/AMPK pathway activation results in negative regulation of one of the most deregulated
signaling
systems
in
cancer,
the
PhosphatidylInositol
3-Kinase
(PI3K)/AKT/mammalian target of the rapamycin (mTOR) pathway6. mTOR is a pivotal protein in mTORC1 and mTORC2 complexes and acts as an integrator of nutrients and growth factor signals, resulting in the control of cell growth in all eukaryotes. These signals act on ribosome biogenesis and the translation of proteins that promote cell growth and division7. The PI3K pathway is not the only regulator of mTOR. Extracellular signalregulated kinase (ERK) and its target, p90 ribosomal S6 kinase (p90RSK), both target mTORC1 activity8, 9. SCH772984, an analog of the orally available clinical candidate SCH900353/MK-8353, has a dual mechanism of action: allosteric inhibition of MEK1/2 binding to ERK, resulting in the lack of ERK phosphorylation and ATP-competitive ERK inhibition, resulting in the absence of phosphorylation of its substrates10. This compound was described as an 5
extremely selective ERK1/2 inhibitor able to suppress the mitogen-activated protein kinase (MAPK) pathway signaling and cell proliferation in many tumor models. In particular, it has a strong activity in cell lines positive for KRAS and BRAF mutations11. Ulixertinib (BVD-523, VRT752271) is a small molecule under phase I clinical evaluation12. It potently and selectively inhibits ERK1/2 kinases in a reversible, ATP-competitive way. Signal transduction, cell survival and cell proliferation are negatively regulated particularly in cell lines harboring MAPK pathway activating mutations. Like SCH772984, ulixertinib inhibits cells growth in BRAF- and in KRAS-mutant tumors13. In the present work, we investigated the activity of SCH772984 and ulixertinib in different NSCLC models with different LKB1 and KRAS status.
6
Methods Cell cultures, drugs, growth assay, siRNA and plasmids NSCLC cell lines (H1299, LU99, H460, A549 and H358) were grown in RPMI-1640 supplemented with 10% FBS. H1299 derived clones (wt and G12C KRAS) were grown in normal medium including 500 µg/mL of G418 (Gibco). LKB1 KO clones in H1299, LU99 and H358 were generated using LKB1 CRISPR/Cas9 KO Plasmid and HDR Plasmid (Santa Cruz Biotechnology) according to the manufacturer's instructions. Cell lines and clones are routinely tested by PCR for mycoplasma contamination and authenticated with the PowerPlex 16 HS System (Promega) every six months by comparing the STR profiles with those deposited in ATCC and/or DSMZ databases. SCH772984 (TargetMol), ulixertinib (TargetMol), ARQ 751 (ArQule) and PIK-75 (Selleckchem) DMSO stock solutions were dissolved in medium just before use. In all the cytotoxicity experiments, cells were continuously treated for 72h. Drug effects were estimated by the CellTiter AQueous MTS as reported14. The mean and SD of at least three independent experiments are presented. EsiRNA were purchased from Sigma Aldrich and transfected by Lipofectamine 2000 (Invitrogen). pcDNA3-FLAG-LKB14 was a gift from Lewis Cantley (Addgene plasmid # 8590). The colony assay was performed as reported15.
Western blotting analyses Proteins were extracted and visualized as reported15. Immunoblotting was carried out with the antibodies listed in the supplementary material.
7
Xenograft studies The IRFMN adheres to the principles declared in the supplementary material. For the xenograft experiments, six-week-old female nude athymic mice (Envigo) were housed at constant temperature and humidity, according to institutional guidelines. SCH772984 (ChemieTek) was dissolved in 50% DMSO, 20% 2-Hydroxypropyl)-beta-cyclodextrin (Carlo Erba Reagents S.r.l) and given i.p. twice a day for 14 days consecutively. ARQ 751 (ArQule) was dissolved in 0.5% methylcellulose (Carlo Erba Reagents S.r.l) and given p.o. for three days a week for 3 times. Additional details are reported in supplementary material.
Genetically engineered murine models of NSCLC and derived cell lines Conditional transgenic models (KRAStm4Tyj/J and LKB1/STK11tm1.1Sjm/J) were purchased from Jackson Laboratories. KRAStm4Tyj/J (KRASG12D/WT) and LKB1/STK11tm1.1Sjm/J (LKB1KO/KO) mice were crossed to generate conditional double KO mice (KRASG12D/WT/LKB1KO/KO) which were genotyped using PCR protocols obtained from the Jackson Laboratories. KRASG12D/WT and KRASG12D/WT/LKB1KO/KO mice were treated intranasally with Adeno-Cre (SignaGen Laboratories) at 6 weeks of age as previously reported16. Four weeks after Adeno-Cre treatment, the animals were randomized to receive SCH772984 or vehicle as for xenograft experiments. At the end of treatment, 6 animals per group were sacrificed and lungs fixed in formalin for histopathological evaluation.
8
Details for isolation of cell lines from lung nodules are reported in supplementary material. Two independent clones from KRASG12D/WT/LKB1KO/KO lung tumors (KL 7 and KL 95) were isolated and their behavior compared to that of a KRASG12D/WT cell line (KP1.9).
MRI scan Before treatment and after 7 days post-treatment, the group of animals for nodule count was analyzed by MRI using a small rodents 7 Tesla BRUKER scanner (Bruker Biospec 70/30), equipped with a 72 volume coil in transmission and a saddle shaped 2x2 surface array for receiving the signal. Details are reported in the supplementary material.
Histopathology Lungs were formalin fixed, paraffin embedded and 4 um thick sections were routinely stained with Hematoxylin and Eosin. Pulmonary proliferative lesions were classified according to international guidelines17. The tumoral burden was assessed by counting the number of pulmonary tumors on an approximatly 50 mm2 of parenchymal area.
Statistical analyses Statistical analyses were done using GraphpadPrism version 7. Differences between groups were considered significant when the p-values were <0.05.
9
Results While exploring drug responses in NSCLC cell lines in vitro, we found striking different responses to the ERK inhibitor SCH772984 in two cell lines, H727 and H1299. In H1299 the drug was completely inactive (IC50 >7 µM) while in H727 there was a very good response, with an IC50 of approximately 0.13 µM (Confidence Interval (CI): 0.07-0.24 µM) (Supplementary Figure 1a). Among the mutations harbored in the two cell lines, our attention was caught on KRAS and LKB1 given H727 cell line is mutated in both KRAS and LKB1 while these proteins were wild-type (wt) in H1299 (COSMIC database, https://cancer.sanger.ac.uk/cosmic). Being H727 cells derived from carcinoid, we no more used this model for subsequent experiments and concentrated our experiments on carcinomas.
Lack of LKB1 expression increases sensitivity to the ERK inhibitor SCH772984 in KRAS wt NSCLC cells by impairing S6 Ribosomal Protein activation through p90RSK protein The H1299 cell line (KRAS wt/LKB1 wt) was genetically manipulated with CRISPR/Cas9 technique to generate a deletion in the LKB1 gene (resulting in lack of protein expression), and we selected two independent LKB1 knock-out (KO) clones (H1299-LKB1 KO 1 and 2) (Figure 1a). SCH772984 treatment gave a marked, statistically significant response in LKB1 deficient cells compared to the parental cell line (Figure 1b). Whereas for the latter it was not possible to calculate an IC50 with the drug concentrations tested, the IC50 of the two LKB1 deficient clones were respectively 2.4 µM (CI: 1.9-3.0 µM) and 2.8 µM (CI: 2.3-3.5
10
µM). These results were confirmed by using the colony formation assay (Figure 1c and 1d). To corroborate these data with a different approach, LKB1 was silenced with specific LKB1 endoribonuclease-prepared siRNA (esiRNA) transfection, which strongly downregulated LKB1 protein expression, while universal negative esiRNA had no effect (Figure 1e). As in H1299-LKB1 KO clones, transient silencing of LKB1 in H1299 cells induced significant sensitization to SCH772984 compared to cells transfected with the negative esiRNA (Figure 1f). In agreement with these data, the re-introduction of LKB1 in the H1299 LKB1 KO clone (Figure 1g) strongly reduced the activity of SCH772984 to the level of parental H1299 cells, while LKB1 overexpression did not alter the behavior of H1299 after drug treatment (Figure 1h). To check whether the absence of LKB1 increased the sensitivity to SCH772984 in vivo, both LKB1 wt and KO cells were subcutaneously implanted into nude mice. Although the LKB1 KO clone took much longer (40 days) to reach randomization weight than the LKB1 wt cell line (19 days), their growth rates from this point were comparable. SCH772984 treatment induced a tumor growth reduction in LKB1 KO clone reaching statistical significance on day 19 (p<0.05) and 21 (p<0.01) after treatment start, with a best treated/control ratio (T/C) of 33% on day 16 (Figure 1i). The same treatment schedule in LKB1 wt expressing tumors had a best T/C of only 77% on day 9 and did not reach a significant reduction at any time during the experiment (Figure 1j). SCH772984 was well tolerated and there was no significant reduction in the animals’ body weights (Supplementary Figure 1b). The drug
11
reached and inhibited its target in both clones, as shown by the similar reduction of ERK phosphorylation 6 hours after drug administration (Figure 1k). To clarify the molecular mechanism responsible for the selective activity of SCH772984 in LKB1 deleted cells, we analyzed the MAPK pathway and mTOR downstream effector activation in cells growing in culture (Figure 2a). ERK phosphorylation was abolished at 6h and reappeared 24h after treatment in both wt and LKB1 deleted cell lines. In both cells the drug induced phosphorylation of AKT. The phosphorylation status of p70S6K, the direct target of mTOR, was qualitatively comparable for both cells. However, the activation of the S6 Ribosomal Protein (S6) was differently affected in these cells. The phosphorylation levels were in fact strongly reduced by SCH772984 in LKB1 KO cells but only slightly perturbed in the LKB1 wt counterpart. Since the activation of p70S6K was comparable, we investigated the role of p90 ribosomal S6 kinase (p90RSK) given it is a ERK target and regulates S6. The phosphorylation at both investigated sites was almost abolished only in cells deleted in LKB1. To further address the role of p90RSK as a mediator of the SCH772984 response, we used CRISPR/Cas9 technology to generate H1299 clones lacking the p90RSK1 protein expression. Figure 2b shows the expression of p90RSK1 in the heterozygous (H1299-RSK KO16) and the homozygous (H1299-RSK KO9 and H1299-RSK KO31) deleted clones. The lack of p90RSK1 resulted in cell responses to the ERK inhibitor (IC50 H1299-RSK KO9 5.70 µM CI: 5.04-6.55 µM and IC50 H1299-RSK KO31 6.03 µM CI: 5.60-6.50 µM) comparable to those obtained by the loss of LKB1 (IC50 4.04 µM, CI: 3.57-4.56 µM) (Figure 2c). The p90RSK1 heterozygous clone’s response was comparable to that observed in the parental cell line (IC50 >5 µM for both). Molecular 12
analyses indicated that homozygous loss of p90RSK1 impaired the phosphorylation of S6 even when the wt LKB1 was expressed (Figure 2d). In vivo too, the inhibition of S6 was noteworthy in the LKB1 KO-derived tumor but not in the parental-derived tumor (Figure 2e).
KRAS mutated NSCLCs deficient in LKB1 are sensitive to the ERK inhibitor SCH772984 To investigate the activity of SCH772984 in cells harboring both a KRAS mutation and an impairment of LKB1, we generated further ad hoc isogenic cell systems. We selected the already available H1299-derived isogenic system expressing wt or G12C KRAS18-21 and applied the CRISPR/Cas9 system, targeting the LKB1 gene. We selected the KRAS(wt)/LKB1 KO and KRAS(G12C)/LKB1 KO clones 22. The presence of a mutant KRAS did not change the response to SCH772984 which remained negligible (IC50 >3 µM). In contrast, treatment of LKB1-deleted clone resulted in a marked response (IC50 KRAS WT/LKB1 KO 0.31 µM CI: 0.24-0.41 µM and IC50 KRAS G12C/LKB1 KO 0.87 µM CI: 0.711.07 µM) (Figure 3a). We then extended our findings in a cell model derived from lung tumors developed in GEMMs expressing mutated KRAS (KP1.9) or mutated KRAS and LKB1 loss (KL7 and KL95). As reported in Figure 3b, the treatment of KL cells resulted in a strong response (IC50 KL7 cells 1.20 µM CI: 1.14-1.26 µM; IC50 KL95 cells 0.49 µM CI: 0.45-0.53 µM) while the same treatment was ineffective in KP cells (IC50 >10 µM). The transient re-introduction of LKB1 in the KL95 clone (Figure 3c) reduced the activity of SCH772984 (Figure 3d).
13
The same cells were implanted s.c. in the syngenic C57Bl/6J mice. The KL tumors treated with SCH772984 showed a tumor weight reduction compared to the control groups reaching statistical significance on days 9, 12 and 15 (p<0.0001) after treatment start, with a best T/C of 39% on day 9 (Figure 3e). The KP tumors had a best T/C of only 77% on day 9 and did not reach a statistically significant reduction at any time in the experiment (Figure 3f). SCH772984 was well tolerated and there was no significant reduction in the animals’ body weights (Supplementary Figure 1c). Also in this in vivo model there was a similar inhibition of ERK phosphorylation in tumor samples from both KP and KL mice, while the reduction in S6 phosphorylation could be appreciated only in KL tumors, thus confirming previous data (Figure 3g). To corroborate these data we moved to the well-characterized Cre-inducible transgenic NSCLC model KRASG12D/WT/LKB1KO/KO. Four weeks after viral induction, mice were randomized to receive SCH772984 or vehicle. By MRI scan we were able to detect a clear effect of the drug (Supplementary Figure 2) and 7 days after treatment start 6 mice per group were sacrificed and lungs histologically examined to determine the tumoral burden. Histological proliferative lesions were represented by multifocal areas of alveolar hyperplasia and multiple adenomas. The mean number of adenomas counted in SCH772984 treated group was 2.5 ± 0.34 compared to 7 ± 0.63 in the vehicle group (p<0.0001) (Figure 3h). To further extend these data in physiologically KRAS mutated cells, LKB1 was selectively knocked down in mutated KRAS NSCLC cell lines proficient in LKB1. Considering the previous data, which strongly suggest that the mTOR pathway is involved in the action of 14
SCH772984, we searched for cells with wt LKB1 and not mutated PI3K/AKT/mTOR pathway. However, all the analyzed NSCLC cell lines harbored a mutation which leads to a constitutive activation of the PI3K/AKT/mTOR pathway which, according to our prediction, could prevent the activity of SCH772984. The LU99 cell line harbors an activating mutation (p.T1025A) in the PIK3CA gene encoding the PI3K catalytic isoform p110α (COSMIC database). This cell line (KRAS mut/LKB1 wt) was used to generate isogenic clones deleted in LKB1 (Figure 4a). As predicted, SCH772984 resulted in an equal lack of response in clones and in the parental cell line (Supplementary Figure 3a). Co-treatment of the LU99 system with the PI3K p110α specific inhibitor PIK-75, using a concentration able to kill less than 15% of cells when used as single agent (Supplementary Figure 3b), restored the sensitivity of LKB1 deficient cells to the ERK inhibitor (IC50 0.28 µM, CI: 0.16-0.42 µM) while no effect was observed for the parental line (IC50 >5 µM) (Figure 4b). To corroborate these results, the PI3K downstream effector AKT was inhibited with ARQ 751 using a concentration of ARQ 751 able to kill less than 15% of cells when used as single agent (Supplementary Figure 3c) but able to inhibit the target as shown by the phosphorylation reduction of the AKT effector PRAS40 (Figure 4c). Again, we restored the sensitivity of LKB1 KO cells to SCH772984 (IC50 0.93 µM, CI: 0.35-1.57 µM) (Figure 4b). From a molecular point of view, as expected, SCH772984 was able to inhibit ERK phosphorylation in both cell lines and a slight S6 inactivation in LKB1 KO cells. The combination SCH772984/ARQ 751 almost completely abolished the S6 activation in the responsive cells only (Figure 4c).
15
We performed the same experiments in the H358 (KRAS mut/LKB1 wt) isogenic system, which is mutated in the PHLPP2 gene (p.D724Y) (COSMIC database). This gene encodes for a phosphatase involved in the regulation of PI3K signalling, in particular acting on ser473 of AKT23. Knocking down LKB1 in H358 (Supplementary Figure 3d) did not significantly alter the sensitivity to SCH772984 (Supplementary Figure 3e). As predicted, co-treatment with the AKT inhibitor ARQ 751, using a dose able to kill less than 15% of cells when used as single agent but able to inhibit the target (Supplementary Figure 3f and 3g) strongly sensitized LKB1 KO (IC50 0.40 µM, CI: 0.23-0.71 µM) but not LKB1 wt cells (IC50 >3 µM) (Figure 3h) to SCH772984. The transient re-introduction of LKB1 in the H358 LKB1 KO clone (Supplementary Figure 3i) strongly reduced the activity of SCH772984 (Supplementary Figure 3j). These findings were extended in vivo using LU99 model where the combination ARQ 751/SCH772984 induced a tumor growth reduction in LKB1 KO clone reaching statistical significance on day 14 (p<0.05) and 16 (p<0.001) after treatment start, with a best T/C of 33% on day 10 (Figure 4d). The LKB1 wt expressing cells had a best T/C of 58% on day 5 and did not reach a significant reduction at any time in the experiment (Figure 4e). It is to note that mice bearing wt cells were sacrificed before the end of the scheduled treatment because the maximum average tumor volume allowed (1500 mg) was reached. All treatments were well tolerated and there was no significant reduction in the animals’ body weights (Supplementary Figure 3k and 3l). Finally, we verified the activity of the ERK inhibitor in two non-manipulated cell lines, A549 and H460, where the expression of LKB1 is naturally lost. Both cell lines, in addition 16
to the LKB1 nonsense mutation (p.Q37*), harbor activating mutations in the KRAS gene (A549: p.G12S; H460: p.Q61H) and mutations activating the PI3K pathway (A549: CPPED1 p.Q188H involved in the regulation of AKT, in particular acting on ser47324; H460: PIK3CA p.E545K; all mutations are described in COSMIC database). As expected, SCH772984 alone was not effective in either cell lines (IC50 >7.5 µM) but the combination of the ERK inhibitor with the AKT inhibitor ARQ 751 induced a cell response (IC50 A549 1.20 µM CI: 0.97-1.47 µM and IC50 H460 1.85 µM CI: 1.38-2.52 µM) (Figure 4f). SCH772984 alone was able to inhibit ERK phosphorylation in both cell lines but had no effect on S6 regulation. The combination SCH772984/ARQ 751 almost completely abolished the activation of S6 (Figure 4g).
Ablation of LKB1 sensitizes NSCLC cells to the ERK inhibitor ulixertinib To corroborate and generalize our findings, avoiding a peculiar mechanism of SCH772984, we treated cells with ulixertinib, a different ERK inhibitor. All the isogenic systems described above were treated with the second ERK inhibitor alone or with the drug combinations that were previously shown to be active. H1299 and the LKB1 KO derived clone were treated with ulixertinib and the lack of LKB1 resulted in a marked response to the drug while cells harboring wt LKB1 were resistant to the treatment (Supplementary Figure 4a). Again, ulixertinib treatment of the H1299 system expressing different mutant isoforms of KRAS and the derived LKB1 KO clones resulted in responses of cells deficient for LKB1 expression, independently from KRAS
17
status, while wt LKB1 cells did not respond to the ERK inhibitor (Supplementary Figure 4b). Similarly, only the KRAS mutated LU99-derived clone deleted in LKB1, but harboring a PIK3CA gene mutation responded to ulixertinib when combined with PIK-75, while ulixertinib alone was not active (Supplementary Figure 4c), in accordance with what was found for SCH772984. The results were the same when AKT was inhibited with ARQ 751 (Supplementary Figure 4d). In the H358 cell line, the combination of ulixertinib with the AKT inhibitor ARQ 751 resulted in a response of the LKB1 KO clone, while the parental cell line was not affected (Supplementary Figure 4e), again similarly to what was seen when SCH772984 was used to inhibit ERK.
18
Discussion This study provides, in a wide panel of experimental systems, evidence that NSCLC tumors lacking LKB1 activity are susceptible to ERK inhibitors activity. This is potentially clinically relevant since the LKB1 gene is mutated, and consequently inactivated, in roughly 30% of sporadic human NSCLCs1. LKB1 phosphorylates and activates AMPK which in turn regulates cellular energy metabolism when the ATP concentration is low25. One of the main activities of AMPK is the inactivation of mTOR when ATP levels are too low to sustain protein synthesis and cell growth6. In this scenario, the lack of LKB1 leads to dysregulation of cellular processes such as cell growth and metabolism under energy stress conditions, resulting in particular sensitivity to treatments that target energy pathways. Our idea, therefore, was to target the MAPK pathway, another signaling pathway involved in cell proliferation, metastatization and metabolism, closely connected to the LKB1/AMPK/mTOR pathway26. Consistent with our hypothesis, LKB1 deficient cells were particularly sensitive to the inhibition of ERK, a protein belonging to the MAPK pathway. Approximately 25-30% of NSCLC patients present with KRAS activating mutations. About 50% of the NSCLC tumors with activating KRAS mutations also harbor LKB1 inactivating mutations2. Current estimates suggest that at least 10% of all NSCLCs are co-mutated for both KRAS and LKB127. LKB1 deficiency in combination with mutant KRAS leads to an aggressive tumor phenotype with high prevalence in mouse models. It has been reported that the KRAS/LKB1 double mutation in murine lung tumors promotes tumor burden, invasion and metastasis28, 29. Our data suggest that ERK inhibition could also be effective in 19
tumors harboring KRAS/LKB1 double mutation, thus increasing the therapeutic strategies for this prognostically unfavorable subgroup of patients for whom no targeted therapy is currently available. We found that ERK inhibition was likely to act through inhibition of the mTOR downstream pathway, as S6 phosphorylation was selectively reduced in cells sensitive to the ERK inhibitors. This is not surprising since there is evidence of connections between the MAPK, PI3K/mTOR and LKB1 pathways3. From this, we hypothesized that constitutive activation of the parallel PI3K/mTOR pathway would abolish the selective activity of ERK inhibitors observed in LKB1 KO cells. The experiments in cell lines with activated PI3K or AKT clearly showed that this was the case. Using specific inhibitors targeting PI3K (PIK-75) and AKT (ARQ 751) at concentrations able to inhibit the targets without inducing cytotoxicity when used as single agent, we showed that LKB1 KO cells, but not LKB1 wt cells, were selectively sensitized to ERK inhibitors. A recently published paper demonstrates that the dual inhibition of mTOR and PI3K can be a promising therapeutic strategy for LKB1-deficient tumors30. These data are in line with our results where we inhibited the PI3K and mTOR target S6 through the inhibition of ERK/p90RSK/S6 axis. These results are potentially and easily translatable to the clinic where the combination of PI3K inhibitors and ERK inhibitors would selectively kill LKB1 deficient (tumor) cells but not LKB1 wt (normal) cells. On the other hand, we were not able to find a panel of PI3K pathway wild-type NSCLC cell lines to further proof that loss of LKB1 is sufficient to sensitize cells with different mutational background to ERK inhibitors. 20
Although SCH772984 is not currently under clinical evaluation, we showed that the results obtained with this inhibitor were also true for another molecule, ulixertinib, an ERK inhibitor already in clinical development12 together with other ERK inhibitors31. In conclusion, our data suggest that the lack of a functional LKB1, now associated with a poor prognosis for patients with NSCLC32, could offer a strong therapeutic advantage. The lack of effect in cells expressing wt LKB1 suggests that treatment of LKB1 mutated tumors with ERK inhibitors should have a low toxicity profile. In this context, we must always consider the status of PI3K/AKT/mTOR pathway, given that the activation of the latter renders unsuccessful the ERK inhibitor treatment. This limitation can be easily overcome considering a combination treatment with, at least, PI3K and AKT inhibitors. Although PI3K inhibitors were described to have several limitations due to the emergence of doselimiting for the presence of adverse effects33, our data open the possibility to reconsider these drugs for the use in combinations with ERK inhibitors, given that these inhibitors should be used at low doses (sufficient to inhibit the target) and hence likely not to induce severe toxicity. On the other hand, the analysis of data available in the cBioPortal
34
revealed that, while the NSCLC LKB1 loss cell lines are 100% mutated in the PI3K/AKT/mTOR pathway, the 60-85% of NSCLC patients mutated in LKB1 do not present additional mutations in genes belonging to the PI3K/AKT/mTOR pathway, rendering the majority of LKB1 mutated patients suitable for a monotherapy treatment. LKB1 mutation in NSCLC is associated with loss of protein. Wt LKB1 expression is regulated both at transcriptional and pos-transcriptional levels35, 36. Any treatment that downregulates LKB1 expression (as we showed using siRNAs in cell cultures) should 21
result in sensitization to ERK inhibitors. Transient and specific (in tumor) LKB1 downregulation in NSCLC is potentially feasible, for example by targeted delivery of siRNAs or miRNAs in the lung, thus increasing the number of the patients who could potentially benefit from this treatment. The selective advantage for NSCLC patients with LKB1 mutated tumors might therefore possibly be extended to other NSCLC patients with wt LKB1.
22
Acknowledgements Prof. Zippelius University Hospital and University of Basel (Switzerland) kindly provided the KRASG12D/WT cell line (KP1.9). ARQ 751 was kindly provided by ArQule.
23
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Figures legend Figure 1: Lack of LKB1 expression causes increased sensitivity to the ERK inhibitor SCH772984 in KRAS wt NSCLC cells. a) expression of LKB1 in H1299 and isogenic H1299-LKB1 KO clones. Ran was used as loading control. b) activity of SCH772984 in H1299 and H1299-LKB1 KO clones. c) activity of SCH772984 in H1299 and H1299-LKB1 KO clone assessed by colony assay. d) representative images of colony assay: H1299 in the left and H1299-LKB1 KO in the right panel. Drug doses are expressed in nM. e) expression of LKB1 in H1299 and isogenic H1299-LKB1 KO clones treated with esiRNA targeting LKB1 (H1299-esiRNA) or scramble (H1299-Neg). Ran was used as loading control. f) activity of esiRNA targeting LKB1 or scramble in H1299. g) expression of LKB1 in H1299 and isogenic H1299-LKB1 KO clone transfected with pCDNA3/LKB1 or pCDNA3 empty vector. Ran was used as loading control. h) activity of SCH772984 in H1299 and H1299-LKB1 KO clone transfected with pCDNA3/LKB1 (L) or pCDNA3 empty vector (p). i) tumor growth inhibition in H1299LKB1 KO clone injected mice (N=8) treated with SCH772984 25 mg/kg or vehicle. *p<0.05 **p<0.01 SCH772984 vs. vehicle j) tumor growth inhibition activity on H1299 injected mice (N=8) treated with SCH772984 25 mg/kg or vehicle. k) activation of ERK in tumors removed from mice 6h after the last treatment. Actin was used as loading control. All in vitro data are depicted as mean ± SD (6 replicates for 3 independent experiments). Statistical analyses are reported in supplementary material.
Figure 2: Molecular analysis after SCH772984 treatment. 29
a) activation of MAPK pathway and mTOR downstream effectors in H1299 and H1299LKB1 KO cells, either untreated or treated with SCH772984 0.5 µM, at the indicated time points. b) expression of p90RSK and LKB1 in H1299, H1299-LKB1 KO and H1299-RSK KO cells. Actin was used as loading control. c) activity of SCH772984 in H1299, H1299-LKB1 KO and H1299-RSK KO clones. Data are depicted as mean ± SD (6 replicates for 3 independent experiments). Statistical analyses are reported in supplementary material. d) activation of ERK, S6 and expression of p90RSK and in H1299, H1299-LKB1 KO and H1299-RSK KO cells, either untreated or treated with SCH772984 0.5 µM, estimated at the indicated time points. e) activation of S6 in tumors removed from mice 24h after the last treatment. Actin was used as loading control.
Figure 3: KRAS mutated NSCLC murine models deficient in LKB1 are sensitive to the ERK inhibitor SCH772984. a) activity of SCH772984 in KRAS(wt) or KRAS(G12C) H1299 and their isogenic LKB1 KO clones. b) activity of SCH772984 in KP or KL cells. c) expression of LKB1 in KL95 cells transiently transfected with pCDNA3/LKB1 or pCDNA3 empty vector. Ran was used as loading control. d) activity of SCH772984 in KL95 cells transfected with pCDNA3 empty vector (upper) or pCDNA3/LKB1 (lower). e) tumor growth inhibition on KL cells injected mice (N=8) treated with SCH772984 25 mg/kg or vehicle. ****p<0.0001 SCH772984 vs. vehicle f) tumor growth inhibition on KP cells injected mice (N=8) treated with SCH772984 25 mg/kg or vehicle. g) activation of ERK and S6 in KP and KL tumors removed from mice
30
6h after the last treatment. Actin was used as loading control. h) mean number of counted masses in lungs of KL mice treated with SCH772984 (N=6) or vehicle (N=6).
Figure 4: KRAS mutated NSCLC human models deficient in LKB1 are sensitive to the ERK inhibitor SCH772984. a) expression of LKB1 in LU99 and in isogenic LU99-LKB1 KO clones. Ran was used as loading control. b) activity of SCH772984 alone or in combination with PIK-75 10 nM or ARQ 751 50 nM in LU99 and LU99-LKB1 KO clone. c) activation of ERK, PRAS40 and S6 in LU99 and LU99-LKB1 KO cells after 24h of treatment with SCH772984 0.5 µM and ARQ 751 50 nM. Ran was used as loading control. d) tumor growth inhibition in LU99-LKB1 KO clone injected mice (N=8) treated with SCH772984 25 mg/kg, ARQ 751 50 mg/kg, SCH772984/ARQ 751 or vehicle. *p<0.05; ***p<0.001 SCH772984/ARQ 751 vs. vehicle. e) tumor growth inhibition activity on LU99 injected mice (N=8) treated with SCH772984 25 mg/kg, ARQ 751 50 mg/kg, SCH772984/ARQ 751 or vehicle. f) activity of SCH772984 alone or in combination with ARQ 751 50 nM in A549 and H460 cells. g) activation of ERK, PRAS40 and S6 in A549 and H460 cells after 24h of treatment with SCH772984 0.5 µM and ARQ 751 50 nM. Ran was used as loading control. All in vitro data are depicted as mean ± SD (6 replicates for 3 independent experiments). Statistical analyses are reported in supplementary material.
31
H1299 H1299-LKB1 KO 1 H1299-LKB1 KO 2
120
c
90
60
d
1000 % cell viability
b
Cell viability (%)
a
H1299 H1299-LKB1 KO 1
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10
- LKB1 1 0.0
30
- RAN
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2
4
6
8
f
- RAN
0.6
0.8
g
120 Cell viability (%)
- LKB1
0.4
SCH772984 (µ µM)
SCH772984 (µ µM)
e
0.2
90
H1299-Neg H1299-esiRNA H1299-LKB1 KO
LKB1 pCDNA3
60
+
+ -
+
+ - LKB1
30 0
1
2
3
4
5
- actin
SCH772984 (µ µM)
j
i
120
k 2500
90 H1299-p H1299-L H1299-LKB1 KO-p H1299-LKB1 KO-L
60
1500 1000 500
* **
0
30 0
1
2
3
SCH772984 (µ µM)
Figure 1
4
H1299 Vehicle SCH
3
3
Vehicle SCH
2000
Tumor volume (mm )
2500 Tumor volume (mm )
Cell viability (%)
h
2000 1500 1000
- p-ERK
500
- ERK
0 0
7
14
21
0
7
14
21
SCH772984
SCH772984 Days after treatment start
H1299 LKB1 KO
- actin Days after treatment start
c
- p-ERK - ERK
H1299 H1299-LKB1 KO H1299-RSK KO9 H1299-RSK KO16 H1299-RSK KO31
60
0
2
6
8
10
24h
6h
H1299 RSK KO ctr
6h
ctr
- p-AKT
24h
H1299 LKB1 KO
H1299
6h
d
- AKT
4
SCH772984 (µ µM)
ctr
- p90RSK
90
30
- p-p90RSK thr359/ser363 - p-p90RSK ser380
120 Cell viability (%)
24h
6h
24h
6h
ctr
H1299
ctr
H1299 LKB1 KO
24h
a
- p-ERK - p-p70S6K - ERK
- p70S6K
- p90RSK
- p-S6
- p-S6
- S6
- S6 - actin
b e
H1299
H1299 LKB1 KO
- p90RSK - LKB1
- p-S6
- actin
- S6 - actin
Figure 2
b
90 WT WT - LKB1 KO G12C G12C - LKB1 KO
60 30
c
120 90 60
LKB1 pCDNA3
+
+ -
30
0
- LKB1 - ran
0
1 2 3 SCH772984 (µ µM)
4
0
2
4
6
f 800
400
********
****
0 10
SCH772984
800
h
- ERK - p-S6
400
- S6
15
0
5
10
SCH772984 Days after treatment start
Figure 3
KL95
SCH772984 10
0 5
KP1.9
- p-ERK
Vehicle SCH
3
Tumor volume (mm )
3
g 1200
Vehicle SCH
0
10
SCH772984 (µ µM)
e 1200
8
Days after treatment start
15
- actin
2
0
Tumor volume (mm )
d
KP 1.9 KL 7 KL 95
Nodules for 50 mm
Cell viability (%)
120
Cell viability (%)
a
****
8 6 4 2 0 Vehicle
SCH
e
3
- LKB1 - actin
Vehicle
1500
SCH ARQ
1000
SCH-ARQ
500
* ***
SCH772984 ARQ751
5
10
15
20
0
LU99 LU99 ARQ LU99 PIK LU99-LKB1 KO LU99-LKB1 KO ARQ LU99-LKB1 KO PIK
5
10
15
20
25
Days after treatment start
120 Cell viability (%)
Cell viability (%)
120
30
500
SCH772984 ARQ751
f
60
1000
25
Days after treatment start
150
90
Vehicle SCH ARQ SCH-ARQ
1500
0
0 0
b
2000 3
2000
Tumor volume (mm )
d Tumor volume (mm )
a
90 A549 A549 ARQ H460 H460 ARQ
60 30 0
0 0
2
4
0
6
2
c ARQ751 SCH772984
LU99 -
+
+ -
g
LU99-LKB1 KO + +
-
+
+ -
ARQ751 SCH772984
+ +
6
8
A549 -
+
+ -
H460 + +
-
+
+ -
+ +
- p-ERK
- p-ERK
- ERK
- ERK
- p-PRAS40
- p-PRAS40
- PRAS40
- PRAS40
- p-S6 - S6 - ran
Figure 4
4
SCH772984 (µ µM)
SCH772984 (µ µM)
- p-S6 - S6 - ran