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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 figues and tables: Figure S1. In vitro AZD1152-hQPA reduces proliferation and induces polyploidy across a panel of human AML cancer cell lines. Figure S2. AZD1152 has a modest effect on the growth of the HL-60 xenograft models in SCID mice Figure S3. AZD1152 in combination with Ara-C has a modest effect on the growth of the HL-60 xenograft models in SCID mice Figure S4. AZD2811 affect the neutrophil count in a dose-dependent manner Table S1: Cytogenetics of the different cell lines Table S2: Summary table of the pIC50 and nuclear area.

Supplementary Table 1. Mass Spectrometer and UPLC system parameters, Supplementary Table 2. Optimization parameters for mass spectrometry analysis, Supplementary Table 3. Estimated mouse PK model parameter values for AZD2811, Supplementary Table 4. Summary table of the pIC50 and nuclear area, Supplementary Table 5: PK parameters summary, Supplementary Figure 1. Representative bioluminescent images of the disseminated U2932Luc2 DLBCL xenograft model in vivo taken at day 18 post treatment initiation, Supplementary Figure 2. 96h post treatment, AZD2811 nanoparticle treatment reduces pHH3 levels with no evidence of polyploidy in the OCI-LY19 xenograft model in vivo, Supplementary Figure 3. In SCID mice AZD2811 nanoparticle is well tolerated at the different doses and schedules explored

Supplementary figure 1: Generation of H2073 ASV and H2073 SVD. Supplementary figure 2: H2073 parental and Ex20Ins are dependent on EGFR for survival in vitro. Supplementary figure 3: Effect of TKI on the Sytox Proliferation of H2073 wt, H2073-SVD and H2073-ASV, in vitro. Supplementary figure 4: The H2073 parental NSCLC xenograft model is sensitive to afatinib in vivo. Supplementary figure 5: Osimertinib and AZ5104 given as monotherapy induce robust inhibition of p-EGFR and relevant downstream signalling pathways. Supplementary figure 6: Osimertinib and AZ5104 as monotherapy induce a strong inhibition of the level of pEGFR and the downstream signalling pathways. Supplementary figure 7: Osimertinib, AZ5104, afatinib and erlotinib are well tolerated at the dose explored in the PDX models and AZ5104 induce significant tumor growth inhibition in the LU0387 model when dosed at 50 mg/kg. Supplementary figure 8: Osimertinib, afatinib and erlotinib robustly inhibit pEGFR; however, select proteins representing downstream signalling pathways (pAKT) have enhanced inhibition to osimertinib and pS6R remains suppressed at early timepoints compared to afatinib and erlotinib in the UC Davis PDX model harboring the V769_D770InsASV.

Osimertinib is an oral, third-generation, irreversible epidermal growth factor receptor tyrosine kinase inhibitor (EGFR-TKI) that selectively inhibits both EGFR-TKI–sensitizing and EGFR T790M–resistance mutations with lower activity against wild-type EGFR and has demonstrated efficacy in non–small cell lung cancer (NSCLC) CNS metastases. The sensitizing mutations, the in-frame deletions in exon 19 and the L858R point mutation in exon 21, represent between 80% and 90% of all EGFR mutations. The remaining 10% to 20% are referred to as uncommon activating mutations and are a diverse group of mutations in exons 18 to 21 within the kinase domain of the EGFR gene. Excluding those found as insertion mutations in exon 20, the uncommon mutations involving codons G719, S768, and L861 are the most prevalent.Although the efficacy of EGFR-TKIs for the common EGFR mutations is well established, much less is known about rare EGFR mutations, such as exon 20 insertions, G719X, L861Q, S768I, as most of the data consist of single case reports or small case series.Using available patient-derived xenografts (PDX) and cell lines derived from two of these PDXs that harbor the G719X mutation, we have evaluated in vitro and in vivo the preclinical activity of osimertinib. We report osimertinib inhibits signaling pathways and cellular growth in G719X-mutant cell lines in vitro and demonstrate sustained tumor growth inhibition of PDX harboring the G719X mutation alone or in combination with L861Q and S768I.Together, these data support clinical testing of osimertinib in patients with uncommon EGFR NSCLC.

EGFR exon 20 insertions (Ex20Ins) account for 4% to 10% of EGFR activating mutations in non–small cell lung cancer (NSCLC). EGFR Ex20Ins tumors are generally unresponsive to first- and second-generation EGFR inhibitors, and current standard of care for NSCLC patients with EGFR Ex20Ins is conventional cytotoxic chemotherapy. Therefore, the development of an EGFR TKI that can more effectively target NSCLC with EGFR Ex20Ins mutations represents a major advance for this patient subset. Osimertinib is a third-generation EGFR TKI approved for the treatment of advanced NSCLC harboring EGFR T790M; however, the activity of osimertinib in EGFR Ex20Ins NSCLC has yet to be fully assessed. Using CRISPR-Cas 9 engineered cell lines carrying the most prevalent Ex20Ins mutations, namely Ex20Ins D770_N771InsSVD (22%) or Ex20Ins V769_D770InsASV (17%), and a series of patient-derived xenografts, we have characterized osimertinib and AZ5104 (a circulating metabolite of osimertinib) activities against NSCLC harboring Ex20Ins. We report that osimertinib and AZ5104 inhibit signaling pathways and cellular growth in Ex20Ins mutant cell lines in vitro and demonstrate sustained tumor growth inhibition of EGFR-mutant tumor xenograft harboring the most prevalent Ex20Ins in vivo. The antitumor activity of osimertinib and AZ5104 in NSCLC harboring EGFR Ex20Ins is further described herein using a series of patient-derived xenograft models. Together these data support clinical testing of osimertinib in patients with EGFR Ex20Ins NSCLC. Mol Cancer Ther; 17(5); 885–96. ©2018 AACR.

Barasertib (AZD1152), a pro-drug of the highly potent and selective Aurora B kinase inhibitor AZD2811, showed promising clinical activity in relapsed/refractory diffuse large B-cell lymphoma (DLBCL) patients administered as a 4-day infusion. To improve potential therapeutic benefit of Aurora B kinase inhibition, a nanoparticle formulation of AZD2811 has been developed to address limitations of repeated intravenous infusion. One of the challenges with the use of nanoparticles for chronic treatment of tumors is optimizing dose and schedule required to enable repeat administration to sustain tumor growth inhibition. AZD2811 gives potent cell growth inhibition across a range of DLBCL cells lines in vitro. In vivo, repeat administration of the AZD2811 nanoparticle gave antitumor activity at half the dose intensity of AZD1152. Compared with AZD1152, a single dose of AZD2811 nanoparticle gave less reduction in pHH3, but increased apoptosis and reduction of cells in G1 and G2–M, albeit at later time points, suggesting that duration and depth of target inhibition influence the nature of the tumor cell response to drug. Further exploration of the influence of dose and schedule on efficacy revealed that AZD2811 nanoparticle can be used flexibly with repeat administration of 25 mg/kg administered up to 7 days apart being sufficient to maintain equivalent tumor control. Timing of repeat administration could be varied with 50 mg/kg every 2 weeks controlling tumor control as effectively as 25 mg/kg every week. AZD2811 nanoparticle can be administered with very different doses and schedules to inhibit DLBCL tumor growth, although maximal tumor growth inhibition was achieved with the highest dose intensities.

Supplementary Figure 1: Osimertinib monotherapy induce tumor growth inhibition in a NSCLC EGFR G719A;S768I mutation PDX model in vivo. Supplementary Figure 2: Osimertinib as monotherapy induce a stronger inhibition of the level of pEGFR and the downstream signalling pathways than afatinib. Supplementary Figure 3: MET amplification induces resistance to osimertinib treatment in a NSCLC EGFR G719A mutation PDX model in vivo.

Clathrin inhibition promotes apoptosis of mutant-EGFR expressing cell lines but not WT-EGFR expressing cell lines

Tyrosine kinase inhibitors (TKI) targeting mutant EGFR in non–small cell lung cancer (NSCLC) have been successful to control cancer growth, but acquired resistance inevitably occurs, including mutations directly on EGFR, for example, T790M and C797S. Strategies to prevent such acquired mutations by reducing mutant-EGFR expression have met limited success. Here, we propose a new model of mutant-EGFR trafficking and demonstrate that clathrin inhibition induces rapid degradation across a large panel of endogenous mutant-EGFR (Ex19del, L858R, and Ex20Ins). This panel included mutant-EGFR (T790M) resistant to the first- and second-generation EGFR inhibitors and to the third-generation TKI osimertinib and occurs through both mutational (C797S) and nonmutational EGFR mechanisms. Clathrin-mediated endocytosis inhibition of mutant EGFR induced a macropinocytosis-dependent lysosomal pathway associated with a loss of mutant-EGFR–dependent signaling (pAKT, pERK). Moreover, induction of this macropinocytic pathway led to robust apoptosis-dependent death across all mutant-EGFR cell lines tested, including those resistant to TKIs. We, therefore, propose a novel strategy to target mutant-EGFR refractory to approved existing TKI treatments in NSCLC and where new treatment strategies remain a key area of unmet need.Significance:These findings extend our mechanistic understanding of NSCLC mutant EGFR trafficking biology, the role that trafficking may play in resistance of mutant EGFR to tyrosine kinase inhibitors, and provide new therapeutic and biological insights to tackle this fundamental issue and improve benefit to patients. Cancer Res; 78(12); 3267–79. ©2018 AACR.

XLS file, 240K, Differentially expressed gene set data for the castrated mice.

PDF file - 404K, Supplementary Figure 1: Strategy for inducible recombination in the prostate. Supplementary Figure 2: Analyses of tumor regression following castration. Supplementary Figure 3: Gene Set Enrichment Analysis (GSEA). Supplementary Table 1: List of the primers used in this study. Supplementary Table 2: List of antibodies for used in this study. Supplementary Table 3: Summary of phenotypic analyses. Supplementary Table 7: Summary of preclinical data

DOC file, 347K, GSEA data for the castrated mice.

XLS file - 58K, List of differentially expressed genes between Nkx3.1CE2/+; Ptenf/f; BrafCA/+ and Nkx3.1CE2/+; Ptenf/f mouse prostate tumors (p-value

XLS file - 188K, List of differentially expressed genes between RAP + PD treated and Vehicle treated Nkx3.1CE2/+; Ptenf/f; Braf CA/+ mouse prostate tumors (p-value

PDF file - 31K, Myc pathway enrichment analysis for differentially expressed genes between RAP + PD treated and Vehicle treated Nkx3.1CE2/+; Ptenf/f; BrafCA/+ mouse prostate tumors

DOC file, 296K, GSEA data for the intact mice.

PDF file - 57K, Pathway enrichment analysis for differentially expressed genes between Nkx3.1CE2/+; Ptenf/f; BrafCA/+ and Nkx3.1CE2/+; Ptenf/f mouse prostate tumors

Both the PI3K → Akt → mTOR and mitogen-activated protein kinase (MAPK) signaling pathways are often deregulated in prostate tumors with poor prognosis. Here we describe a new genetically engineered mouse model of prostate cancer in which PI3K-Akt-mTOR signaling is activated by inducible disruption of PTEN, and extracellular signal-regulated kinase 1/2 (ERK1/2) MAPK signaling is activated by inducible expression of a BRAFV600E oncogene. These tissue-specific compound mutant mice develop lethal prostate tumors that are inherently resistant to castration. These tumors bypass cellular senescence and disseminate to lymph nodes, bone marrow, and lungs where they form overt metastases in approximately 30% of the cases. Activation of PI3K → Akt → mTOR and MAPK signaling pathways in these prostate tumors cooperate to upregulate c-Myc. Accordingly, therapeutic treatments with rapamycin and PD0325901 to target these pathways, respectively, attenuate c-Myc levels and reduce tumor and metastatic burden. Together, our findings suggest a generalized therapeutic approach to target c-Myc activation in prostate cancer by combinatorial targeting of the PI3K → Akt → mTOR and ERK1/2 MAPK signaling pathways. Cancer Res; 72(18); 4765–76. ©2012 AACR.

PDF file, 4581K, Supplementary Figure 1 and Table 1-3 Supp. Figure 1: Preclinical analyses of intact Nkx3.1CE2/+; Ptenf/f mice Supp. Table 1: List of primers used in this study Supp. Table 2: List of antibodies used in this study Supp. Table 3: Summary of Tumor weights and proliferation of prostate tumor from intact Nkx3.1CE2/+;Ptenf/f mice.

XLS file, 239K, Differentially expressed gene set data for the intact mice.

PDF file - 36K, Pathway enrichment analysis for differentially expressed genes between Nkx3.1CE2/+; Ptenf/f; BrafCA/+ and Nkx3.1CE2/+; Ptenf/f mouse prostate tumors

Ocean warming is altering the biogeographical distribution of marine organisms. In the tropics, rising sea surface temperatures are restructuring coral reef communities with sensitive species being lost. At the biogeographical divide between temperate and tropical communities, warming is causing macroalgal forest loss and the spread of tropical corals, fishes and other species, termed “tropicalization”. A lack of field research into the combined effects of warming and ocean acidification means there is a gap in our ability to understand and plan for changes in coastal ecosystems. Here, we focus on the tropicalization trajectory of temperate marine ecosystems becoming coral-dominated systems. We conducted field surveys and in situ transplants at natural analogues for present and future conditions under (i) ocean warming and (ii) both ocean warming and acidification at a transition zone between kelp and coral-dominated ecosystems. We show that increased herbivory by warm-water fishes exacerbates kelp forest loss and that ocean acidification negates any benefits of warming for range extending tropical corals growth and physiology at temperate latitudes. Our data show that, as the combined effects of ocean acidification and warming ratchet up, marine coastal ecosystems lose kelp forests but do not gain scleractinian corals. Ocean acidification plus warming leads to overall habitat loss and a shift to simple turf-dominated ecosystems, rather than the complex coral-dominated tropicalized systems often seen with warming alone. Simplification of marine habitats by increased CO2 levels cascades through the ecosystem and could have severe consequences for the provision of goods and services.

Osimertinib is an oral, third-generation, irreversible epidermal growth factor receptor tyrosine kinase inhibitor (EGFR-TKI) that selectively inhibits both EGFR-TKI–sensitizing and EGFR T790M–resistance mutations with lower activity against wild-type EGFR and has demonstrated efficacy in non–small cell lung cancer (NSCLC) CNS metastases. The sensitizing mutations, the in-frame deletions in exon 19 and the L858R point mutation in exon 21, represent between 80% and 90% of all EGFR mutations. The remaining 10% to 20% are referred to as uncommon activating mutations and are a diverse group of mutations in exons 18 to 21 within the kinase domain of the EGFR gene. Excluding those found as insertion mutations in exon 20, the uncommon mutations involving codons G719, S768, and L861 are the most prevalent. Although the efficacy of EGFR-TKIs for the common EGFR mutations is well established, much less is known about rare EGFR mutations, such as exon 20 insertions, G719X, L861Q, S768I, as most of the data consist of single case reports or small case series. Using available patient-derived xenografts (PDX) and cell lines derived from two of these PDXs that harbor the G719X mutation, we have evaluated in vitro and in vivo the preclinical activity of osimertinib. We report osimertinib inhibits signaling pathways and cellular growth in G719X-mutant cell lines in vitro and demonstrate sustained tumor growth inhibition of PDX harboring the G719X mutation alone or in combination with L861Q and S768I. Together, these data support clinical testing of osimertinib in patients with uncommon EGFR NSCLC.

The epidermal growth factor receptor (EGFR) harboring activating mutations is a clinically validated target in non-small-cell lung cancer, and a number of inhibitors of the EGFR tyrosine kinase domain, including osimertinib, have been approved for clinical use. Resistance to these therapies has emerged due to a variety of molecular events including the C797S mutation which renders third-generation C797-targeting covalent EGFR inhibitors considerably less potent against the target due to the loss of the key covalent-bond-forming residue. We describe the medicinal chemistry optimization of a biochemically potent but modestly cell-active, reversible EGFR inhibitor starting point with sub-optimal physicochemical properties. These studies culminated in the identification of compound 12 that showed improved cell potency, oral exposure, and in vivo activity in clinically relevant EGFR-mutant-driven disease models, including an Exon19 deletion/T790M/C797S triple-mutant mouse xenograft model.

A subset of drug-resistant EGFR -mutant MET -amplified lung cancer switches oncogenic dependence to MET and is treatable with MET inhibitor monotherapy.

There has been huge progress in the discovery of targeted cancer therapies in recent years. However, even for the most successful and impactful cancer drugs which have been approved, both innate and acquired mechanisms of resistance are commonplace. These emerging mechanisms of resistance have been studied intensively, which has enabled drug discovery scientists to learn how it may be possible to overcome such resistance in subsequent generations of treatments. In some cases, novel drug candidates have been able to supersede previously approved agents; in other cases they have been used sequentially or in combinations with existing treatments. This review summarizes the current field in terms of the challenges and opportunities that cancer resistance presents to drug discovery scientists, with a focus on small molecule therapeutics. As part of this review, common themes and approaches have been identified which have been utilized to successfully target emerging mechanisms of resistance. This includes the increase in target potency and selectivity, alternative chemical scaffolds, change of mechanism of action (covalents, PROTACs), increases in blood-brain barrier permeability (BBBP), and the targeting of allosteric pockets. Finally, wider approaches are covered such as monoclonal antibodies (mAbs), bispecific antibodies, antibody drug conjugates (ADCs), and combination therapies.

Tyrosine kinase inhibitors (TKI) targeting mutant EGFR in non–small cell lung cancer (NSCLC) have been successful to control cancer growth, but acquired resistance inevitably occurs, including mutations directly on EGFR, for example, T790M and C797S. Strategies to prevent such acquired mutations by reducing mutant-EGFR expression have met limited success. Here, we propose a new model of mutant-EGFR trafficking and demonstrate that clathrin inhibition induces rapid degradation across a large panel of endogenous mutant-EGFR (Ex19del, L858R, and Ex20Ins). This panel included mutant-EGFR (T790M) resistant to the first- and second-generation EGFR inhibitors and to the third-generation TKI osimertinib and occurs through both mutational (C797S) and nonmutational EGFR mechanisms. Clathrin-mediated endocytosis inhibition of mutant EGFR induced a macropinocytosis-dependent lysosomal pathway associated with a loss of mutant-EGFR–dependent signaling (pAKT, pERK). Moreover, induction of this macropinocytic pathway led to robust apoptosis-dependent death across all mutant-EGFR cell lines tested, including those resistant to TKIs. We, therefore, propose a novel strategy to target mutant-EGFR refractory to approved existing TKI treatments in NSCLC and where new treatment strategies remain a key area of unmet need. Significance:These findings extend our mechanistic understanding of NSCLC mutant EGFR trafficking biology, the role that trafficking may play in resistance of mutant EGFR to tyrosine kinase inhibitors, and provide new therapeutic and biological insights to tackle this fundamental issue and improve benefit to patients. Cancer Res; 78(12); 3267–79. ©2018 AACR.

Barasertib (AZD1152), a highly potent and selective aurora kinase B inhibitor, gave promising clinical activity in elderly acute myeloid leukemia (AML) patients. However, clinical utility was limited by the requirement for a 7-day infusion. Here we assessed the potential of a nanoparticle formulation of the selective Aurora kinase B inhibitor AZD2811 (formerly known as AZD1152-hQPA) in preclinical models of AML. When administered to HL-60 tumor xenografts at a single dose between 25 and 98.7 mg/kg, AZD2811 nanoparticle treatment delivered profound inhibition of tumor growth, exceeding the activity of AZD1152. The improved antitumor activity was associated with increased phospho-histone H3 inhibition, polyploidy, and tumor cell apoptosis. Moreover, AZD2811 nanoparticles increased antitumor activity when combined with cytosine arabinoside. By modifying dose of AZD2811 nanoparticle, therapeutic benefit in a range of preclinical models was further optimized. At high-dose, antitumor activity was seen in a range of models including the MOLM-13 disseminated model. At these higher doses, a transient reduction in bone marrow cellularity was observed demonstrating the potential for the formulation to target residual disease in the bone marrow, a key consideration when treating AML. Collectively, these data establish that AZD2811 nanoparticles have activity in preclinical models of AML. Targeting Aurora B kinase with AZD2811 nanoparticles is a novel approach to deliver a cell-cycle inhibitor in AML, and have potential to improve on the clinical activity seen with cell-cycle agents in this disease. Mol Cancer Ther; 16(6); 1031–40. ©2017 AACR.

Barasertib (AZD1152), a pro-drug of the highly potent and selective Aurora B kinase inhibitor AZD2811, showed promising clinical activity in relapsed/refractory diffuse large B-cell lymphoma (DLBCL) patients administered as a 4-day infusion. To improve potential therapeutic benefit of Aurora B kinase inhibition, a nanoparticle formulation of AZD2811 has been developed to address limitations of repeated intravenous infusion. One of the challenges with the use of nanoparticles for chronic treatment of tumors is optimizing dose and schedule required to enable repeat administration to sustain tumor growth inhibition. AZD2811 gives potent cell growth inhibition across a range of DLBCL cells lines in vitro. In vivo, repeat administration of the AZD2811 nanoparticle gave antitumor activity at half the dose intensity of AZD1152. Compared with AZD1152, a single dose of AZD2811 nanoparticle gave less reduction in pHH3, but increased apoptosis and reduction of cells in G1 and G2–M, albeit at later time points, suggesting that duration and depth of target inhibition influence the nature of the tumor cell response to drug. Further exploration of the influence of dose and schedule on efficacy revealed that AZD2811 nanoparticle can be used flexibly with repeat administration of 25 mg/kg administered up to 7 days apart being sufficient to maintain equivalent tumor control. Timing of repeat administration could be varied with 50 mg/kg every 2 weeks controlling tumor control as effectively as 25 mg/kg every week. AZD2811 nanoparticle can be administered with very different doses and schedules to inhibit DLBCL tumor growth, although maximal tumor growth inhibition was achieved with the highest dose intensities.

EGFR exon 20 insertions (Ex20Ins) account for 4% to 10% of EGFR activating mutations in non–small cell lung cancer (NSCLC). EGFR Ex20Ins tumors are generally unresponsive to first- and second-generation EGFR inhibitors, and current standard of care for NSCLC patients with EGFR Ex20Ins is conventional cytotoxic chemotherapy. Therefore, the development of an EGFR TKI that can more effectively target NSCLC with EGFR Ex20Ins mutations represents a major advance for this patient subset. Osimertinib is a third-generation EGFR TKI approved for the treatment of advanced NSCLC harboring EGFR T790M; however, the activity of osimertinib in EGFR Ex20Ins NSCLC has yet to be fully assessed. Using CRISPR-Cas 9 engineered cell lines carrying the most prevalent Ex20Ins mutations, namely Ex20Ins D770_N771InsSVD (22%) or Ex20Ins V769_D770InsASV (17%), and a series of patient-derived xenografts, we have characterized osimertinib and AZ5104 (a circulating metabolite of osimertinib) activities against NSCLC harboring Ex20Ins. We report that osimertinib and AZ5104 inhibit signaling pathways and cellular growth in Ex20Ins mutant cell lines in vitro and demonstrate sustained tumor growth inhibition of EGFR-mutant tumor xenograft harboring the most prevalent Ex20Ins in vivo. The antitumor activity of osimertinib and AZ5104 in NSCLC harboring EGFR Ex20Ins is further described herein using a series of patient-derived xenograft models. Together these data support clinical testing of osimertinib in patients with EGFR Ex20Ins NSCLC. Mol Cancer Ther; 17(5); 885–96. ©2018 AACR.

Exon 20 insertions (Ex20Ins) have been identified in approximately 5% of epidermal growth factor receptor (EGFR)-mutated lung tumours in patients presenting with non-small cell lung cancer (NSCLC). Several small molecule tyrosine kinase inhibitors (TKIs) have been reported to have pre-clinical activity against such insertions including afatinib, poziotinib, osimertinib, nazartinib, AP32788/TAK-788 and TAS6417. However, there remains a lack of approved treatments for patients with Ex20Ins with early approved EGFR agents appearing to be ineffective in this setting. Poziotinib, osimertinib and AP32788/TAK-788 are undergoing clinical evaluation in patients whose tumours carry Ex20Ins and in some cases clinical responses have been reported giving hope that such insertions can be targeted by small molecules. There is however a need for comparable data across such compounds that would enable understanding of the relative activity of these compounds between Ex20Ins and the wild-type form of EGFR. As many of the Exon 20 insertions are not part of the ATP binding pocket achieving selectivity over wild type EGFR is highly challenging and may limit the clinical utility of agents due to dose limiting EGFR wild-type driven toxicity. A selection of TKIs were profiled for Ex20Ins and wild-type EGFR activity using biochemical, in vitro cellular phosphorylation and proliferation assays. This has enabled us to differentiate the Ex20Ins versus wild-type EGFR selectivity profiles of a range of pre-clinical, clinical and proprietary compounds. As part of this evaluation we utilized a CRISPR CAS9 approach in H2073 EGFR wild-type NSCLC cell line, where we have established cellular disease models against the most prevalent insertions including D770-N771insSVD (22%). Finally, we will show anti-tumour efficacy data for a selection of these inhibitors along with a potential combination approach of osimertinib and cetuximab. EGFR D770-N771InsSVD cell phospho IC50 (µM)EGFR WT (H2073) cell phospho IC50 (µM)Fold-EGFR WT margin (cell)afatinib0.0060.00330.5osimertinib0.0940.414.4poziotinib0.00340.00351TAS64170.0230.0672.9AZ62810.0972.223 Citation Format: Richard A. Ward, Ambra Bianco, Nicola Colclough, Darren Cross, Emanuela M. Cuomo, M. Raymond V. Finlay, Martina Fitzek, Nicolas Floc’h, Sladjana Gagrica, Beverley Hammond, Matthew J. Martin, Darren McKerrecher, Daniel J. O’Neill, Jonathan P. Orme, Paul D. Smith, Anna D. Staniszewska, Jelena Urosevic, Nicky Whalley, James W. Yates. Comparative activity profiling of tyrosine kinase inhibitors (TKIs) against exon 20 insertions and the wild-type form of epidermal growth factor receptor (EGFR) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4813.

Two first-generation (erlotinib & gefitinib), two second generation (afatinib & dacomitinib) and a third generation (osimertinib) EGFR-TKIs are currently available for the management of EGFR mutation-positive NSCLC. All these TKIs are effective in patients with NSCLC whose tumors harbor the in-frame deletions in exon 19 and the L858R point mutation in exon 21. These two mutations represent 90% of all EGFR mutations. Osimertinib, when used in the front-line setting, has shown more favorable treatment outcomes than first-generation EGFR-TKIs. In approximately 50% of patients, resistance to first and second generation TKI is mediated by the acquisition of the ‘gatekeeper’ mutation T790M. Currently, osimertinib is the only registered EGFR TKI that is active against exon 19 deletions and L858R mutation, regardless of the presence of T790M mutation. While the efficacy of EGFR TKIs for the common EGFR mutations is well established, much less is known about rare EGFR mutations such as exon 20 insertions, G719X, L861Q, S768I, as most of the data consist of single case reports or small case series. This work describes the therapeutic potential of osimertinib in tumors harboring the G719X mutation alone or in combination with L861Q and S768I. Using available patient-derived xenografts (PDX) and cell lines derived from two of these PDX which habor the G719X mutation, we have evaluated in vitro and in vivo the pre-clinical activity of osimertinib. We show that osimertinib inhibits signalling pathways and cellular growth of G719X cell lines in vitro. This translates into sustained tumor growth inhibition in vivo in 3 out of 4 PDX explored (87%, p The work presented herein demonstrates that osimertinib has the potential to improve upon the current treatment options for NSCLC patients whose tumors harbor a G719X mutation, and warrants further clinical investigation. Citation Format: Nicolas Floch, Sue Bickerton, Matthew J. Martin, Darren A. Cross, Paul D. Smith. Osimertinib, an irreversible next generation EGFR tyrosine kinase inhibitor, exerts anti-tumor activity in various preclinical NSCLC models harboring G719X mutant-EGFR [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1330.

Current treatments for castration-resistant prostate cancer (CRPC) that target androgen receptor (AR) signaling improve patient survival, yet ultimately fail. Here, we provide novel insights into treatment response for the antiandrogen abiraterone by analyses of a genetically engineered mouse (GEM) model with combined inactivation of Trp53 and Pten, which are frequently comutated in human CRPC. These NPp53 mice fail to respond to abiraterone and display accelerated progression to tumors resembling treatment-related CRPC with neuroendocrine differentiation (CRPC-NE) in humans. Cross-species computational analyses identify master regulators of adverse response that are conserved with human CRPC-NE, including the neural differentiation factor SOX11, which promotes neuroendocrine differentiation in cells derived from NPp53 tumors. Furthermore, abiraterone-treated NPp53 prostate tumors contain regions of focal and/or overt neuroendocrine differentiation, distinguished by their proliferative potential. Notably, lineage tracing in vivo provides definitive and quantitative evidence that focal and overt neuroendocrine regions arise by transdifferentiation of luminal adenocarcinoma cells. These findings underscore principal roles for TP53 and PTEN inactivation in abiraterone resistance and progression from adenocarcinoma to CRPC-NE by transdifferentiation. Significance: Understanding adverse treatment response and identifying patients likely to fail treatment represent fundamental clinical challenges. By integrating analyses of GEM models and human clinical data, we provide direct genetic evidence for transdifferentiation as a mechanism of drug resistance as well as for stratifying patients for treatment with antiandrogens. Cancer Discov; 7(7); 736–49. ©2017 AACR. See related commentary by Sinha and Nelson, p. 673. This article is highlighted in the In This Issue feature, p. 653

Up to 20% of non small-cell lung cancers are driven by activating mutations in EGFR (EGFRm). While EGFR tyrosine kinase inhibitors (TKIs) have proven highly effective in the clinic (e.g. gefitinib in first line EGFRm and osimertinib in T790M post-TKI resistance), acquired resistance mechanisms present an obstacle to curing EGFR-driven disease. To-date, approximately 20% of osimertinib-resistant cases in the 2nd line setting are EGFRm/T790M/C797S, and there is currently no approved therapy for these “triple-mutant” (TM) patients. We have obsrerved that the ALK inhibitor brigatinib shows moderate, equipotent activity against both EGFR-TM and the single activating mutant. We used this information to screen a panel of compounds to identify brigatinib-based combinations that could effectively target EGFR-TM-expressing cells. Among the most potent combination partners were selumetinib and other inhibitors of the MAP kinase pathway. These agents were able to synergistically impair growth of TM cells when combined with brigatinib in vitro, and accordingly show prolonged suppression of downstream pathway biomarkers in combination- vs. brigatinib monotherapy-treated cells. Further, we have shown effective combinations can dramatically enhance apoptotic cell death compared to brigatinib treatment alone. Critically, neither monotherapy treatment with effective combination partners, nor combinations based on osimertinib showed growth inhibition in the EGFR triple-mutant setting. Taken together, these data suggest novel therapeutic strategies for patients in this critical area of unmet need that warrant further preclinical testing. Citation Format: Matthew J. Martin, Nicolas Floc'h, Chrysiis Michaloglou, M Raymond Finlay, Richard A. Ward, Paul D. Smith, Darren A. Cross. A screen to target EGFR (C797S) in osimertinib-resistant lung cancer identifies brigatinib-based combinations that synergistically inhibit cell growth [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 916.

EGFR exon 20 insertion (Ex20Ins) mutations represent a combination of in-frame insertions and/or duplications that account for 4-10% of all EGFR mutants in non-small cell lung cancer (NSCLC). To date, more than one hundred different Ex20Ins mutations have been identified. With the notable exception of the rare A763_Y764insFQEA insertion ( Using CRISPR-Cas 9 genome editing technology, we engineered cellular disease relevant models carrying the most prevalent Ex20Ins mutations, namely Ex20Ins D770_N771InsSVD (22%) or Ex20Ins V769_D770InsASV (17%). Using these models, we show that osimertinib and AZ5104 inhibit signalling pathways and cellular growth of Ex20Ins CRISPR-Cas9 engineered cell lines in vitro. This translates into sustained tumor growth inhibition in vivo in both the Ex20Ins SVD (65%, p EGFR signal. Two patients from the AURA Phase 2 osimertinib trials with plasma positive Ex20Ins (concurrent with Ex19del and T790M) had durable PFS responses of 6.4 and 13.9 months, supporting the premise that osimertinib has the potential to be clinically beneficial in tumors harboring Ex20Ins. The work presented herein demonstrates that osimertinib has the potential to improve upon the current treatment options for NSCLC patients whose tumors harbour an Ex20Ins mutation, and warrants its further clinical investigation. Citation Format: Nicolas Floc'h, Susan Ashton, Ambra Bianco, Nicola Colclough, Darren AE Cross, Maria Emanuela Cuomo, M. Raymond V. Finlay, Matthew J Martin, Ludovic Menard, Darren McKerrecher, Daniel J O'Neill, Jonathan P Orme, Anna D Staniszewska, Richard A Ward, James W T Yates. Osimertinib, an irreversible mutant selective EGFR tyrosine kinase inhibitor, exerts anti tumor activity in NSCLC harbouring exon 20 insertion mutant-EGFR [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2079. doi:10.1158/1538-7445.AM2017-2079

9030 Background: EGFR exon 20 insertions (Ex20Ins), the 3rd most common EGFR activating mutation, are generally unresponsive to 1st and 2nd generation EGFR-TKIs. Development of EGFR-TKIs that effectively target NSCLC with Ex20Ins mutations represents a major unmet need. Osimertinib is an EGFR TKI approved for the treatment of advanced NSCLC harboring EGFR T790M, but the potential of osimertinib remains to be fully assessed in patients (pts) with Ex20Ins NSCLC. Methods: CRISPR engineered Ex20Ins cell line xenografts representing the two most common Ex20Ins (D770_N771InsSVD and V769_D770InsASV) and pt derived xenograft (PDX) of 3 EGFR Ex20Ins (V769_D770InsASV, M766_A767insASV, H773_V774insNPH) were used for in vivo experiments. Xenografts were treated by oral gavage with vehicle, erlotinib (50 mg/kg/day) or afatinib (20 mg/kg/day), osimertinib metabolite AZ5104 (50 mg/kg/day) and osimertinib (25 mg/kg/day) and assessed for tumor growth inhibition (TGI). Immunoblotting was performed for EGFR and relevant signaling pathways. A pt from whom the V769_D770InsASV Ex20ins PDX was derived was treated on a UC Davis IRB approved protocol with osimertinib at 160 mg PO once-daily (QD). Results: At completion of treatment, QD administration of osimertinib or AZ5104 induced significant TGI in xenografts across the 4 EGFR Ex20ins tested (range 60-95% TGI, p < 0.001 compared to control for all models) that was superior to either afatinib or erlotinib. Robust decrease in p-EGFR, p-ERK, p-Akt, p-Stat3 was observed with osimertinib treatment. The patient corresponding to the V769_D770InsASV Ex20ins PDX treated with osimertinib exhibited clinical improvement and tumor shrinkage; unfortunately he was found to have interstitial pneumonitis that necessitated drug discontinuation. Conclusions: Osimertinib at clinically representative doses has in vivo activity across multiple EGFR Ex20ins that comprising the most common Ex20ins detected in patients (~50% prevalence); metabolite AZ5104 may contribute to efficacy. Tumor shrinkage was observed in a patient with lung cancer harboring an Ex20ins treated for a limited time with osimertinib. Based on this in vivo xenograft and pt data, osimertinib warrants further study in pts with EGFR Ex20ins NSCLC.

Exposure to hepatitis C virus (HCV) typically results in chronic infection that leads to progressive liver disease ranging from mild inflammation to severe fibrosis and cirrhosis as well as primary liver cancer. HCV triggers innate immune signaling within the infected hepatocyte, a first step in mounting of the adaptive response against HCV infection. Persistent inflammation is strongly associated with liver tumorigenesis. The goal of our work was to investigate the initiation of the inflammatory processes triggered by HCV viral proteins in their host cell and their possible link with HCV-related liver cancer. We report a dramatic upregulation of the lymphotoxin signaling pathway and more specifically of lymphotoxin-β in tumors of the FL-N/35 HCV-transgenic mice. Lymphotoxin expression is accompanied by activation of NF-κB, neosynthesis of chemokines and intra-tumoral recruitment of mononuclear cells. Spectacularly, IKKβ inactivation in FL-N/35 mice drastically reduces tumor incidence. Activation of lymphotoxin-β pathway can be reproduced in several cellular models, including the full length replicon and HCV-infected primary human hepatocytes. We have identified NS5B, the HCV RNA dependent RNA polymerase, as the viral protein responsible for this phenotype and shown that pharmacological inhibition of its activity alleviates activation of the pro-inflammatory pathway. These results open new perspectives in understanding the inflammatory mechanisms linked to HCV infection and tumorigenesis., Author Summary Hepatitis C affects nearly 200 million people worldwide. It results from the failure of the immune system to control the hepatitis C virus (HCV) replication and spread, leading to progressive liver disease that can culminate in fibrosis, cirrhosis and cancer. The inflammatory cells that infiltrate the diseased liver functionally contribute to fibrotic disease and cancer development by the release of potent soluble mediators that regulate cell survival and proliferation, angiogenesis, tissue remodelling, metabolism and genomic integrity. The goal of our work was to study the mechanisms of the initiation of the inflammatory process linked to HCV infection. We have shown that the presence of a single viral protein, namely NS5B, the RNA dependent RNA polymerase, promotes pro-inflammatory signaling. Moreover, inhibition of this pathway in HCV transgenic mice fully protects the animals from HCV-linked liver cancer. Our study contributes to a better understanding of the inflammatory mechanisms linked to HCV infection and thereby to tumorigenesis.

Both the PI3K → Akt → mTOR and mitogen-activated protein kinase (MAPK) signaling pathways are often deregulated in prostate tumors with poor prognosis. Here we describe a new genetically engineered mouse model of prostate cancer in which PI3K-Akt-mTOR signaling is activated by inducible disruption of PTEN, and extracellular signal-regulated kinase 1/2 (ERK1/2) MAPK signaling is activated by inducible expression of a BRAFV600E oncogene. These tissue-specific compound mutant mice develop lethal prostate tumors that are inherently resistant to castration. These tumors bypass cellular senescence and disseminate to lymph nodes, bone marrow, and lungs where they form overt metastases in approximately 30% of the cases. Activation of PI3K → Akt → mTOR and MAPK signaling pathways in these prostate tumors cooperate to upregulate c-Myc. Accordingly, therapeutic treatments with rapamycin and PD0325901 to target these pathways, respectively, attenuate c-Myc levels and reduce tumor and metastatic burden. Together, our findings suggest a generalized therapeutic approach to target c-Myc activation in prostate cancer by combinatorial targeting of the PI3K → Akt → mTOR and ERK1/2 MAPK signaling pathways. Cancer Res; 72(18); 4765–76. ©2012 AACR.

Although the prognosis for clinically localized prostate cancer is now favorable, there are still no curative treatments for castration-resistant prostate cancer (CRPC) and, therefore, it remains fatal. In this study, we investigate a new therapeutic approach for treatment of CRPC, which involves dual targeting of a major signaling pathway that is frequently deregulated in the disease. We found that dual targeting of the Akt and mTOR signaling pathways with their respective inhibitors, MK-2206 and ridaforolimus (MK-8669), is highly effective for inhibiting CRPC in preclinical studies in vivo using a refined genetically engineered mouse model of the disease. The efficacy of the combination treatment contrasts with their limited efficacy as single agents, since delivery of MK-2206 or MK-8669 individually had a modest impact in vivo on the overall tumor phenotype. In human prostate cancer cell lines, although not in the mouse model, the synergistic actions of MK-2206 and ridaforolimus (MK-8669) are due in part to limiting the mTORC2 feedback activation of Akt. Moreover, the effects of these drugs are mediated by inhibition of cellular proliferation via the retinoblastoma (Rb) pathway. Our findings suggest that dual targeting of the Akt and mTOR signaling pathways using MK-2206 and ridaforolimus (MK-8669) may be effective for treatment of CRPC, particularly for patients with deregulated Rb pathway activity. Cancer Res; 72(17); 4483–93. ©2012 AACR.