Search

Purpose: KIT is the major oncogenic driver of gastrointestinal stromal tumors (GIST). Imatinib, sunitinib, and regorafenib are approved therapies; however, efficacy is often limited by the acquisition of polyclonal secondary resistance mutations in KIT, with those located in the activation (A) loop (exons 17/18) being particularly problematic. Here, we explore the KIT-inhibitory activity of ponatinib in preclinical models and describe initial characterization of its activity in patients with GIST.Experimental Design: The cellular and in vivo activities of ponatinib, imatinib, sunitinib, and regorafenib against mutant KIT were evaluated using an accelerated mutagenesis assay and a panel of engineered and GIST-derived cell lines. The ponatinib–KIT costructure was also determined. The clinical activity of ponatinib was examined in three patients with GIST previously treated with all three FDA-approved agents.Results: In engineered and GIST-derived cell lines, ponatinib potently inhibited KIT exon 11 primary mutants and a range of secondary mutants, including those within the A-loop. Ponatinib also induced regression in engineered and GIST-derived tumor models containing these secondary mutations. In a mutagenesis screen, 40 nmol/L ponatinib was sufficient to suppress outgrowth of all secondary mutants except V654A, which was suppressed at 80 nmol/L. This inhibitory profile could be rationalized on the basis of structural analyses. Ponatinib (30 mg daily) displayed encouraging clinical activity in two of three patients with GIST.Conclusion:Ponatinib possesses potent activity against most major clinically relevant KIT mutants and has demonstrated preliminary evidence of activity in patients with refractory GIST. These data strongly support further evaluation of ponatinib in patients with GIST. Clin Cancer Res; 20(22); 5745–55. ©2014 AACR.

Summary of plasma protein binding (% unbound) of AZD3229

Legends for supplementary figures and tables

Supplementary methods, patient information and figure legends

Fraction Absorbed (Fabs) as a Function of AZD3229 Dose using GI-SIM tool

Purpose: Non–small cell lung cancers (NSCLCs) harboring ALK gene rearrangements (ALK+) typically become resistant to the first-generation anaplastic lymphoma kinase (ALK) tyrosine kinase inhibitor (TKI) crizotinib through development of secondary resistance mutations in ALK or disease progression in the brain. Mutations that confer resistance to second-generation ALK TKIs ceritinib and alectinib have also been identified. Here, we report the structure and first comprehensive preclinical evaluation of the next-generation ALK TKI brigatinib.Experimental Design: A kinase screen was performed to evaluate the selectivity profile of brigatinib. The cellular and in vivo activities of ALK TKIs were compared using engineered and cancer-derived cell lines. The brigatinib–ALK co-structure was determined.Results: Brigatinib potently inhibits ALK and ROS1, with a high degree of selectivity over more than 250 kinases. Across a panel of ALK+ cell lines, brigatinib inhibited native ALK (IC50, 10 nmol/L) with 12-fold greater potency than crizotinib. Superior efficacy of brigatinib was also observed in mice with ALK+ tumors implanted subcutaneously or intracranially. Brigatinib maintained substantial activity against all 17 secondary ALK mutants tested in cellular assays and exhibited a superior inhibitory profile compared with crizotinib, ceritinib, and alectinib at clinically achievable concentrations. Brigatinib was the only TKI to maintain substantial activity against the most recalcitrant ALK resistance mutation, G1202R. The unique, potent, and pan-ALK mutant activity of brigatinib could be rationalized by structural analyses.Conclusions: Brigatinib is a highly potent and selective ALK inhibitor. These findings provide the molecular basis for the promising activity being observed in ALK+, crizotinib-resistant patients with NSCLC being treated with brigatinib in clinical trials. Clin Cancer Res; 22(22); 5527–38. ©2016 AACR.

Purpose:The emergence of secondary mutations is a cause of resistance to current KIT inhibitors used in the treatment of patients with gastrointestinal stromal tumors (GIST). AZD3229 is a selective inhibitor of wild-type KIT and a wide spectrum of primary and secondary mutations seen in patients with GIST. The objective of this analysis is to establish the pharmacokinetic–pharmacodynamic (PKPD) relationship of AZD3229 in a range of mouse GIST tumor models harboring primary and secondary KIT mutations, and to benchmark AZD3229 against other KIT inhibitors.Experimental Design:A PKPD model was developed for AZD3229 linking plasma concentrations to inhibition of phosphorylated KIT using data generated from several in vivo preclinical tumor models, and in vitro data generated in a panel of Ba/F3 cell lines.Results:AZD3229 drives inhibition of phosphorylated KIT in an exposure-dependent manner, and optimal efficacy is observed when >90% inhibition of KIT phosphorylation is sustained over the dosing interval. Integrating the predicted human pharmacokinetics into the mouse PKPD model predicts that an oral twice daily human dose greater than 34 mg is required to ensure adequate coverage across the mutations investigated. Benchmarking shows that compared with standard-of-care KIT inhibitors, AZD3229 has the potential to deliver the required target coverage across a wider spectrum of primary or secondary mutations.Conclusions:We demonstrate that AZD3229 warrants clinical investigation as a new treatment for patients with GIST based on its ability to inhibit both ATP-binding and A-loop mutations of KIT at clinically relevant exposures.

Supplementary Tables. Table S1. In Vitro Kinase IC50 Values (nM) for Native and Mutant Recombinant KIT. Table S2. Summary of Ba/F3 KIT Cell Lines Generated in this Study. Table S3. Summary of IC50 Viability Values in Ba/F3 KIT Cells. Table S4. Summary of KIT Driven Ba/F3 In Vivo Studies.

Supplementary Figures. Figure S1. Chemical structures of imatinib, sunitinib, regorafenib and ponatinib. Figure S2. Expression and activation of KIT in engineered Ba/F3 cells. Figure S3. Ponatinib inhibits the phosphorylation of exon 11primary activating, and secondary resistant mutant forms of KIT. Figure S4. Secondary mutants reduce ponatinib potency in Ex9 ins and V560D cell lines. Figure S5. Illustration of the optimal fit of ponatinib to KIT. Figure S6. Impact of compound treatment on KIT signaling in GIST-derived cell lines.

Supplementary Table 1. Brigatinib in vitro activity (IC50s) in a kinase panel (N=289); Supplementary Figure 1. Chemical structures of brigatinib, crizotinib, ceritinib, and alectinib; Supplementary Figure 2. Brigatinib-mediated inhibition of native or mutant ALK-, ROS1-,FLT3-, and EGFR-driven tumor activity in vitro; Supplementary Figure 3. Cellular and in vivo activity of brigatinib in ROS1 harboring models, compared with crizotinib; Supplementary Figure 4. Comparison of IGF-1R and INSR inhibitory activity of brigatinib with NVP-TAE684; Supplementary Figure 5. Inhibition of ALK phosphorylation in ALK+ cellular models by brigatinib, compared with crizotinib; Supplementary Figure 6. Inhibition of ALK phosphorylation and downstream signaling, and tolerability of brigatinib in vivo, compared with crizotinib; Supplementary Figure 7. TKI activity against parental, and native and mutant EML4-ALK driven Ba/F3 cells; Supplementary Figure 8. TKI antitumor activity against native and mutant EML4-ALK tumors, TKI plasma levels, and tolerability, in vivo.

Purpose:The emergence of secondary mutations is a cause of resistance to current KIT inhibitors used in the treatment of patients with gastrointestinal stromal tumors (GIST). AZD3229 is a selective inhibitor of wild-type KIT and a wide spectrum of primary and secondary mutations seen in patients with GIST. The objective of this analysis is to establish the pharmacokinetic–pharmacodynamic (PKPD) relationship of AZD3229 in a range of mouse GIST tumor models harboring primary and secondary KIT mutations, and to benchmark AZD3229 against other KIT inhibitors.Experimental Design:A PKPD model was developed for AZD3229 linking plasma concentrations to inhibition of phosphorylated KIT using data generated from several in vivo preclinical tumor models, and in vitro data generated in a panel of Ba/F3 cell lines.Results:AZD3229 drives inhibition of phosphorylated KIT in an exposure-dependent manner, and optimal efficacy is observed when >90% inhibition of KIT phosphorylation is sustained over the dosing interval. Integrating the predicted human pharmacokinetics into the mouse PKPD model predicts that an oral twice daily human dose greater than 34 mg is required to ensure adequate coverage across the mutations investigated. Benchmarking shows that compared with standard-of-care KIT inhibitors, AZD3229 has the potential to deliver the required target coverage across a wider spectrum of primary or secondary mutations.Conclusions:We demonstrate that AZD3229 warrants clinical investigation as a new treatment for patients with GIST based on its ability to inhibit both ATP-binding and A-loop mutations of KIT at clinically relevant exposures.

The clinical efficacy of epidermal growth factor receptor (EGFR)–targeted therapy in EGFR-mutant non–small cell lung cancer is limited by the development of drug resistance. One mechanism of EGFR inhibitor resistance occurs through amplification of the human growth factor receptor (MET) proto-oncogene, which bypasses EGFR to reactivate downstream signaling. Tumors exhibiting concurrent EGFR mutation and MET amplification are historically thought to be codependent on the activation of both oncogenes. Hence, patients whose tumors harbor both alterations are commonly treated with a combination of EGFR and MET tyrosine kinase inhibitors (TKIs). Here, we identify and characterize six patient-derived models of EGFR-mutant, MET-amplified lung cancer that have switched oncogene dependence to rely exclusively on MET activation for survival. We demonstrate in this MET-driven subset of EGFR TKI-refractory cancers that canonical EGFR downstream signaling was governed by MET, even in the presence of sustained mutant EGFR expression and activation. In these models, combined EGFR and MET inhibition did not result in greater efficacy in vitro or in vivo compared to single-agent MET inhibition. We further identified a reduced EGFR:MET mRNA expression stoichiometry as associated with MET oncogene dependence and single-agent MET TKI sensitivity. Tumors from 10 of 11 EGFR inhibitor–resistant EGFR-mutant, MET-amplified patients also exhibited a reduced EGFR:MET mRNA ratio. Our findings reveal that a subset of EGFR-mutant, MET-amplified lung cancers develop dependence on MET activation alone, suggesting that such patients could be treated with a single-agent MET TKI rather than the current standard-of-care EGFR and MET inhibitor combination regimens.

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

As technology advances, testing of an integrated chip is quite complex because of scaling of channel length. Testing methodologies of automatic test equipment are majorly important to test any chip at circuit level. As huge number of test patterns are used to test a circuit, test compression is critically required therefore reducing the test duration and also number of test patterns. Decompression method generates seeding for linear feedback shift register (LFSR) continually therefore skipping the LFSR lock out for limited number clock cycles. This required separate controlling for scan chain and decompressor. This paper studied effectiveness of decompression, number of parallel inputs at LFSR and also scan chains. A state skipping LFSR proposed with basic exclusive or (XOR) gates thereby number of test patterns are reduced and also delay reduced through hardware. The proposed method simulated on Xilinx 14.7 ISE and implemented on Vertx-7 FPGA as target device. The state skipping method reduces approximately 30% of test compression in terms of test cycles when compared to conventional LFSR.

In this article, we report the discovery of a series of 5-azaquinazolines as selective IRAK4 inhibitors. From modestly potent quinazoline 4, we introduced a 5-aza substitution to mask the 4-NH hydrogen bond donor (HBD). This allowed us to substitute the core with a 2-aminopyrazole, which showed large gains in cellular potency despite the additional formal HBD. Further optimization led to 6-cyanomethyl-5-azaquinazoline 13, a selective IRAK4 inhibitor, which proved efficacious in combination with ibrutinib, while showing very little activity as a single agent up to 100 mg/kg. This contrasted to previously reported IRAK4 inhibitors that exhibited efficacy in the same model as single agents and was attributed to the enhanced specificity of 13 toward IRAK4.

In this article, we report our efforts towards improving in vitro human clearance in a series of 5-azaquinazolines through a series of C4 truncations and C2 expansions. Extensive DMPK studies enabled us to tackle high Aldehyde Oxidase (AO) metabolism and unexpected discrepancies in human hepatocyte and liver microsomal intrinsic clearance. Our efforts culminated with the discovery of 5-azaquinazoline 35, which also displayed exquisite selectivity for IRAK4, and showed synergistic in vitro activity against MyD88/CD79 double mutant ABC-DLBCL in combination with the covalent BTK inhibitor acalabrutinib.

Background and purpose Savolitinib (AZD6094, HMPL-504, volitinib) is an oral, potent, and highly MET receptor TK inhibitor. This series of studies aimed to develop a pharmacokinetic-pharmacodynamic (PK/PD) model to link inhibition of MET phosphorylation (pMET) by savolitinib with anti-tumour activity. Experimental approach Cell line-derived xenograft (CDX) experiments using human lung cancer (EBC-1) and gastric cancer (MKN-45) cells were conducted in athymic nude mice using a variety of doses and schedules of savolitinib. Tumour pMET changes and growth inhibition were calculated after 28 days. Population PK/PD techniques were used to construct a PK/PD model for savolitinib. Key results Savolitinib showed dose- and dose frequency-dependent anti-tumour activity in the CDX models, with more frequent, lower dosing schedules (e.g., twice daily) being more effective than intermittent, higher dosing schedules (e.g., 4 days on/3 days off or 2 days on/5 days off). There was a clear exposure-response relationship, with maximal suppression of pMET of >90%. Data from additional CDX and patient-derived xenograft (PDX) models overlapped, allowing calculation of a single EC50 of 0.38 ng·ml-1 . Tumour growth modelling demonstrated that prolonged, high levels of pMET inhibition (>90%) were required for tumour stasis and regression in the models. Conclusion and implications High and persistent levels of MET inhibition by savolitinib were needed for optimal monotherapy anti-tumour activity in preclinical models. The modelling framework developed here can be used to translate tumour growth inhibition from the mouse to human and thus guide choice of clinical dose and schedule.

While the treatment of gastrointestinal stromal tumors (GISTs) has been revolutionized by the application of targeted tyrosine kinase inhibitors capable of inhibiting KIT-driven proliferation, diverse mutations to this kinase drive resistance to established therapies. Here we describe the identification of potent pan-KIT mutant kinase inhibitors that can be dosed without being limited by the tolerability issues seen with multitargeted agents. This effort focused on identification and optimization of an existing kinase scaffold through the use of structure-based design. Starting from a series of previously reported phenoxyquinazoline and quinoline based inhibitors of the tyrosine kinase PDGFRα, potency against a diverse panel of mutant KIT driven Ba/F3 cell lines was optimized, with a particular focus on reducing activity against a KDR driven cell model in order to limit the potential for hypertension commonly seen in second and third line GIST therapies. AZD3229 demonstrates potent single digit nM growth inhibition across a broad cell panel, with good margin to KDR-driven effects. Selectivity over KDR can be rationalized predominantly by the interaction of water molecules with the protein and ligand in the active site, and its kinome selectivity is similar to the best of the approved GIST agents. This compound demonstrates excellent cross-species pharmacokinetics, shows strong pharmacodynamic inhibition of target, and is active in several in vivo models of GIST.

Herein we report the optimization of a series of pyrrolopyrimidine inhibitors of interleukin-1 receptor associated kinase 4 (IRAK4) using X-ray crystal structures and structure based design to identify and optimize our scaffold. Compound 28 demonstrated a favorable physicochemical and kinase selectivity profile and was identified as a promising in vivo tool with which to explore the role of IRAK4 inhibition in the treatment of mutant MYD88L265P diffuse large B-cell lymphoma (DLBCL). Compound 28 was shown to be capable of demonstrating inhibition of NF-κB activation and growth of the ABC subtype of DLBCL cell lines in vitro at high concentrations but showed greater effects in combination with a BTK inhibitor at lower concentrations. In vivo, the combination of compound 28 and ibrutinib led to tumor regression in an ABC-DLBCL mouse model.

In this article, we report the discovery of a series of 5-azaquinazolines as selective IRAK4 inhibitors. From modestly potent quinazoline

Gastrointestinal stromal tumor (GIST) is the most common human sarcoma driven by mutations in KIT or platelet-derived growth factor α (PDGFRα). Although first-line treatment, imatinib, has revolutionized GIST treatment, drug resistance due to acquisition of secondary KIT/PDGFRα mutations develops in a majority of patients. Second- and third-line treatments, sunitinib and regorafenib, lack activity against a plethora of mutations in KIT/PDGFRα in GIST, with median time to disease progression of 4 to 6 months and inhibition of vascular endothelial growth factor receptor 2 (VEGFR2) causing high-grade hypertension. Patients with GIST have an unmet need for a well-tolerated drug that robustly inhibits a range of KIT/PDGFRα mutations. Here, we report the discovery and pharmacological characterization of AZD3229, a potent and selective small-molecule inhibitor of KIT and PDGFRα designed to inhibit a broad range of primary and imatinib-resistant secondary mutations seen in GIST. In engineered and GIST-derived cell lines, AZD3229 is 15 to 60 times more potent than imatinib in inhibiting KIT primary mutations and has low nanomolar activity against a wide spectrum of secondary mutations. AZD3229 causes durable inhibition of KIT signaling in patient-derived xenograft (PDX) models of GIST, leading to tumor regressions at doses that showed no changes in arterial blood pressure (BP) in rat telemetry studies. AZD3229 has a superior potency and selectivity profile to standard of care (SoC) agents-imatinib, sunitinib, and regorafenib, as well as investigational agents, avapritinib (BLU-285) and ripretinib (DCC-2618). AZD3229 has the potential to be a best-in-class inhibitor for clinically relevant KIT/PDGFRα mutations in GIST.

TPS178 Background: In metastatic hormone-sensitive prostate cancer (mHSPC), abiraterone combined with androgen deprivation therapy (ADT) is highly effective in improving patient outcomes. However, around 20% of patients progress to castration-resistant disease within 12 months of treatment; these patients have shorter survival and limited treatment options. Aberrant activation of the PI3K/AKT pathway, predominately due to PTEN loss, is common in prostate cancer, especially in later-stage disease. The androgen receptor signaling and AKT pathway are reciprocally cross-regulated such as that inhibition of one leads to upregulation of the other. Therefore, combining abiraterone therapy with AKT inhibition may be beneficial in patients with mHSPC who have PTEN deficiency. In preclinical studies, capivasertib, a selective oral pan-AKT inhibitor, inhibited proliferation of models of hormone-sensitive and castration-resistant prostate cancer with PTEN loss. The results of the IPATential150 phase 3 study (NCT03072238) demonstrated that another oral AKT inhibitor, ipatasertib, prolonged radiographic progression-free survival (rPFS) when combined with abiraterone compared with placebo plus abiraterone in metastatic castration-resistant prostate cancer, particularly among patients with PTEN loss. CAPItello-281 (NCT04493853) is a global, multicenter, phase 3 trial to evaluate capivasertib in combination with abiraterone, on a background of ADT, as a treatment for de novo mHSPC patients with PTEN-deficient tumors. Methods: Eligible patients for this double-blind, randomized trial are men aged 18 years or older with confirmed newly diagnosed previously untreated metastatic hormone-sensitive prostate adenocarcinoma with immunohistochemically confirmed PTEN deficiency and Eastern Cooperative Oncology Group (ECOG) performance status 0 or 1. Following screening, approximately 1000 patients will be randomized 1:1 to receive either capivasertib (400 mg) or placebo (twice daily; 4 days on, 3 days off) in combination with abiraterone (1000 mg once daily) and ADT until radiographic progression or intolerable toxicity. The primary endpoint is rPFS. Secondary endpoints include overall survival, time to start of first subsequent therapy or death, symptomatic skeletal event-free survival, time to pain progression and safety profile. Enrolment started in July 2020. Acknowledgments: We thank Julia Grigorieva, PhD, of Oxford PharmaGenesis, Philadelphia, USA, for providing medical writing assistance. Funding: The CAPItello-281 trial is funded and overseen by AstraZeneca. Clinical trial information: NCT04493853.

85 Background: Androgen receptor (AR) targeting therapies prolong survival of patients with metastatic castration-resistant prostate cancer (mCRPC); however, in many cases, resistance develops, resulting in disease progression. Activation of the PI3K/AKT/mTOR signaling pathway is common in mCRPC and contributes to resistance, mostly due to loss of PTEN, which occurs in 40–60% of patients. Preclinical studies have demonstrated reciprocal regulation between the AR and PI3K/AKT/mTOR pathways and significant anti-tumor activity when both pathways are inhibited, particularly in models with PTEN-loss. Thus, a rationale exists to inhibit both pathways in mCRPC patients. We report interim results of a phase 1 multicohort study (NCT04087174) to confirm the acceptable dose of capivasertib, a potent, selective pan-AKT inhibitor in combination with the androgen synthesis inhibitor abiraterone acetate (AA) in mCRPC patients. Methods: Patients who had received at least one prior systemic therapy (chemotherapy or novel hormonal agent) for mCRPC were given AA (1000 mg, once daily) with capivasertib (400 mg, twice daily, 4 days on/3 days off) until unacceptable toxicity or disease progression. Dose-limiting toxicity in the first 28 days of treatment and adverse events were recorded. Results: 15 patients, median age 67 (range 49–82) years, were recruited in the USA and Spain. Twelve patients had received prior chemotherapy; 7 had two or more prior lines. Seven patients had received prior AA and 10 had received prior enzalutamide. No dose-limiting toxicities were recorded. Eight patients reported at least one grade ≥ 3 adverse event (AE). Grade ≥ 3 AEs in 7 patients were related to capivasertib: allergic reaction to medication, asthenia, type 2 diabetes mellitus, diarrhea and fatigue were each reported in 1 patient, maculopapular rash − in 2 patients, both hypokalemia and acquired Fanconi syndrome − in 1 patient. Acute kidney injury was reported in 4 patients but was not considered related to capivasertib. The most common AEs of any grade related to capivasertib were: diarrhea, 6/15 patients (40%); maculopapular rash, 5/15 (33%); fatigue, 4/15 (27%); hyperglycemia/type 2 diabetes mellitus, 4/15 (27%); nausea, 3/15 (20%); hypokalemia, 2/15 (13%); hypophosphatemia, 2/15 (13%). Capivasertib was discontinued in 4/15 patients (27%) due to AEs. Between initial screening and day 29 of treatment, 5 patients had reduced (> 20%) PSA levels, with 3 patients showing sustained falls in PSA over 12 weeks. Conclusions: In this phase 1 study combined capivasertib and AA exhibits an acceptable safety and tolerability profile. Further data on the clinical efficacy and safety of the combination are being collected in the phase 3 CAPItello-281 trial. Acknowledgments: We thank Adam Errington, PhD, of Oxford PharmaGenesis, for medical writing assistance. Funding: This trial is funded by AstraZeneca. Clinical trial information: NCT04087174.

MET-amplification has been identified as a resistance mechanism in >5% of patients with non-small cell lung cancer (NSCLC) and 25% of those that progress on 1st/2nd or 3rd generation epidermal growth factor receptor-tyrosine kinase inhibitors (EGFR-TKIs). There are no approved therapies specifically indicated to treat EGFR mutation-positive (EGFRm)/MET amplified NSCLC patients. We report preclinical data of savolitinib (AZD6094, HMPL-504, volitinib, a potent and selective MET inhibitor) plus osimertinib (3rd generation, irreversible, oral EGFR-TKI that potently and selectively inhibits both EGFR sensitizing mutation and EGFR T790M) in an EGFRm/MET amplified model. Combination efficacy of savolitinib and osimertinib was tested in an EGFRm (L858R) and MET-amplified LG1208 PDX model, resistant to osimertinib. As single agents, and at doses that deliver clinically relevant exposures, osimertinib (10 mg/kg) and savolitinib (15 mg/kg) showed minimal (32% tumor growth inhibition [TGI]) and moderate (81% TGI) anti-tumor activity, respectively. Upon combination, at a fixed dose of osimertinib (10 mg/kg), anti-tumor activity increased with savolitinib dose, ranging from 1 mg/kg (99.8 % TGI) to 15 mg/kg (77.9% regression). Pharmacokinetic-pharmacodynamic (PKPD) modelling was also conducted linking exposure of savolitinib to inhibition of phosphorylated MET (pMET), and osimertinib to inhibition of pEGFR. A combination effect on pEGFR was observed whereby the maximum inhibition of pEGFR increased from approximately 50% with no savolitinib to approximately 90% with 15 mg/kg of savolitinib. The exposure-dependent effect of savolitinib on pEGFR was accounted for in the PKPD model by incorporating an interaction term such that the inhibition of pEGFR is modelled as a function of savolitinib and osimertinib exposure. This PKPD model was successfully used to link to the anti-tumor activity and provide biomarker-led rationale for the combination benefit seen for efficacy in this model. Overall, the PKPD analysis describes the biomarker changes linking to efficacy and provides supporting insight into the exposure requirements for combination dosing in the clinic. Savolitinib plus osimertinib was tested in the TATTON study (NCT02143466) where patients with EGFRm, MET-amplified NSCLC with prior progression on EGFR-TKIs had encouraging responses to this combination therapy. A phase 2 trial, SAVANNAH (NCT03778229), is testing the combination of osimertinib and savolitinib in patients with EGFRm, MET-amplified NSCLC with prior progression on osimertinib only. Citation Format: Rhys Jones, Awlin Schuller, Klas Petersson, Ghada Ahmed, Stein Schalkwijk, Areya Tabatabai, Andrew Mortlock, Rana Anjum. Using a pharmacodynamic biomarker led approach to explore and quantify the combination effect of savolitinib and osimertinib in the LG1208 NSCLC PDX model [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3017.

We have developed a series of orally efficacious IRAK4 inhibitors, based on a scaffold hopping strategy and using rational structure based design. Efforts to tackle low permeability and high efflux in our previously reported pyrrolopyrimidine series (Scott et al., 2017) led to the identification of pyrrolotriazines which contained one less formal hydrogen bond donor and were intrinsically more lipophilic. Further optimisation of substituents on this pyrrolotriazine core culminated with the discovery of 30 as a promising in vivo probe to assess the potential of IRAK4 inhibition for the treatment of MyD88 mutant DLBCL in combination with a BTK inhibitor. When tested in an ABC-DLBCL model with a dual MyD88/CD79 mutation (OCI-LY10), 30 demonstrated tumour regressions in combination with ibrutinib.

Herein we report the optimization of a series of pyrrolopyrimidine inhibitors of interleukin-1 receptor associated kinase 4 (IRAK4) using X-ray crystal structures and structure based design to identify and optimize our scaffold. Compound 28 demonstrated a favorable physicochemical and kinase selectivity profile and was identified as a promising in vivo tool with which to explore the role of IRAK4 inhibition in the treatment of mutant MYD88

Purpose: KIT is the major oncogenic driver of gastrointestinal stromal tumors (GIST). Imatinib, sunitinib, and regorafenib are approved therapies; however, efficacy is often limited by the acquisition of polyclonal secondary resistance mutations in KIT, with those located in the activation (A) loop (exons 17/18) being particularly problematic. Here, we explore the KIT-inhibitory activity of ponatinib in preclinical models and describe initial characterization of its activity in patients with GIST. Experimental Design: The cellular and in vivo activities of ponatinib, imatinib, sunitinib, and regorafenib against mutant KIT were evaluated using an accelerated mutagenesis assay and a panel of engineered and GIST-derived cell lines. The ponatinib–KIT costructure was also determined. The clinical activity of ponatinib was examined in three patients with GIST previously treated with all three FDA-approved agents. Results: In engineered and GIST-derived cell lines, ponatinib potently inhibited KIT exon 11 primary mutants and a range of secondary mutants, including those within the A-loop. Ponatinib also induced regression in engineered and GIST-derived tumor models containing these secondary mutations. In a mutagenesis screen, 40 nmol/L ponatinib was sufficient to suppress outgrowth of all secondary mutants except V654A, which was suppressed at 80 nmol/L. This inhibitory profile could be rationalized on the basis of structural analyses. Ponatinib (30 mg daily) displayed encouraging clinical activity in two of three patients with GIST. Conclusion:Ponatinib possesses potent activity against most major clinically relevant KIT mutants and has demonstrated preliminary evidence of activity in patients with refractory GIST. These data strongly support further evaluation of ponatinib in patients with GIST. Clin Cancer Res; 20(22); 5745–55. ©2014 AACR.

cKIT belongs a family of transmembrane tyrosine kinase growth factor receptors and gain of function mutations result in constitutive c-KIT activation that provide an important pathogenic role in gastrointestinal stromal tumors (GIST). AZD3229 is a highly potent and selective oral best-in-class KIT inhibitor that is active against a wide spectrum of primary and secondary KIT mutations that are known to confer resistance to standard of care agents. AZD3229 demonstrates rapid, extensive and sustained inhibition of KIT signaling in preclinical xenograft models with KIT primary exon 11 del mutations and primary/secondary ATP-binding pocket mutations (GIST 430/654A and patient-derived xenograft (PDX) model of KIT V654A, HGiXF-106) and A-loop mutations (PDX model of KIT Y823D, HGiXF-105 and Ba/F3 KIT D816H). In these models, AZD3229 results in anti-tumour activity (tumour regressions ranging from -60% to -99 % TGI) when dosed orally up to 20 mg/kg BID. The objective of this PK-PD modeling work was to (1) establish the PK-PD-Efficacy relationship for AZD3229 across the four cell-lines and PDX mouse models tested; (2) to confirm the extent and duration of pKIT inhibition required for optimal efficacy, as defined by tumor regressions. Inhibition of KIT phosphorylation observed with AZD3229 shows a direct relationship to free mouse plasma concentration and an estimated in vivo EC90 was estimated for each model (Ba/F3 D816H = 20 nM; GIST 430/V654A= 43 nM; HGiX-106 = 76 nM; HGiX-105 = 4 nM). No change in the extent of inhibition of KIT phosphorylation was observed on repeat dosing. Optimal efficacy is observed when sustained inhibition of pKIT, >90% is achieved over the dosing interval. Conclusion: Exploring the in vivo activity and PK-PD relationship in multiple cell-line derived xenograft and PDX models harboring different mutations provides a benchmark from which to anchor human dose predictions that partly reflect the spectrum of responses likely to be seen in GIST patients. Citation Format: Venkatesh Pilla Reddy, Aaron Smith, Michael Grondine, Rana Anjum, Rhys D. Jones. Exploring the pharmacokinetic-pharmacodynamic relationships of AZD3229, a novel and selective inhibitor of cKIT, in a range of mouse xenograft models of gastrointestinal stromal tumors [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 1328.

In the treatment of echinoderm microtubule-associated protein-like 4 (EML4)-anaplastic lymphoma kinase positive (ALK+) non-small-cell lung cancer (NSCLC), secondary mutations within the ALK kinase domain have emerged as a major resistance mechanism to both first- and second-generation ALK inhibitors. This report describes the design and synthesis of a series of 2,4-diarylaminopyrimidine-based potent and selective ALK inhibitors culminating in identification of the investigational clinical candidate brigatinib. A unique structural feature of brigatinib is a phosphine oxide, an overlooked but novel hydrogen-bond acceptor that drives potency and selectivity in addition to favorable ADME properties. Brigatinib displayed low nanomolar IC50s against native ALK and all tested clinically relevant ALK mutants in both enzyme-based biochemical and cell-based viability assays and demonstrated efficacy in multiple ALK+ xenografts in mice, including Karpas-299 (anaplastic large-cell lymphomas [ALCL]) and H3122 (NSCLC). Brigatinib represents the most clinically advanced phosphine oxide-containing drug candidate to date and is currently being evaluated in a global phase 2 registration trial.

Purpose: Non–small cell lung cancers (NSCLCs) harboring ALK gene rearrangements (ALK+) typically become resistant to the first-generation anaplastic lymphoma kinase (ALK) tyrosine kinase inhibitor (TKI) crizotinib through development of secondary resistance mutations in ALK or disease progression in the brain. Mutations that confer resistance to second-generation ALK TKIs ceritinib and alectinib have also been identified. Here, we report the structure and first comprehensive preclinical evaluation of the next-generation ALK TKI brigatinib.Experimental Design: A kinase screen was performed to evaluate the selectivity profile of brigatinib. The cellular and in vivo activities of ALK TKIs were compared using engineered and cancer-derived cell lines. The brigatinib–ALK co-structure was determined.Results: Brigatinib potently inhibits ALK and ROS1, with a high degree of selectivity over more than 250 kinases. Across a panel of ALK+ cell lines, brigatinib inhibited native ALK (IC50, 10 nmol/L) with 12-fold greater potency than crizotinib. Superior efficacy of brigatinib was also observed in mice with ALK+ tumors implanted subcutaneously or intracranially. Brigatinib maintained substantial activity against all 17 secondary ALK mutants tested in cellular assays and exhibited a superior inhibitory profile compared with crizotinib, ceritinib, and alectinib at clinically achievable concentrations. Brigatinib was the only TKI to maintain substantial activity against the most recalcitrant ALK resistance mutation, G1202R. The unique, potent, and pan-ALK mutant activity of brigatinib could be rationalized by structural analyses.Conclusions: Brigatinib is a highly potent and selective ALK inhibitor. These findings provide the molecular basis for the promising activity being observed in ALK+, crizotinib-resistant patients with NSCLC being treated with brigatinib in clinical trials. Clin Cancer Res; 22(22); 5527–38. ©2016 AACR.

N-alpha-tosyl-l-phenylalanyl chloromethyl ketone (TPCK) has anti-tumorigenic properties, but its direct cellular targets are unknown. Previously, we showed TPCK inhibited the PDKl-dependent AGC kinases RSK, Akt and S6K1 without inhibiting PKA, ERK1/2, PI3K, and PDK1 itself. Here we show TPCK-inhibition of the RSK-related kinases MSK1 and 2, which can be activated independently of PDK1. Mass spectrometry analysis of RSK1, Aktl, S6K1 and MSK1 immunopurified from TPCK-treated cells identified TPCK adducts on cysteines located in conserved activation loop Phenylalanine–Cysteine (Phe–Cys) motifs. Mutational analysis of the Phe–Cys residues conferred partial TPCK resistance. These studies elucidate a primary mechanism by which TPCK inhibits several AGC kinases, inviting consideration of TPCK-like compounds in chemotherapy given their potential for broad control of cellular growth, proliferation and survival.

Activating gene rearrangements of anaplastic lymphoma kinase (ALK) have been identified as driver mutations in non-small-cell lung cancer, inflammatory myofibroblastic tumors, and other cancers. Crizotinib, a dual MET/ALK inhibitor, has demonstrated promising clinical activity in patients with non-small-cell lung cancer and inflammatory myofibroblastic tumors harboring ALK translocations. Inhibitors of driver kinases often elicit kinase domain mutations that confer resistance, and such mutations have been successfully predicted using in vitro mutagenesis screens. Here, this approach was used to discover an extensive set of ALK mutations that can confer resistance to crizotinib. Mutations at 16 residues were identified, structurally clustered into five regions around the kinase active site, which conferred varying degrees of resistance. The screen successfully predicted the L1196M, C1156Y, and F1174L mutations, recently identified in crizotinib-resistant patients. In separate studies, we demonstrated that crizotinib has relatively modest potency in ALK-positive non-small-cell lung cancer cell lines. A more potent ALK inhibitor, TAE684, maintained substantial activity against mutations that conferred resistance to crizotinib. Our study identifies multiple novel mutations in ALK that may confer clinical resistance to crizotinib, suggests that crizotinib's narrow selectivity window may underlie its susceptibility to such resistance and demonstrates that a more potent ALK inhibitor may be effective at overcoming resistance.

To gain insights into the tumor microenvironment in primary and secondary CNS lymphomas, we applied LC/MS and GC/MS for differential metabolomic profiling of the cerebrospinal fluid (CSF) of CNS lymphoma patients compared to control subjects. Among 145 analytes identified, the majority were involved in energy metabolism; one of the most significantly upregulated metabolites in CNS lymphoma was lactate (1.8 fold, p Thus far we have applied these metabolic imaging approaches to preclinical murine models involving diffusely invasive, intracranial, patient-derived xenografts of CNS DLBCL in RAG-/- mice, to detect tumor-associated lactate production generated by infiltrating lymphoma. We demonstrated that each of these MRS approaches detect highly invasive lymphoma that is undetectable by conventional gadolinium-enhanced T1, T2 sequences, or diffusion-weighted imaging. Because of its ability to detect real-time changes in metabolic pathways, we focused on the application of HP13C pyruvate metabolic imaging as a non-invasive imaging tool for NF-kB pathway inhibition in CNS lymphoma using these models. We evaluated the metabolic response to AZ1495, a novel, CNS penetrant, orally-bioavailable inhibitor of IRAK4 kinase, comparing MYD88 wild type vs. MYD88 L265P mutant tumor models. Using a 14.1T imaging system for MR acquisition, we demonstrated similar tumor-associated production of HP 13C lactate in both MYD88wt and MY88 mutant tumors at 3 weeks post-implantation. We determined that while AZ1495 did not significantly impact lactate production in MYD88wt lymphoma, this agent significantly down-regulated tumor-associated HP pyruvate to lactate conversion (>47%) within 2 days in MYD88 mutant CNS lymphoma (p 2-fold down-modulation of NF-kB gene expression at 4h of AZ1495 therapy, including transcripts encoding LDH-A as well as the catalytic subunit of PI3K, suggesting interaction with the B cell receptor pathway. Combination AZ1495 plus ibrutinib starting d+5 was synergistic in survival prolongation compared to AZ1495 monotherapy (p Disclosures Gao: Glaxo Smith Kline: Employment. Drew:AstraZeneca: Employment. Degorce:Astra Zeneca: Employment. Mayo:Astra Zeneca: Employment. Dillman:Astra Zeneca: Employment. Anjum:Astra Zeneca: Employment. Bloecher:Astra Zeneca: Employment. Rubenstein:Celgene: Research Funding; Genentech: Research Funding.

CLIP-170 conformational changes are regulated by phosphorylation on S309 and S311 residues resulting in diminished binding of CLIP-170 for growing MT ends and p150Glued., Cytoplasmic linker protein (CLIP)-170 is a microtubule (MT) plus-end-tracking protein that regulates MT dynamics and links MT plus ends to different intracellular structures. We have shown previously that intramolecular association between the N and C termini results in autoinhibition of CLIP-170, thus altering its binding to MTs and the dynactin subunit p150Glued (J. Cell Biol. 2004: 166, 1003–1014). In this study, we demonstrate that conformational changes in CLIP-170 are regulated by phosphorylation that enhances the affinity between the N- and C-terminal domains. By using site-directed mutagenesis and phosphoproteomic analysis, we mapped the phosphorylation sites in the third serine-rich region of CLIP-170. A phosphorylation-deficient mutant of CLIP-170 displays an “open” conformation and a higher binding affinity for growing MT ends and p150Glued as compared with nonmutated protein, whereas a phosphomimetic mutant confined to the “folded back” conformation shows decreased MT association and does not interact with p150Glued. We conclude that phosphorylation regulates CLIP-170 conformational changes resulting in its autoinhibition.

Constitutive activation of one or more kinase signaling pathways is a hallmark of many cancers. Here we extend the previously described mass spectrometry-based KAYAK approach by monitoring kinase activities from multiple signaling pathways simultaneously. This improved single-reaction strategy, which quantifies the phosphorylation of 90 synthetic peptides in a single mass spectrometry run, is compatible with nanogram to microgram amounts of cell lysate. Furthermore, the approach enhances kinase monospecificity through substrate competition effects, faithfully reporting the signatures of many signaling pathways after mitogen stimulation or of basal pathway activation differences across a panel of well-studied cancer cell lines. Hierarchical clustering of activities from related experiments groups peptides phosphorylated by similar kinases together and, when combined with pathway alteration using pharmacological inhibitors, distinguishes underlying differences in potency, off-target effects and genetic backgrounds. Finally, we introduce a strategy to identify the kinase, and even associated protein complex members, responsible for phosphorylation events of interest.

Summary The viability of vertebrate cells depends on a complex signaling interplay between survival factors and cell-death effectors. Subtle changes in the equilibrium between these regulators can result in abnormal cell proliferation or cell death, leading to various pathological manifestations [1, 2]. Death-associated protein kinase (DAPK) is a multidomain calcium/calmodulin (CaM)-dependent Ser/Thr protein kinase with an important role in apoptosis regulation and tumor suppression [3–6]. The molecular signaling mechanisms regulating this kinase, however, remain unclear. Here, we show that DAPK is phosphorylated upon activation of the Ras-extracellular signal-regulated kinase (ERK) pathway [7]. This correlates with the suppression of the apoptotic activity of DAPK. We demonstrate that DAPK is a novel target of p90 ribosomal S6 kinases (RSK) 1 and 2, downstream effectors of ERK1/2 [8–11]. Using mass spectrometry, we identified Ser-289 as a novel phosphorylation site in DAPK, which is regulated by RSK. Mutation of Ser-289 to alanine results in a DAPK mutant with enhanced apoptotic activity, whereas the phosphomimetic mutation (Ser289Glu) attenuates its apoptotic activity. Our results suggest that RSK-mediated phosphorylation of DAPK is a unique mechanism for suppressing the proapoptotic function of this death kinase in healthy cells as well as Ras/Raf-transformed cells.

In primary central nervous system lymphoma (PCNSL), L265P mutation of the myeloid differentiation 88 (MYD88) protein is the most common, and induces a constitutive activation of IRAK4 kinase, IRAK1 phosphorylation and increased NF-kB signaling, offering a unique window for therapeutic development. We previously show that AZ1495, a potent inhibitor of IRAK4, significantly delayed progression and improved OS in L265P-mutated PCNSL models [ASH 2016], highlighting the clinical potential of IRAK inhibition. In the clinic however, early monitoring of such targeted therapies remains challenging using conventional imaging, as treatment often results in non-readily detectable anatomical changes. Here, we questioned the potential of a new neuroimaging method, hyperpolarized 13C magnetic resonance spectroscopic imaging (HP-13C MRSI), to non-invasively monitor IRAK inhibition in PCNSL for the first time. Two PCNSL patient-derived xenografts models were studied: one harboring the L265P MYD88 mutation (MYD88mut), and the other, wild type for this protein (MYD88wt). Using HP-13C MRSI, a high production of HP [1-13C] lactate was detected in both MYD88wt and MYD88mut (0.5 ± 0.04, n=2 MYD88wt; 0.61 ± 0.2, n=4 MYD88mut), indicating PCNSL presence throughout the brain. At day 2 and 4 of AZ1495 treatment, the HP [1-13C] lactate-to-pyruvate ratio remains unchanged in MYD88wt animals at all treatment time points (p=0.29 and p=0.29 at day 2 and 4 when compared to baseline, respectively) On the other hand, this ratio significantly decreased in MYD88mut animals at all time points (36.6% at day 2, p=0.07 and 56.3% at day 4, p=0.0006), in line with the previously reported effect of AZ1495 treatment on MYD88mut PCNSL only. To conlude, we showed that HP-13C MRSI can detect MYD88-mutation-specific modulations of HP lactate production following IRAK inhibition. Because HP-13C MRSI is clinically translatable and expanding rapidly, this study is of high significance for future clinical trials, to enhance monitoring of PCNSL progression and response of targeted therapies.

Summary: An efficient immune response comprises a highly intricate, integrated circuitry involving both the cellular and the humoral arms of the immune system of the host interacting with the rapidly proliferating microcosm of the tumor. The mechanism of tumor rejection involving multiple arms of the immune system was reviewed in a spontaneously regressing rat histiocytoma, AK-5, an autologous tumor–host system. Intraperitoneal tumor transplantation leads to death in all animals, whereas subcutaneously (s.c.) transplanted tumor undergoes regression in 70% of animals. Regression of the tumor occurs by both apoptosis and necrosis, and natural killer (NK) cells were identified as the chief effectors mediating tumor cell death in vivo. A type 1 helper T cell (Th1)-driven cytokine cascade played a crucial role in enhancing cellular functions at the tumor site and obtaining a sufficient immune response for tumor rejection. The s.c. tumor-bearing hosts were shown to produce a factor which induced apoptosis in tumor cells, mediating tumor rejection. This review emphasizes the daunting complexities and interesting liaisons between the host immune system and the tumor, highlighting the work from our laboratory, and stressing that it is the interaction of several factors in concert or antagonizing each other that is responsible for the spontaneous regression of a tumor.

Among the clinical subtypes of diffuse large B-cell lymphoma, central nervous system (CNS) lymphomas, both primary (PCNSL) and secondary (SCNSL), are generally associated with the worst prognosis. Moreover, PCNSL typically exhibits multifocal dissemination, prompting its description as a whole brain disease. An accumulation of genomic data suggests that aberrant NF-kB pro-survival signaling may be a critical factor in the pathogenesis of CNS lymphoma. To date, however, there has been an absence of mechanistic studies to interrogate the mediators and significance of NF-kB activation in CNS lymphoma invasion. Recently our lab derived the first panel of xenografts yet established from PCNSL patients (n=7), and defined their invasive properties in murine models. These novel model systems provide a unique opportunity to study key regulatory mechanisms as well as novel targeted therapies of CNS lymphoma invasion and growth within the brain. We determined that the intracranial growth patterns of the xenografts can be classified based upon reproducible patterns of invasiveness. Two xenografts derived from SCNSL failed to infiltrate brain parenchyma or to migrate to the contralateral hemisphere; instead these tumors exhibited solely a meningeal dissemination pattern, largely growing in the ventricles. By contrast, five of the xenografts exhibited diffuse brain invasion into bilateral hemispheres. In addition, we noted that CNS lymphoma xenografts with an invasive phenotype can be sub-divided phenotypically into moderately vs. highly invasive tumors. After standard injection of 200,000 tumor cells/brain, the highly invasive lymphomas were reproducibly lethal to mice within 3 weeks post-implantation and yielded ~ 1X106 lymphoma cells/brain at necropsy. Under identical conditions, the moderately-invasive lymphoma xenografts were lethal to mice within 1-3 months and yielded between 3-5 X106lymphoma cells/brain. Notably each of the highly invasive CNS lymphoma xenografts were derived from CNS lymphomas that contained the activating L265P mutation of MYD88 (activator of NF-kB via IRAK1/4) and/or Y196H mutation of CD79B (activator of NF-kB), whereas the non-invasive xenografts contained wild type MYD88 and CD79B. We analyzed RNA-seq expression data of these xenograft samples to evaluate relationship of individual genes and signaling pathways with the distinct subclasses of CNS lymphomas in terms of invasiveness. Upregulated signatures in the pro-invasive phenotype are significantly enriched with NF-kB target genes (http://lymphochip.nih.gov/signaturedb/) (FDR Given that the L265P mutation of MYD88 represents a candidate key driver of NF-kB activation in CNS lymphoma, we evaluated the preclinical efficacy of AZ1495, a novel and potent inhibitor of IRAKs. We demonstrated CNS penetration of AZ1495 after PO administration to mice with intracranial lymphoma xenografts, yielding a mean brain/plasma partition coefficient of 0.8. AZ1495 potently antagonized NF-kB activation as demonstrated by p-p65 immunoblot analysis of tumor lysates isolated from murine CNS lymphoma xenografts, within 3 hrs of administration. AZ1495 monotherapy resulted in > 2-fold delay in both intracranial plus spinal progression in a dissemination model of CNS lymphoma using L265P mutant OCI-LY10 cells engineered for bioluminescence (p2-fold increase in median OS in a patient-derived model of L265P mutant PCNSL. These results support the preclinical utility of these models to dissect impact of NF-kB inhibition on CNS lymphoma progression as well as the clinical potential of IRAK antagonism as a means to target NF-kB activation in L265P mutant CNS lymphomas. Disclosures Anjum: Astra Zeneca: Employment. Drew:Astra Zeneca: Employment. Degorce:Astra Zeneca: Employment. Dillman:Astra Zeneca: Employment. Mayo:Astra Zeneca: Employment. Rubenstein:Celgene: Research Funding; Genentech: Research Funding.

Curcumin, the active ingredient of the rhizome of Curcuma longa has anti-inflammatory, antioxidant and antiproliferative activities. Although its precise mode of action remains elusive, studies have shown that chemopreventive action of curcumin might be due to its ability to induce apoptosis in cancer cells. Curcumin was shown to be responsible for the inhibition of AK-5 tumor (a rat histiocytoma) growth by inducing apoptosis in AK-5 tumor cells via caspase activation. This study was designed to investigate the mechanism leading to the induction of apoptosis in AK-5 tumor cells. Curcumin treatment resulted in the hyperproduction of reactive oxygen species (ROS), loss of mitochondrial membrane potential (delta psi(m)) and cytochrome c release to the cytosol, with the concomitant exposure of phosphatidylserine (PS) residues on the cell surface. This study suggests redox signalling and caspase activation as the mechanisms responsible for the induction of curcumin mediated apoptosis in AK-5 tumor cells.

Ceramide, a product of sphingomyelin metabolism, is a novel lipid second messenger that mediates diverse cellular functions. The present study demonstrates the activation of caspase-3/CPP-32β, during apoptosis induced by cell permeable exogenous ceramides, in AK-5 tumor, a spontaneously regressing rat histiocytoma. The apoptotic events were suppressed by the caspase-3 specific tetrapeptide inhibitor DEVD-CHO but not by the caspase-1 inhibitor YVAD-CMK. In cells overexpressing Bcl-2, a significant decrease in cell death was observed after exogenous addition of ceramides. Furthermore the processing of caspase-3 to its active form upon apoptotic stimulus, and the subsequent cleavage of the substrate PARP, suggested a central role for caspase-3 in the ceramide mediated apoptosis in AK-5 tumor cells.

Circadian rhythms in Drosophila rely on cyclic regulation of the period (per) and timeless (tim) clock genes. The molecular cycle requires rhythmic phosphorylation of PER and TIM proteins, which is mediated by several kinases and phosphatases such as Protein Phosphatase-2A (PP2A) and Protein Phosphatase-1 (PP1). Here, we used mass spectrometry to identify 35 “phospho-occupied” serine/threonine residues within PER, 24 of which are specifically regulated by PP1/PP2A. We found that cell culture assays were not good predictors of protein function in flies and so we generated per transgenes carrying phosphorylation site mutations and tested for rescue of the per01 arrhythmic phenotype. Surprisingly, most transgenes restore wild type rhythms despite carrying mutations in several phosphorylation sites. One particular transgene, in which T610 and S613 are mutated to alanine, restores daily rhythmicity, but dramatically lengthens the period to ∼30 hrs. Interestingly, the single S613A mutation extends the period by 2–3 hours, while the single T610A mutation has a minimal effect, suggesting these phospho-residues cooperate to control period length. Conservation of S613 from flies to humans suggests that it possesses a critical clock function, and mutational analysis of residues surrounding T610/S613 implicates the entire region in determining circadian period. Biochemical and immunohistochemical data indicate defects in overall phosphorylation and altered timely degradation of PER carrying the double or single S613A mutation(s). The PER-T610A/S613A mutant also alters CLK phosphorylation and CLK-mediated output. Lastly, we show that a mutation at a previously identified site, S596, is largely epistatic to S613A, suggesting that S613 negatively regulates phosphorylation at S596. Together these data establish functional significance for a new domain of PER, demonstrate that cooperativity between phosphorylation sites maintains PER function, and support a model in which specific phosphorylated regions regulate others to control circadian period., Author Summary Circadian rhythms coordinate an organism's activities with its environment to ensure appropriate physiology and behavior at the relevant times of day. In Drosophila melanogaster, the central molecular clock is regulated by transcriptional and translational feedback loops that drive rhythmic waves of gene expression. Additionally, the clock is controlled by post-translational modifications, including phosphorylation of a core negative regulator, PERIOD (PER). Using a proteomic approach, we identified two key phosphorylation sites within PER that cooperate to establish proper daily rhythms. Mutation of both residues results in a period of ∼30 hrs, whereas single mutant phenotypes are less severe suggesting these sites normally cooperate to control the pace of the clock. Biochemical and immunohistochemical data demonstrate that altered PER phosphorylation and changes in protein stability give rise to behavioral defects. Other mutations in the vicinity of the two sites above also alter circadian period, thereby confirming a new regulatory domain of PER. Our results also suggest that the phospho-residues identified in this study regulate phosphorylation in another important domain of PER. Together our data establish that cooperativity between neighboring phospho-sites as well as interactions between different phosphorylated domains of PER control its stability and overall circadian periodicity.

Most kinases are capable of recognizing and phosphorylating peptides containing short, linear sequence motifs. To measure the activation state of many kinases from the same cell lysate, we created a multiplexed, mass-spectrometry-based in vitro kinase assay. Ninety chemically synthesized peptides derived from well-characterized peptide substrates and in vivo phosphorylation sites with either known or previously unidentified upstream kinases were reacted individually in a plate format with crude cell lysates and ATP. Phosphorylation rates were directly measured based on the addition of 90 same-sequence, site-specific phosphopeptides enriched in stable isotopes to act as ideal quantitative internal standards for analysis by liquid chromatography coupled to tandem mass spectrometry. This approach concurrently measured up to 90 site-specific peptide phosphorylation rates, reporting a diagnostic fingerprint for activated kinase pathways. We applied this unique kinome-activity profiling strategy in a variety of cellular settings, including mitogen stimulation, cell cycle, pharmacological inhibition of pathways, and to a panel of breast cancer cell lines. Finally, we identified the source of activity for a peptide (derived from a PI3K regulatory subunit) from our library. This peptide substrate demonstrated mitotic and tyrosine-specific phosphorylation, which was confirmed to be a novel Src family kinase site in vivo.

The 90 kDa ribosomal S6 kinase (RSK) family of proteins is a group of highly conserved Ser/Thr kinases that regulate diverse cellular processes, such as cell growth, cell motility, cell survival and cell proliferation. RSKs are downstream effectors of the Ras-extracellular signal-regulated kinase (ERK)/mitogen-activated protein kinase (MAPK) signalling cascade. Significant advances in the field of RSK and ERK/MAPK signalling have occurred in the past few years, including biological insights and the discovery of novel substrates and new RSK regulatory mechanisms. Collectively, these data expand the current models of RSK signalling and highlight potential directions of research in RSK-mediated survival, growth, proliferation and migration.

Tuberous sclerosis complex (TSC) is a genetic disorder caused by mutations in either of the two tumor suppressor genes TSC1 or TSC2 , which encode hamartin and tuberin, respectively. Tuberin and hamartin form a complex that inhibits signaling by the mammalian target of rapamycin (mTOR), a critical nutrient sensor and regulator of cell growth and proliferation. Phosphatidylinositol 3-kinase (PI3K) inactivates the tumor suppressor complex and enhances mTOR signaling by means of phosphorylation of tuberin by Akt. Importantly, cellular transformation mediated by phorbol esters and Ras isoforms that poorly activate PI3K promote tumorigenesis in the absence of Akt activation. In this study, we show that phorbol esters and activated Ras also induce the phosphorylation of tuberin and collaborates with the nutrient-sensing pathway to regulate mTOR effectors, such as p70 ribosomal S6 kinase 1 (S6K1). The mitogen-activated protein kinase (MAPK)-activated kinase, p90 ribosomal S6 kinase (RSK) 1, was found to interact with and phosphorylate tuberin at a regulatory site, Ser-1798, located at the evolutionarily conserved C terminus of tuberin. RSK1 phosphorylation of Ser-1798 inhibits the tumor suppressor function of the tuberin/hamartin complex, resulting in increased mTOR signaling to S6K1. Together, our data unveil a regulatory mechanism by which the Ras/MAPK and PI3K pathways converge on the tumor suppressor tuberin to inhibit its function.

The tuberous sclerosis complex (TSC) is a genetic disorder that is caused through mutations in either one of the two tumor suppressor genes, TSC1 and TSC2, that encode hamartin and tuberin, respectively. Interaction of hamartin with tuberin forms a heterodimer that inhibits signaling by the mammalian target of rapamycin to its downstream targets: eukaryotic initiation factor 4E-binding protein 1 (4E-BP1) and ribosomal protein S6 kinase 1 (S6K1). During mitogenic sufficiency, the phosphoinositide 3-kinase (PI3K)/Akt pathway phosphorylates tuberin on Ser-939 and Thr-1462 that inhibits the tumor suppressor function of the TSC complex. Here we show that tuberin-hamartin heterodimers block protein kinase C (PKC)/MAPK- and phosphatidic acid-mediated signaling toward mammalian target of rapamycin-dependent targets. We also show that two TSC2 mutants derived from TSC patients are defective in repressing phorbol 12-myristate 13-acetate-induced 4E-BP1 phosphorylation. PKC/MAPK signaling leads to phosphorylation of tuberin at sites that overlap with and are distinct from Akt phosphorylation sites. Phosphorylation of tuberin by phorbol 12-myristate 13-acetate was reduced by treatment of cells with either bisindolylmaleimide I or UO126, inhibitors of PKC and MAPK/MEK (MAPK/ERK kinase), respectively, but not by wortmannin (an inhibitor of PI3K). This work reveals that both PI3K-independent and -dependent mechanisms modulate tuberin phosphorylation in vivo.

Bax, a proapoptotic member of the Bcl-2 family of proteins, resides in the cytosol and translocates to the mitochondrial membrane upon induction of apoptosis. It has been proposed that Bax does not translocate to mitochondria under normal physiological conditions, due to interaction between amino (ART) and carboxy (TM) terminal domains. Here, we report the physiological consequences of introducing a matrix targeting mitochondrial signal sequence (Su9) at the amino terminus of Bax and its mutants lacking ART, TM, or both segments. In vitro mitochondrial protein import assays of the fusion proteins suggests localization to the mitochondrial matrix. When expressed in Cos-1 cells, Su9 could target Bax to mitochondria in the absence of an apoptotic stimulus. However, mitochondrial localization did not result in apoptosis. When ART, TM, or both segments of Bax were deleted, expression of fusion proteins containing Su9 resulted in apoptosis via cytochrome c release. Cell death was inhibited by the pan-caspase inhibitor zVAD-fmk. We thus demonstrate that an effective mitochondrial matrix targeting signal can override the inhibition of import of Bax to the organelle, presumed to arise as a result of interaction between ART and TM segments, in the absence of apoptotic stimulus. We also demonstrate the ability of truncated variants of Bax to cause apoptosis when targeted to mitochondria by cytochrome c release from an ectopic environment.

Curcumin, the yellow pigment of turmeric (Curcuma longa), used commonly as a spice, has been shown to possess anticarcinogenic activity. Curcumin inhibited AK-5 tumor growth and induced apoptosis in AK-5 cells. Curcumin induced apoptosis is mediated through the activation of caspase-3, which is specifically inhibited by the tetrapeptide Ac-DEVD-CHO. In addition, curcumin induced tumor cell death is caused through the generation of reactive oxygen intermediates which is inhibited by N-acetyl-l-cysteine. Our studies suggest that the apoptotic process induced by curcumin is the mechanism mediating AK-5 tumor cell death.