Supplementary Figures - PDF file 770K, Supplementary Figure 1. JQ1 treatment induces apoptosis proliferation arrest in a subset of NSCLC cells Supplementary Figure 2. JQ1 treatment promotes a depletion of 57kDa MYC in a time dependent manner in NSCLC cells Supplementary Figure 3. FOSL1 depletion is induced by JQ1 treatment regardless of the status of LKB1 Supplementary Figure 4. JQ1 treatment in mutant kras mouse induce apoptosis

Supplementary Methods - PDF file 201K, Supplementary Methods

PDF file - 806K, Supplemental Figure 1. MRI scans at the indicated time points showing tumor burden in EML4-ALK lung cancer mice treated by crizotinib. Multiple scans from a total of 4 mice are shown. Supplemental Figure 2. Representative MRI images showing that EML4-ALK F1174L mutant lung cancers respond to 17-DMAG and TAE684. Note the development of acquired resistance to 17-DMAG and TAE684 after prolonged treatment. Supplemental Figure 3. Acquired resistance to 17-DMAG and TAE684 in mice bearing tumors driven by the EML4-ALK F1174L mutant can be overcome by the combination of the two drugs.

PDF file - 11671KB, Supplemental Figure 1. Response of Kras mouse models to MEKi/PI3Ki. Supplemental Figure 2. MEKi/PI3Ki induces G1 arrest and inhibits proliferation of KRAS mutant NSCLC cell lines. Supplemental Figure 3. Combined MEK and PI3K inhibition is necessary for maximal reduction in cell proliferation. Supplemental Figure 4. Apoptotic response of KRAS mutant NSCLC cell lines in response to MEKi/PI3Ki. Supplemental Figure 5. Mutational status of TP53 or STK11/LKB1 does not correlate with apoptotic response to MEKi/PI3Ki. Supplemental Figure 6. Secreted Gaussia luciferase allows for precise quantitation of tumor growth and treatment response. Supplemental Figure 7. Tumor response of KRAS NSCLC xenografts to MEKi/PI3Ki. Supplemental Figure 8. Apoptosis induced by MEKi/PI3Ki is BAX and caspase-3 dependent. Supplemental Figure 9. Modulation of MEK/ERK and PI3Ki/AKT transcriptional output does not correlate with apoptotic sensitivity of KRAS mutant NSCLC cancer cells. Supplemental Figure 10. Modulation of RalGDS signaling does not correlate with apoptotic sensitivity of KRAS mutant NSCLC cell lines. Supplemental Figure 11. Sensitivity to MEKi/PI3Ki does not correlate with BH3 priming. Supplemental Figure 12. siRNA mediated knockdown of PUMA and BIM protects from apoptosis induced by MEKi/PI3Ki. Supplemental Figure 13. Inhibition of BCL-2 alone does not restore apoptotic response in insensitive KRAS mutant NSCLC cell lines. Supplemental Figure 14. Individual protein expression levels of BCL-2 family members do not correlate with sensitivity to MEKi/PI3Ki. Supplemental Figure 15. Inducible ectopic expression of BIM. Supplemental Figure 16. Restoration of apoptosis by ABT-263 leads to MEKi/PI3Ki-induced regression in vivo. Supplemental Figure 17. In vitro derived MEKi/PI3Ki resistant cells. Supplemental Figure 18. Cell lines derived from resistant Kras p53L/L tumors. Supplemental Figure 19. PUMA and BIM mRNA levels do not differ between cell lines derived from treatment naive and resistant Kras p53L/L tumors. Supplemental Table 1. Mutational status of KRAS mutant NSCLC cell lines. Supplemental Table 2. Knockdown efficiency of pLKO shRNA hairpins used in lentiviral screen.

PDF file - 1.4MB, Supplemental Figure 1. MET is inducible in MET, TD/MET, and TL/MET models Supplemental Figure 2. Quantification of hMET expression in mouse models Supplemental Figure 3. Comparison of the tumor counts in mutant EGFR lung tumors with or without expression of hMET. Supplemental Figure 4. Representative MRI images prior to and after treatment in TL and TL/MET mice. Supplemental Figure 5. Xenografts of HCC827GR6 cells ectopically expressing EGFR E746_A750del/T790M are sensitive to the combination of WZ4002 and 17-DMAG. Supplemental Figure 6. The WZ4002/crizotinib or WZ4002/17-DMAG combinations suppress phosphorylation of EGFR, Akt, and Erk in TD and TD/MET to a greater extent than individual treatments

PDF file - 329K, Procedures for xenografts and a list of antibodies used for IHC and Western blots

Although several groups have demonstrated that concomitant use of MEK and phosphoinositide 3-kinase (PI3K) inhibitors (MEKi/PI3Ki) can induce dramatic tumor regressions in mouse models of KRAS-mutant non– small cell lung cancer (NSCLC), ongoing clinical trials investigating this strategy have been underwhelming to date. While efficacy may be hampered by a narrow therapeutic index, the contribution of biologic heterogeneity in the response of KRAS-mutant NSCLCs to MEKi/PI3Ki has been largely unexplored. In this study, we find that most human KRAS-mutant NSCLC cell lines fail to undergo marked apoptosis in response to MEKi/PI3Ki, which is key for tumor responsiveness in vivo. This heterogeneity of apoptotic response occurs despite relatively uniform induction of growth arrest. Using a targeted short hairpin RNA screen of BCL-2 family members, we identify BIM, PUMA, and BCL-XL as key regulators of the apoptotic response induced by MEKi/PI3Ki, with decreased expression of BIM and PUMA relative to BCL-XL in cell lines with intrinsic resistance. In addition, by modeling adaptive resistance to MEKi/PI3Ki both in vitro and in vivo, we find that, upon the development of resistance, tumors have a diminished apoptotic response due to downregulation of BIM and PUMA. These results suggest that the inability to induce apoptosis may limit the effectiveness of MEKi/PI3Ki for KRAS-mutant NSCLCs by contributing to intrinsic and adaptive resistance to this therapy. Cancer Res; 74(11); 3146–56. 2014 AACR.

Abstract Although several groups have demonstrated that concomitant use of MEK and phosphoinositide 3-kinase (PI3K) inhibitors (MEKi/PI3Ki) can induce dramatic tumor regressions in mouse models of KRAS-mutant non–small cell lung cancer (NSCLC), ongoing clinical trials investigating this strategy have been underwhelming to date. While efficacy may be hampered by a narrow therapeutic index, the contribution of biologic heterogeneity in the response of KRAS-mutant NSCLCs to MEKi/PI3Ki has been largely unexplored. In this study, we find that most human KRAS-mutant NSCLC cell lines fail to undergo marked apoptosis in response to MEKi/PI3Ki, which is key for tumor responsiveness in vivo. This heterogeneity of apoptotic response occurs despite relatively uniform induction of growth arrest. Using a targeted short hairpin RNA screen of BCL-2 family members, we identify BIM, PUMA, and BCL-XL as key regulators of the apoptotic response induced by MEKi/PI3Ki, with decreased expression of BIM and PUMA relative to BCL-XL in cell lines with intrinsic resistance. In addition, by modeling adaptive resistance to MEKi/PI3Ki both in vitro and in vivo, we find that, upon the development of resistance, tumors have a diminished apoptotic response due to downregulation of BIM and PUMA. These results suggest that the inability to induce apoptosis may limit the effectiveness of MEKi/PI3Ki for KRAS-mutant NSCLCs by contributing to intrinsic and adaptive resistance to this therapy. Cancer Res; 74(11); 3146–56. ©2014 AACR.

Abstract Purpose: To extend the results of a phase III trial in patients with non–small cell lung cancer with adenocarcinomas harboring EML4-ALK fusion. Experimental Design: We conducted a co-clinical trial in a mouse model comparing the ALK inhibitor crizotinib to the standard-of-care cytotoxic agents docetaxel or pemetrexed. Results: Concordant with the clinical outcome in humans, crizotinib produced a substantially higher response rate compared with chemotherapy, associated with significantly longer progression-free survival. Overall survival was also prolonged in crizotinib- compared with chemotherapy-treated mice. Pemetrexed produced superior overall survival compared with docetaxel, suggesting that this agent may be the preferred chemotherapy in the ALK population. In addition, in the EML4-ALK–driven mouse lung adenocarcinoma model, HSP90 inhibition can overcome both primary and acquired crizotinib resistance. Furthermore, HSP90 inhibition, as well as the second-generation ALK inhibitor TAE684, demonstrated activity in newly developed lung adenocarcinoma models driven by crizotinib-insensitive EML4-ALK L1196M or F1174L. Conclusions: Our findings suggest that crizotinib is superior to standard chemotherapy in ALK inhibitor–naïve disease and support further clinical investigation of HSP90 inhibitors and second-generation ALK inhibitors in tumors with primary or acquired crizotinib resistance. Clin Cancer Res; 20(5); 1204–11. ©2013 AACR.

Abstract KRAS activating mutations are detected in 30% Non-Small Cell Lung Cancer (NSCLC) cases, and represent the subset of patients with the worst prognosis. Current efforts to develop treatments for this type of lung cancer are partially focused on inhibiting the Raf/MEK/ERK signaling cascade downstream of KRAS. Inhibition of the Raf/MEK/ERK pathway results in death of many mutant KRAS-driven lung cancer cell lines in vitro, and we have previously reported a murine active Kras lung cancer model that showed significant tumor regression upon treatment with the MEK inhibitor, selumetinib, when it was combined with other agents, such as PI3K/mTOR dual inhibitor BEZ235 or the cytotoxic agent docetaxel. The activity of the latter combination was validated in a recent phase II clinical trial in NSCLC patients harboring KRAS mutations. However, the responses were not durable in either human patients or mice, as resistance develop rapidly following chronic MEK inhibition. We have developed two approaches to reduce or delay the emergence of resistant clones. First, we have identified a number of novel treatment strategies that would directly improve the initial efficacy of MEK/ERK inhibition to dramatically enhance the initial response to MEK inhibition. Second, we have designed chemical screening strategies that identify compounds that act against active Kras lung cancer independently from the Raf/MEK/ERK pathway. We are using the first approach to delay the development of chronic resistance, while using the second approach to overcome resistance to MEK/ERK inhibition. Citation Format: Zhao Chen, Ellen Weisberg, Katherine Cheng, Andrew L. Kung, James Bradner, James Griffin, Kwok-Kin Wong. Improve MEK/ERK targeting in mutant KRAS lung cancer. [abstract]. In: Proceedings of the AACR Special Conference on RAS Oncogenes: From Biology to Therapy; Feb 24-27, 2014; Lake Buena Vista, FL. Philadelphia (PA): AACR; Mol Cancer Res 2014;12(12 Suppl):Abstract nr B01. doi: 10.1158/1557-3125.RASONC14-B01

Abstract Purpose: Amplification of MYC is one of the most common genetic alterations in lung cancer, contributing to a myriad of phenotypes associated with growth, invasion, and drug resistance. Murine genetics has established both the centrality of somatic alterations of Kras in lung cancer, as well as the dependency of mutant Kras tumors on MYC function. Unfortunately, drug-like small-molecule inhibitors of KRAS and MYC have yet to be realized. The recent discovery, in hematologic malignancies, that bromodomain and extra-terminal (BET) bromodomain inhibition impairs MYC expression and MYC transcriptional function established the rationale of targeting KRAS-driven non–small cell lung cancer (NSCLC) with BET inhibition. Experimental Design: We performed functional assays to evaluate the effects of JQ1 in genetically defined NSCLC cell lines harboring KRAS and/or LKB1 mutations. Furthermore, we evaluated JQ1 in transgenic mouse lung cancer models expressing mutant kras or concurrent mutant kras and lkb1. Effects of bromodomain inhibition on transcriptional pathways were explored and validated by expression analysis. Results: Although JQ1 is broadly active in NSCLC cells, activity of JQ1 in mutant KRAS NSCLC is abrogated by concurrent alteration or genetic knockdown of LKB1. In sensitive NSCLC models, JQ1 treatment results in the coordinate downregulation of the MYC-dependent transcriptional program. We found that JQ1 treatment produces significant tumor regression in mutant kras mice. As predicted, tumors from mutant kras and lkb1 mice did not respond to JQ1. Conclusion: Bromodomain inhibition comprises a promising therapeutic strategy for KRAS-mutant NSCLC with wild-type LKB1, via inhibition of MYC function. Clinical studies of BET bromodomain inhibitors in aggressive NSCLC will be actively pursued. Clin Cancer Res; 19(22); 6183–92. ©2013 AACR.

KRAS is the most commonly mutated oncogene, yet no effective targeted therapies exist for KRAS mutant cancers. We developed a pooled shRNA-drug screen strategy to identify genes that, when inhibited, cooperate with MEK inhibitors to effectively treat KRAS mutant cancer cells. The anti-apoptotic BH3 family gene BCL-XL emerged as a top hit through this approach. ABT-263 (navitoclax), a chemical inhibitor that blocks the ability of BCL-XL to bind and inhibit pro-apoptotic proteins, in combination with a MEK inhibitor led to dramatic apoptosis in many KRAS mutant cell lines from different tissue types. This combination caused marked in vivo tumor regressions in KRAS mutant xenografts and in a genetically engineered KRAS-driven lung cancer mouse model, supporting combined BCL-XL/MEK inhibition as a potential therapeutic approach for KRAS mutant cancers.

Abstract Amplification of MYC is one of the most common genetic alterations in lung cancer, contributing to a myriad of phenotypes associated with growth, invasion and drug resistance. Murine genetics has established both the centrality of somatic alterations of Kras in lung cancer, as well as dependency of Kras-dependent tumors on c-Myc function. Unfortunately, drug-like small-molecule inhibitors of KRAS and c-Myc have yet to be realized. The recent discovery in hematologic malignancies that bromodomain inhibition impairs MYC expression and MYC-dependent transcriptional function prompted the possibility of targeting KRAS-driven NSCLC with a potent, prototypical BET bromodomain inhibitor, JQ1. Here, we report that NSCLC cells harboring the KRAS mutation are sensitive to JQ1 while NSCLC cells with concurrent mutant KRAS and LKB1 mutations are resistant to JQ1. In sensitive NSCLC models, JQ1 treatment results in the coordinate downregulation of the MYC-dependent transcriptional program. Furthermore, we evaluated JQ1 in transgenic mouse lung cancer models expressing mutant kras or concurrent mutant kras and lkb1. We found that JQ1 treatment produces significant tumor regression in mutant kras mice. As predicted, tumors from mutant kras and lkb1 mice did not respond to JQ1. Together, these data provide a compelling rationale for the study of BET bromodomain inhibitors in a common, genetically-defined population of patients with aggressive NSCLC. Citation Format: Takeshi Shimamura, Zhao Chen, Margaret Soucheray, Julian Carretero, Eiki Kikuchi, Jeremy H. Tchaicha, Yandhi Gao, Katherine A. Cheng, Travis J. Cohoon, Jun Qi, Esra A. Akbay, Alec C. Kimmelman, Andrew L. Kung, James E. Bradner, Kwok Kin Wong. Efficacy of BET bromodomain inhibition in Kras-positive non-small cell lung cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1126. doi:10.1158/1538-7445.AM2013-1126

Abstract Although KRAS is the most commonly mutated oncogene in human cancer, no effective therapies exist for KRAS mutant cancers. Attempts to target KRAS directly or to target single effector pathways downstream of KRAS have met limited success. Previously, our laboratory and others showed that simultaneous targeting of more than one KRAS effector pathway, specifically the MEK-ERK and PI3K-AKT pathways, can cause dramatic responses in KRAS-driven genetically-engineered mouse tumor models. These findings support the promise of targeted therapy combinations for KRAS mutant cancers and have led to clinical trials evaluating combined PI3K/MEK inhibition. We evaluated the efficacy of combined PI3K/MEK inhibition in a panel of 30 KRAS mutant cell lines. Roughly half of all cell lines showed limited sensitivity to this combination, suggesting that combined PI3K/MEK inhibition may only be effective in a subset of KRAS mutant cancers and underscoring the need for additional combination therapy strategies. To develop new targeted therapy combinations for KRAS mutant cancers, we designed a pooled shRNA-drug screen strategy aimed at rapidly identifying genes that, when inhibited, synergize with MEK inhibitors to decrease the viability of KRAS mutant cancer cells. Using this approach, we identified the anti-apoptotic protein BCLXL as a potential target for combination therapy with MEK inhibitors. When tested in vitro against a panel of 30 KRAS mutant cell lines, simultaneous pharmacologic inhibition of BCLXL (using the BH3 mimetic ABT-263) and MEK led to pronounced apoptosis and reduced viability in most cell lines, including many cell lines that were insensitive to combined PI3K/MEK inhibition. In vivo, the combination of ABT-263 and a MEK inhibitor led to marked tumor regressions in KRAS mutant xenograft models and to dramatic and sustained tumor regressions (>70% reduction in tumor size) in a KRAS-driven genetically-engineered mouse model of lung cancer. These findings suggest that combined inhibition of BCLXL and MEK is a promising targeted therapy combination for potential evaluation in clinical trials for patients with KRAS mutant cancers. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 875. doi:1538-7445.AM2012-875

Abstract Tyrosine kinase inhibitors (TKI) that target the EGF receptor (EGFR) are effective in most non–small cell lung carcinoma (NSCLC) patients whose tumors harbor activating EGFR kinase domain mutations. Unfortunately, acquired resistance eventually emerges in these chronically treated cancers. Two of the most common mechanisms of acquired resistance to TKIs seen clinically are the acquisition of a secondary “gatekeeper” T790M EGFR mutation that increases the affinity of mutant EGFR for ATP and activation of MET to offset the loss of EGFR signaling. Although up to one-third of patient tumors resistant to reversible EGFR TKIs harbor concurrent T790M mutation and MET amplification, potential therapies for these tumors have not been modeled in vivo. In this study, we developed a preclinical platform to evaluate potential therapies by generating transgenic mouse lung cancer models expressing EGFR-mutant Del19-T790M or L858R-T790M, each with concurrent MET overexpression. We found that monotherapy targeting EGFR or MET alone did not produce significant tumor regression. In contrast, combination therapies targeting EGFR and MET simultaneously were highly efficacious against EGFR TKI–resistant tumors codriven by Del19-T790M or L858R-T790M and MET. Our findings therefore provide an in vivo model of intrinsic resistance to reversible TKIs and offer preclinical proof-of-principle that combination targeting of EGFR and MET may benefit patients with NSCLC. Cancer Res; 72(13); 3302–11. ©2012 AACR.

In parallel with an ongoing human clinical trial, genetically engineered mouse models of lung cancer with different genetic alterations are treated with chemotherapeutic agents; the results have implications for the clinical trial. The idea of 'co-clinical' trials has been put forward as way of evaluating novel therapies. By testing a drug simultaneously in human clinical and mouse preclinical trials, the thinking is, the two sets of data can be combined to extract extra information. To demonstrate the potential of this approach, genetically engineered mouse models were used to mirror a randomized phase II clinical trial of the chemotherapeutic docetaxel in KRAS-driven lung cancer, comparing its action alone with that in combination with a MEK inhibitor. In the mouse model, tumours with Kras or Kras and p53 mutations were more responsive to the combination than to docetaxel alone, whereas mice carrying a deletion of Lkb1 in addition to activated Kras remained relatively unresponsive. This has important implications for the ongoing clinical trial, suggesting that patients should be tested for LKB1 mutations. Targeted therapies have demonstrated efficacy against specific subsets of molecularly defined cancers1,2,3,4. Although most patients with lung cancer are stratified according to a single oncogenic driver, cancers harbouring identical activating genetic mutations show large variations in their responses to the same targeted therapy1,3. The biology underlying this heterogeneity is not well understood, and the impact of co-existing genetic mutations, especially the loss of tumour suppressors5,6,7,8,9, has not been fully explored. Here we use genetically engineered mouse models to conduct a ‘co-clinical’ trial that mirrors an ongoing human clinical trial in patients with KRAS-mutant lung cancers. This trial aims to determine if the MEK inhibitor selumetinib (AZD6244)10 increases the efficacy of docetaxel, a standard of care chemotherapy. Our studies demonstrate that concomitant loss of either p53 (also known as Tp53) or Lkb1 (also known as Stk11), two clinically relevant tumour suppressors6,9,11,12, markedly impaired the response of Kras-mutant cancers to docetaxel monotherapy. We observed that the addition of selumetinib provided substantial benefit for mice with lung cancer caused by Kras and Kras and p53 mutations, but mice with Kras and Lkb1 mutations had primary resistance to this combination therapy. Pharmacodynamic studies, including positron-emission tomography (PET) and computed tomography (CT), identified biological markers in mice and patients that provide a rationale for the differential efficacy of these therapies in the different genotypes. These co-clinical results identify predictive genetic biomarkers that should be validated by interrogating samples from patients enrolled on the concurrent clinical trial. These studies also highlight the rationale for synchronous co-clinical trials, not only to anticipate the results of ongoing human clinical trials, but also to generate clinically relevant hypotheses that can inform the analysis and design of human studies.

Cells that are deficient in homologous recombination, such as those that lack functional breast cancer-associated 1 (BRCA1) or BRCA2, are hypersensitive to inhibition of poly(ADP-ribose) polymerase (PARP). However, BRCA-deficient tumors represent only a small fraction of adult cancers, which might restrict the therapeutic utility of PARP inhibitor monotherapy. Cyclindependent kinase 1 (Cdk1) phosphorylates BRCA1, and this is essential for efficient formation of BRCA1 foci. Here we show that depletion or inhibition of Cdk1 compromises the ability of cells to repair DNA by homologous recombination. Combined inhibition of Cdk1 and PARP in BRCA-wild-type cancer cells resulted in reduced colony formation, delayed growth of human tumor xenografts and tumor regression with prolonged survival in a mouse model of lung adenocarcinoma. Inhibition of Cdk1 did not sensitize nontransformed cells or tissues to inhibition of PARP. Because reduced Cdk1 activity impaired BRCA1 function and consequently, repair by homologous recombination, inhibition of Cdk1 represents a plausible strategy for expanding the utility of PARP inhibitors to BRCA-proficient cancers.