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Osimertinib is an EGFR tyrosine kinase inhibitor (TKI) with proven clinical efficacy; however, acquired resistance presents an obstacle to curing EGFR-driven disease. Recent studies have shown that drug-tolerant persister cells (DTP) have a distinct transcriptional profile that may confer specific vulnerabilities. By definition these cells avoid apoptosis, yet little is known about how their survival is regulated. We found that paradoxically, the proapoptotic gene BIM was upregulated in osimertinib DTPs, and cotreatment with BH3 mimetics could trigger DTP cell death. Furthermore, cIAP proteins, antiapoptotic members of the extrinsic pathway, were significantly elevated in DTPs. cIAP antagonists could block DTP formation as an up-front combination, and could eliminate preformed DTPs. Critically, when treated at the time of maximal osimertinib response, cIAP or MCL1 inhibitor treatment could significantly attenuate the regrowth of EGFRm cell line mouse xenografts. Finally, we show that apoptosis can be maximized in cell lines with acquired osimertinib resistance by combining BH3 or SMAC mimetics with agents that target the resistance driver in these models. Taken together, these data suggest novel therapeutic strategies at the point of minimal residual disease or full osimertinib resistance for patients in this critical area of unmet need. Significance: These studies uncover strategies to use targeted agents that activate apoptosis in non–small cell lung cancer cells that survive initial EGFR TKI treatment.

Osimertinib is an EGFR tyrosine kinase inhibitor (TKI) with proven clinical efficacy; however, acquired resistance presents an obstacle to curing EGFR-driven disease. Recent studies have shown that drug-tolerant persister cells (DTP) have a distinct transcriptional profile that may confer specific vulnerabilities. By definition these cells avoid apoptosis, yet little is known about how their survival is regulated. We found that paradoxically, the proapoptotic gene BIM was upregulated in osimertinib DTPs, and cotreatment with BH3 mimetics could trigger DTP cell death. Furthermore, cIAP proteins, antiapoptotic members of the extrinsic pathway, were significantly elevated in DTPs. cIAP antagonists could block DTP formation as an up-front combination, and could eliminate preformed DTPs. Critically, when treated at the time of maximal osimertinib response, cIAP or MCL1 inhibitor treatment could significantly attenuate the regrowth of EGFRm cell line mouse xenografts. Finally, we show that apoptosis can be maximized in cell lines with acquired osimertinib resistance by combining BH3 or SMAC mimetics with agents that target the resistance driver in these models. Taken together, these data suggest novel therapeutic strategies at the point of minimal residual disease or full osimertinib resistance for patients in this critical area of unmet need.Significance:These studies uncover strategies to use targeted agents that activate apoptosis in non–small cell lung cancer cells that survive initial EGFR TKI treatment.

Supplementary Table 1. Cell lines used. Supplementary Table 2. Antibodies used. Supplementary Figure S1. Osimertinib drug tolerant cells re-aquire apoptotic capacity after drug holiday. Supplementary Figure S2. BH3 mimetics can trigger apoptosis in established DTPs. Supplementary Figure S3. DTPs upregulate cIAP proteins and are sensitive to SMAC mimetic treatment. Supplementary Figure S4. SMAC mimetics inhibit DTP growth in vivo. Supplementary Figure S5. BH3 and SMAC mimetics enhance apoptosis induced by agents targeting resistance drivers. Supplementary Figure S6. Measuring transcript levels for anti-apoptotic genes in osimertinib DTPs.

Supplementary Figure S1. Vistusertib (AZD2014) causes a decrease in RB phosphorylation in the absence of cell cycle arrest. Supplementary Figure S2. Different mTOR inhibitors in combination with palbociclib cause a decrease in cell viability. Supplementary Figure S3. The effects of vistusertib and palbociclib in the CTC-174 model in vivo. Supplementary Figure S4. Analysis of cell cycle and cell morphology. Supplementary Figure S5. The effects of vistusertib (AZD2014) and palbociclib on P21 expression levels. Supplementary Figure S6. Analysis of apoptosis in vitro and in vivo. Supplementary Figure S7. Robust inhibition of mTOR signalling by short-term treatment with RAD001 or vistusertib (AZD2014) does not affect ER binding. Supplementary Table S1. Selected genes and primer IDs. Supplementary Table S2. Supplementary Table S3. Palbociclib resistant cell lines are sensitive to vistusertib (AZD2014).

Tumor cell line panel composition and growth inhibition sensitivity (GI50) to AZD8835.

Details on PK / PD / efficacy modeling in BT474 xenograft model

Primers used for targeted gene profiling on selected RAS/MAPK pathway genes either on Fluidigm or by qRT-PCR on Roche Lightcycler 480

Supplementary Figure 3

Supp Fig 1 Tumour efficacy Supp Fig 2 In vitro gene expression modulation Supp Fig 3 In vivo biomarker analysis Supp Fig 4 HCC70 In vivo FDG uptake imaging Supp Fig 5 U87MG in vivo FDG uptake imaging

Supplementary Figure 2

The cyclin dependent kinase (CDK)–retinoblastoma (RB)–E2F pathway plays a critical role in the control of cell cycle in estrogen receptor–positive (ER+) breast cancer. Small-molecule inhibitors of CDK4/6 have shown promise in this tumor type in combination with hormonal therapies, reflecting the particular dependence of this subtype of cancer on cyclin D1 and E2F transcription factors. mTOR inhibitors have also shown potential in clinical trials in this disease setting. Recent data have suggested cooperation between the PI3K/mTOR pathway and CDK4/6 inhibition in preventing early adaptation and eliciting growth arrest, but the mechanisms of the interplay between these pathways have not been fully elucidated. Here we show that profound and durable inhibition of ER+ breast cancer growth is likely to require multiple hits on E2F-mediated transcription. We demonstrate that inhibition of mTORC1/2 does not affect ER function directly, but does cause a decrease in cyclin D1 protein, RB phosphorylation, and E2F-mediated transcription. Combination of an mTORC1/2 inhibitor with a CDK4/6 inhibitor results in more profound effects on E2F-dependent transcription, which translates into more durable growth arrest and a delay in the onset of resistance. Combined inhibition of mTORC1/2, CDK4/6, and ER delivers even more profound and durable regressions in breast cancer cell lines and xenografts. Furthermore, we show that CDK4/6 inhibitor–resistant cell lines reactivate the CDK–RB–E2F pathway, but remain sensitive to mTORC1/2 inhibition, suggesting that mTORC1/2 inhibitors may represent an option for patients that have relapsed on CDK4/6 therapy. Mol Cancer Ther; 17(5); 908–20. ©2018 AACR.

55 FGFR2 modified dynamic biomarker after treatment by AZD4547

Supplementary figures S1: volcano plot of insensitive cell lines treated by AZD4547 S2:Immunohistochemistry of p-S6 and p-ERK tissue sections from xenograft treated with AZD4547 S3:FGFR pathway modulation in xenograft models treated with AZD4547 S4: ANC3A xenograft tumour growth inhibition after treated with AZD4547 and/or AZD5363 S5: AZD4547 dynamic biomarkers in ANC3A model after 14 days treatment with AKT and FGFR inhibitors

KRAS mutation status of cell lines included in in vitro combination screen

Pharmacokinetics modelling of AZD2014 intermittent dosing schedule.

Cell line panel used to identify AZD4547 dynamic transcript biomarkers.

The RAS-regulated RAF–MEK1/2–ERK1/2 (RAS/MAPK) signaling pathway is a major driver in oncogenesis and is frequently dysregulated in human cancers, primarily by mutations in BRAF or RAS genes. The clinical benefit of inhibitors of this pathway as single agents has only been realized in BRAF-mutant melanoma, with limited effect of single-agent pathway inhibitors in KRAS-mutant tumors. Combined inhibition of multiple nodes within this pathway, such as MEK1/2 and ERK1/2, may be necessary to effectively suppress pathway signaling in KRAS-mutant tumors and achieve meaningful clinical benefit. Here, we report the discovery and characterization of AZD0364, a novel, reversible, ATP-competitive ERK1/2 inhibitor with high potency and kinase selectivity. In vitro, AZD0364 treatment resulted in inhibition of proximal and distal biomarkers and reduced proliferation in sensitive BRAF-mutant and KRAS-mutant cell lines. In multiple in vivo xenograft models, AZD0364 showed dose- and time-dependent modulation of ERK1/2-dependent signaling biomarkers resulting in tumor regression in sensitive BRAF- and KRAS-mutant xenografts. We demonstrate that AZD0364 in combination with the MEK1/2 inhibitor, selumetinib (AZD6244 and ARRY142886), enhances efficacy in KRAS-mutant preclinical models that are moderately sensitive or resistant to MEK1/2 inhibition. This combination results in deeper and more durable suppression of the RAS/MAPK signaling pathway that is not achievable with single-agent treatment. The AZD0364 and selumetinib combination also results in significant tumor regressions in multiple KRAS-mutant xenograft models. The combination of ERK1/2 and MEK1/2 inhibition thereby represents a viable clinical approach to target KRAS-mutant tumors.

Supplementary Figure 6

The challenge of developing effective pharmacodynamic biomarkers for preclinical and clinical testing of FGFR signaling inhibition is significant. Assays that rely on the measurement of phospho-protein epitopes can be limited by the availability of effective antibody detection reagents. Transcript profiling enables accurate quantification of many biomarkers and provides a broader representation of pathway modulation. To identify dynamic transcript biomarkers of FGFR signaling inhibition by AZD4547, a potent inhibitor of FGF receptors 1, 2, and 3, a gene expression profiling study was performed in FGFR2-amplified, drug-sensitive tumor cell lines. Consistent with known signaling pathways activated by FGFR, we identified transcript biomarkers downstream of the RAS-MAPK and PI3K/AKT pathways. Using different tumor cell lines in vitro and xenografts in vivo, we confirmed that some of these transcript biomarkers (DUSP6, ETV5, YPEL2) were modulated downstream of oncogenic FGFR1, 2, 3, whereas others showed selective modulation only by FGFR2 signaling (EGR1). These transcripts showed consistent time-dependent modulation, corresponding to the plasma exposure of AZD4547 and inhibition of phosphorylation of the downstream signaling molecules FRS2 or ERK. Combination of FGFR and AKT inhibition in an FGFR2-mutated endometrial cancer xenograft model enhanced modulation of transcript biomarkers from the PI3K/AKT pathway and tumor growth inhibition. These biomarkers were detected on the clinically validated nanoString platform. Taken together, these data identified novel dynamic transcript biomarkers of FGFR inhibition that were validated in a number of in vivo models, and which are more robustly modulated by FGFR inhibition than some conventional downstream signaling protein biomarkers. Mol Cancer Ther; 15(11); 2802–13. ©2016 AACR.

HCC1428-eveR and HCC1428-LTED-eveR cell lines resistant to everolimus remain sensitive to dual mTORC1/2 inhibitor AZD2014.

Supplementary materials and methods

Gene symbol and primer ID used for targeted profiling on Fluidigm arrays

Differential gene expression and statistical analysis of cell lines treated with AZD4547 or DMSO over time

Loss of the tumor suppressor PTEN confers a tumor cell dependency on the PI3Kβ isoform. Achieving maximal inhibition of tumor growth through PI3K pathway inhibition requires sustained inhibition of PI3K signaling; however, efficacy is often limited by suboptimal inhibition or reactivation of the pathway. To select combinations that deliver comprehensive suppression of PI3K signaling in PTEN-null tumors, the PI3Kβ inhibitor AZD8186 was combined with inhibitors of kinases implicated in pathway reactivation in an extended cell proliferation assay. Inhibiting PI3Kβ and mTOR gave the most effective antiproliferative effects across a panel of PTEN-null tumor cell lines. The combination of AZD8186 and the mTOR inhibitor vistusertib was also effective in vivo controlling growth of PTEN-null tumor models of TNBC, prostate, and renal cancers. In vitro, the combination resulted in increased suppression of pNDRG1, p4EBP1, as well as HMGCS1 with reduced pNDRG1 and p4EBP1 more closely associated with effective suppression of proliferation. In vivo biomarker analysis revealed that the monotherapy and combination treatment consistently reduced similar biomarkers, while combination increased nuclear translocation of the transcription factor FOXO3 and reduction in glucose uptake. These data suggest that combining the PI3Kβ inhibitor AZD8186 and vistusertib has potential to be an effective combination treatment for PTEN-null tumors. Mol Cancer Ther; 17(11); 2309–19. ©2018 AACR.

Supplementary Table 1: Cell lines; Supplementary Table 2: Antibodies; Supplementary Table 3: Gene lists

Supplementary Figure 1

Supplementary data on cell lines

Pharmacokinetics and PK/PD relationships for AZD2014

The PIK3CA gene, encoding the p110α catalytic unit of PI3Kα, is one of the most frequently mutated oncogenes in human cancer. Hence, PI3Kα is a target subject to intensive efforts in identifying inhibitors and evaluating their therapeutic potential. Here, we report studies with a novel PI3K inhibitor, AZD8835, currently in phase I clinical evaluation. AZD8835 is a potent inhibitor of PI3Kα and PI3Kδ with selectivity versus PI3Kβ, PI3Kγ, and other kinases that preferentially inhibited growth in cells with mutant PIK3CA status, such as in estrogen receptor–positive (ER+) breast cancer cell lines BT474, MCF7, and T47D (sub-μmol/L GI50s). Consistent with this, AZD8835 demonstrated antitumor efficacy in corresponding breast cancer xenograft models when dosed continuously. In addition, an alternative approach of intermittent high-dose scheduling (IHDS) was explored given our observations that higher exposures achieved greater pathway inhibition and induced apoptosis. Indeed, using IHDS, monotherapy AZD8835 was able to induce tumor xenograft regression. Furthermore, AZD8835 IHDS in combination with other targeted therapeutic agents further enhanced antitumor activity (up to 92% regression). Combination partners were prioritized on the basis of our mechanistic insights demonstrating signaling pathway cross-talk, with a focus on targeting interdependent ER and/or CDK4/6 pathways or alternatively a node (mTOR) in the PI3K-pathway, approaches with demonstrated clinical benefit in ER+ breast cancer patients. In summary, AZD8835 IHDS delivers strong antitumor efficacy in a range of combination settings and provides a promising alternative to continuous dosing to optimize the therapeutic index in patients. Such schedules merit clinical evaluation. Mol Cancer Ther; 15(5); 877–89. ©2016 AACR.

supplementary figures and tables legend

Supplementary Figure 5

Supplementary table 1: Biochemical and cellular activity of AZD2014. Supplementary table 2: Biochemical activity of AZD2014. Supplementary table 3: Anti proliferative activity of AZD2014 in a panel of ER+, endocrine resistant and LTED cell lines. Supplementary table 4: Activity of AZD2014 in a number of ER+ breast cancer in vivo models.

Cell lines defined as sensitive to AZD0364 from 747 cell panel screen

Differential gene expression and two statistical analysis of ANC3A xenograft after treatment by AZD4547 and/or AZD5363 compared to vehicle group, and Combination group compared to singled agent.

Response of 747 cell lines to treatment with AZD0364 for 72 hours.

Statistical analysis of gene modulation between FGFR1, 2 and 3 dysregulated cell lines.

Supplementary Figure Legends

Supplemtary figures 1-9

Supplementary Table results

Supplementary Table Methods