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GNE-900 potentiates the cytotoxicity of gemcitabine in HCT-116 isogenic variants, irrespective of NHEJ competence.

Kinase selectivity profile evaluated at 100 nM GNE-900

Pharmacokinetics of GNE-900 in female CD-1 mice or male Sprague-Dawley rats.

Representative data from a cell-based checkpoint abrogation assay.

GNE-900 can potentiate the cytotoxicity of gemcitabine, SN-38 and pemetrexed.

Representative histograms from high content imaging studies.

GNE-900 is more potent at chemopotentiation in p53 null HCT-116.

Checkpoint kinase 1 (ChK1) is a serine/threonine kinase that functions as a central mediator of the intra-S and G2–M cell-cycle checkpoints. Following DNA damage or replication stress, ChK1-mediated phosphorylation of downstream effectors delays cell-cycle progression so that the damaged genome can be repaired. As a therapeutic strategy, inhibition of ChK1 should potentiate the antitumor effect of chemotherapeutic agents by inactivating the postreplication checkpoint, causing premature entry into mitosis with damaged DNA resulting in mitotic catastrophe. Here, we describe the characterization of GNE-900, an ATP-competitive, selective, and orally bioavailable ChK1 inhibitor. In combination with chemotherapeutic agents, GNE-900 sustains ATR/ATM signaling, enhances DNA damage, and induces apoptotic cell death. The kinetics of checkpoint abrogation seems to be more rapid in p53-mutant cells, resulting in premature mitotic entry and/or accelerated cell death. Importantly, we show that GNE-900 has little single-agent activity in the absence of chemotherapy and does not grossly potentiate the cytotoxicity of gemcitabine in normal bone marrow cells. In vivo scheduling studies show that optimal administration of the ChK1 inhibitor requires a defined lag between gemcitabine and GNE-900 administration. On the refined combination treatment schedule, gemcitabine's antitumor activity against chemotolerant xenografts is significantly enhanced and dose-dependent exacerbation of DNA damage correlates with extent of tumor growth inhibition. In summary, we show that in vivo potentiation of gemcitabine activity is mechanism based, with optimal efficacy observed when S-phase arrest and release is followed by checkpoint abrogation with a ChK1 inhibitor. Mol Cancer Ther; 12(10); 1968–80. ©2013 AACR.

Representative data from enzyme assays evaluating the relative potency of GNE-900 against ChK1 or ChK2.

Optimization of a series of aryl urea RAF inhibitors led to the identification of type II pan-RAF inhibitor GNE-0749 (7), which features a fluoroquinazolinone hinge-binding motif. By minimizing reliance on common polar hinge contacts, this hinge binder allows for a greater contribution of RAF-specific residue interactions, resulting in exquisite kinase selectivity. Strategic substitution of fluorine at the C5 position efficiently masked the adjacent polar NH functionality and increased solubility by impeding a solid-state conformation associated with stronger crystal packing of the molecule. The resulting improvements in permeability and solubility enabled oral dosing of 7. In vivo evaluation of 7 in combination with the MEK inhibitor cobimetinib demonstrated synergistic pathway inhibition and significant tumor growth inhibition in a KRAS mutant xenograft mouse model.

Structure-based design was utilized to optimize 6,6-diaryl substituted dihydropyrone and hydroxylactam to obtain inhibitors of lactate dehydrogenase (LDH) with low nanomolar biochemical and single-digit micromolar cellular potencies. Surprisingly the replacement of a phenyl with a pyridyl moiety in the chemical structure revealed a new binding mode for the inhibitors with subtle conformational change of the LDHA active site. This led to the identification of a potent, cell-active hydroxylactam inhibitor exhibiting an in vivo pharmacokinetic profile suitable for mouse tumor xenograft study.

Optimization of a series of aryl urea RAF inhibitors led to the identification of type II pan-RAF inhibitor GNE-0749 (

Although RAF monomer inhibitors (type I.5, BRAF(V600)) are clinically approved for the treatment of BRAFV600-mutant melanoma, they are ineffective in non-BRAFV600 mutant cells1–3. Belvarafenib is a potent and selective RAF dimer (type II) inhibitor that exhibits clinical activity in patients with BRAFV600E- and NRAS-mutant melanomas. Here we report the first-in-human phase I study investigating the maximum tolerated dose, and assessing the safety and preliminary efficacy of belvarafenib in BRAFV600E- and RAS-mutated advanced solid tumours (NCT02405065, NCT03118817). By generating belvarafenib-resistant NRAS-mutant melanoma cells and analysing circulating tumour DNA from patients treated with belvarafenib, we identified new recurrent mutations in ARAF within the kinase domain. ARAF mutants conferred resistance to belvarafenib in both a dimer- and a kinase activity-dependent manner. Belvarafenib induced ARAF mutant dimers, and dimers containing mutant ARAF were active in the presence of inhibitor. ARAF mutations may serve as a general resistance mechanism for RAF dimer inhibitors as the mutants exhibit reduced sensitivity to a panel of type II RAF inhibitors. The combination of RAF plus MEK inhibition may be used to delay ARAF-driven resistance and suggests a rational combination for clinical use. Together, our findings reveal specific and compensatory functions for the ARAF isoform and implicate ARAF mutations as a driver of resistance to RAF dimer inhibitors. The development, characterization and phase I clinical testing of the RAF inhibitor belvarafenib in cancer and a new resistance mechanism mediated by ARAF mutations are described.

A series of trisubstituted hydroxylactams was identified as potent enzymatic and cellular inhibitors of human lactate dehydrogenase A. Utilizing structure-based design and physical property optimization, multiple inhibitors were discovered with

Activating mutations in protein kinases drive many cancers. While how recurring point mutations affect kinase activity has been described, the effect of in-frame deletions is not well understood. We show that oncogenic deletions within the β3-αC loop of HER2 and BRAF are analogous to the recurrent EGFR exon 19 deletions. We identify pancreatic carcinomas with BRAF deletions mutually exclusive with KRAS mutations. Crystal structures of BRAF deletions reveal the truncated loop restrains αC in an active "in" conformation, imparting resistance to inhibitors like vemurafenib that bind the αC "out" conformation. Characterization of loop length explains the prevalence of five amino acid deletions in BRAF, EGFR, and HER2 and highlights the importance of this region for kinase activity and inhibitor efficacy.

Targeting KRAS mutant tumors through inhibition of individual downstream pathways has had limited clinical success. Here we report that RAF inhibitors exhibit little efficacy in KRAS mutant tumors. In combination drug screens, MEK and PI3K inhibitors synergized with pan-RAF inhibitors through an RAS-GTP-dependent mechanism. Broad cell line profiling with RAF/MEK inhibitor combinations revealed synergistic efficacy in KRAS mutant and wild-type tumors, with KRASG13D mutants exhibiting greater synergy versus KRASG12 mutant tumors. Mechanistic studies demonstrate that MEK inhibition induced RAS-GTP levels, RAF dimerization and RAF kinase activity resulting in MEK phosphorylation in synergistic tumor lines regardless of KRAS status. Taken together, our studies uncover a strategy to rewire KRAS mutant tumors to confer sensitivity to RAF kinase inhibition.

Optimization of 5-(2,6-dichlorophenyl)-3-hydroxy-2-mercaptocyclohex-2-enone using structure-based design strategies resulted in inhibitors with considerable improvement in biochemical potency against human lactate dehydrogenase A (LDHA). These potent inhibitors were typically selective for LDHA over LDHB isoform (4–10 fold) and other structurally related malate dehydrogenases, MDH1 and MDH2 (>500 fold). An X-ray crystal structure of enzymatically most potent molecule bound to LDHA revealed two additional interactions associated with enhanced biochemical potency.

Checkpoint kinase 1 (ChK1) is activated in response to DNA damage, acting to temporarily block cell cycle progression and allow for DNA repair. It is envisaged that inhibition of ChK1 will sensitize tumor cells to treatment with DNA-damaging therapies, and may enhance the therapeutic window. High throughput screening identified carboxylate-containing diarylpyrazines as a prominent hit series, but with limited biochemical potency and no cellular activity. Through a series of SAR investigations and X-ray crystallographic analysis the critical role of polar contacts with conserved waters in the kinase back pocket was established. Structure-based design, guided by in silico modeling, transformed the series to better satisfy these contacts and the novel 1,7-diazacarbazole class of inhibitors was discovered. Here we present the genesis of this novel series and the identification of GNE-783, a potent, selective and orally bioavailable inhibitor of ChK1.

Activating mutations within the epidermal growth factor receptor (EGFR) kinase domain, commonly L858R or deletions within exon 19, increase EGFR-driven cell proliferation and survival and are correlated with impressive responses to the EGFR inhibitors erlotinib and gefitinib in nonsmall cell lung cancer patients. Approximately 60% of acquired resistance to these agents is driven by a single secondary mutation within the EGFR kinase domain, specifically substitution of the gatekeeper residue threonine-790 with methionine (T790M). Due to dose-limiting toxicities associated with inhibition of wild-type EGFR (wtEGFR), we sought inhibitors of T790M-containing EGFR mutants with selectivity over wtEGFR. We describe the evolution of HTS hits derived from Jak2/Tyk2 inhibitors into selective EGFR inhibitors. X-ray crystal structures revealed two distinct binding modes and enabled the design of a selective series of novel diaminopyrimidine-based inhibitors with good potency against T790M-containing mutants of EGFR, high selectivity over wtEGFR, broad kinase selectivity, and desirable physicochemical properties.

Numerous oncogenic mutations occur within the BRAF kinase domain (BRAF(KD)). Here we show that stable BRAF-MEK1 complexes are enriched in BRAF(WT) and KRAS mutant (MT) cells but not in BRAF(MT) cells. The crystal structure of the BRAF(KD) in a complex with MEK1 reveals a face-to-face dimer sensitive to MEK1 phosphorylation but insensitive to BRAF dimerization. Structure-guided studies reveal that oncogenic BRAF mutations function by bypassing the requirement for BRAF dimerization for activity or weakening the interaction with MEK1. Finally, we show that conformation-specific BRAF inhibitors can sequester a dormant BRAF-MEK1 complex resulting in pathway inhibition. Taken together, these findings reveal a regulatory role for BRAF in the MAPK pathway independent of its kinase activity but dependent on interaction with MEK.

A novel class of 3-hydroxy-2-mercaptocyclohex-2-enone-containing inhibitors of human lactate dehydrogenase (LDH) was identified through a high-throughput screening approach. Biochemical and surface plasmon resonance experiments performed with a screening hit (LDHA IC50 = 1.7 μM) indicated that the compound specifically associated with human LDHA in a manner that required simultaneous binding of the NADH co-factor. Structural variation of this screening hit resulted in significant improvements in LDHA biochemical inhibition activity (best IC50 = 0.18 μM). Two crystal structures of optimized compounds bound to human LDHA were obtained and explained many of the observed structure–activity relationships. In addition, an optimized inhibitor exhibited good pharmacokinetic properties after oral administration to rats (F = 45%).

Checkpoint kinase 1 (ChK1) is a serine/threonine kinase that functions as a central mediator of the intra-S and G2–M cell-cycle checkpoints. Following DNA damage or replication stress, ChK1-mediated phosphorylation of downstream effectors delays cell-cycle progression so that the damaged genome can be repaired. As a therapeutic strategy, inhibition of ChK1 should potentiate the antitumor effect of chemotherapeutic agents by inactivating the postreplication checkpoint, causing premature entry into mitosis with damaged DNA resulting in mitotic catastrophe. Here, we describe the characterization of GNE-900, an ATP-competitive, selective, and orally bioavailable ChK1 inhibitor. In combination with chemotherapeutic agents, GNE-900 sustains ATR/ATM signaling, enhances DNA damage, and induces apoptotic cell death. The kinetics of checkpoint abrogation seems to be more rapid in p53-mutant cells, resulting in premature mitotic entry and/or accelerated cell death. Importantly, we show that GNE-900 has little single-agent activity in the absence of chemotherapy and does not grossly potentiate the cytotoxicity of gemcitabine in normal bone marrow cells. In vivo scheduling studies show that optimal administration of the ChK1 inhibitor requires a defined lag between gemcitabine and GNE-900 administration. On the refined combination treatment schedule, gemcitabine's antitumor activity against chemotolerant xenografts is significantly enhanced and dose-dependent exacerbation of DNA damage correlates with extent of tumor growth inhibition. In summary, we show that in vivo potentiation of gemcitabine activity is mechanism based, with optimal efficacy observed when S-phase arrest and release is followed by checkpoint abrogation with a ChK1 inhibitor. Mol Cancer Ther; 12(10); 1968–80. ©2013 AACR.

A 2-amino-5-aryl-pyrazine was identified as an inhibitor of human lactate dehydrogenase A (LDHA) via a biochemical screening campaign. Biochemical and biophysical experiments demonstrated that the compound specifically interacted with human LDHA. Structural variation of the screening hit resulted in improvements in LDHA biochemical inhibition and pharmacokinetic properties. A crystal structure of an improved compound bound to human LDHA was also obtained and it explained many of the observed structure-activity relationships.

A novel 2-thio-6-oxo-1,6-dihydropyrimidine-containing inhibitor of human lactate dehydrogenase (LDH) was identified by high-throughput screening (IC50=8.1 μM). Biochemical, surface plasmon resonance, and saturation transfer difference NMR experiments indicated that the compound specifically associated with human LDHA in a manner that required simultaneous binding of the NADH co-factor. Structural variation of the screening hit resulted in significant improvements in LDHA biochemical inhibition activity (best IC50=0.48 μM). A crystal structure of an optimized compound bound to human LDHA was obtained and explained many of the observed structure-activity relationships.

The single bromodomain of the closely related transcriptional regulators CBP/EP300 is a target of much recent interest in cancer and immune system regulation. A co-crystal structure of a ligand-efficient screening hit and the CBP bromodomain guided initial design targeting the LPF shelf, ZA loop, and acetylated lysine binding regions. Structure–activity relationship studies allowed us to identify a more potent analogue. Optimization of permeability and microsomal stability and subsequent improvement of mouse hepatocyte stability afforded 59 (GNE-272, TR-FRET IC50 = 0.02 μM, BRET IC50 = 0.41 μM, BRD4(1) IC50 = 13 μM) that retained the best balance of cell potency, selectivity, and in vivo PK. Compound 59 showed a marked antiproliferative effect in hematologic cancer cell lines and modulates MYC expression in vivo that corresponds with antitumor activity in an AML tumor model.

Inhibitors targeting the activating mutants of the epidermal growth factor receptor (EGFR) have found success in the treatment of EGFR mutant positive non-small-cell lung cancer. A secondary point mutation (T790M) in the inhibitor binding site has been linked to the acquired resistance against those first generation therapeutics. Herein, we describe the lead optimization of a series of reversible, pan-mutant (L858R, del746–750, T790M/L858R, and T790M/del746–750) EGFR inhibitors. By use of a noncovalent double mutant (T790M/L858R and T790M/del746–750) selective EGFR inhibitor (2) as a starting point, activities against the single mutants (L858R and del746–750) were introduced through a series of structure-guided modifications. The in vitro ADME-PK properties of the lead molecules were further optimized through a number of rational structural changes. The resulting inhibitor (21) exhibited excellent cellular activity against both the single and double mutants of EGFR, demonstrating target engagement in vivo...

The rapid advancement of a series of noncovalent inhibitors of T790M mutants of EGFR is discussed. The optimization of pyridone 1, a nonselective high-throughput screening hit, to potent molecules with high levels of selectivity over wtEGFR and the broader kinome is described herein.

The Ras, Raf, mitogen-activated protein kinase kinase (MEK) and extracellular signal-regulated kinase (ERK) signaling cascade is critically involved in cellular signaling with activating mutations in Ras and Raf present in many human tumors. Each constituent of this pathway is considered an important target for pharmaceutical intervention. The terminal kinase ERK is known to phosphorylate p90RSK among myriad substrates, yet robust plate-based high-throughput cellular assays monitoring such activity are not commercially available. In this study, we have utilized the Meso Scale Discovery platform to develop a plate-based assay to monitor the level of phosphorylation of p90RSK. This method is highly robust and can be used to evaluate a large number of inhibitors of ERK, MEK, or Raf in a variety of cellular backgrounds. Furthermore, this assay can be used to quantify the level of phospho-p90RSK in tumor lysates to function as a valuable pharmacodynamic readout.

Although effective in BRAF V600 mutant melanoma, Type 1.5 RAF inhibitors such as vemurafinib and dabrafenib have not proven to be successful in KRAS mutant cancers, neither as single agent nor in combination with MEK inhibitors. Through large-scale cellular compound combination screening we found that Type II RAF inhibitors such as AZ-628 do show synergistic activity with MEK inhibitors in multiple KRAS mutant indications, including NSCLC, colorectal cancer and ovarian cancer. The combination of Type II RAF inhibitors and MEK inhibitors demonstrates robust and durable abrogation of MAPK signaling both on canonical markers of MAPK activity such as pERK and pRSC as well as transcriptional output. We also observe synergistic in vivo tumor growth inhibition in two independent models of KRAS mutant cancer by this combination treatment. We found that treatment with MEK inhibitors alone drives the increase of active RAS-GTP levels and induces CRAF:BRAF hetero-dimerization. These induced dimers are active and able to phosphorylate MEK in vitro. This increased dimerization renders cells sensitive to Type 2 RAF inhibitors. We find that this effect is not limited to KRAS mutant cells, as a subset of KRAS wild-type cells show increased RAS-GTP levels upon MEK inhibitor treatment. These cells also show synergistic sensitivity to Type 2 RAF inhibition. Additionally, we observed significantly higher synergy and higher RAS-GTP levels in KRAS G13D mutant cells, which have intrinsically high GDP exchange and low intrinsic GTP hydrolysis. Finally, we show that GDC-0941 and GDC-0032, two broad PI3K inhibitors, also induce RAS-GTP levels in cells independent of PIK3CA or KRAS mutation status. We subsequently observed a synergistic sensitivity to Type 2 RAF inhibitors in these PI3K inhibitor-treated cells. Overall, we demonstrate that pharmacologic inhibition of MEK or PI3K increases RAS-GTP levels and drives increased CRAF:BRAF hetero-dimerization. This in turn sensitizes cells to Type 2 RAF inhibition, leading to a synergistic drug effect. Combination of these inhibitors may be a viable therapeutic approach in KRAS mutant cancer, and may be especially effective in KRAS G13D-carrying tumors. Citation Format: Christiaan N. Klijn, Ivana Yen, Frances Shanahan, Mark Merchant, Christine Orr, Thomas Hunsaker, Matthew Durk, Hank La, Xiaoling Zhang, Scott Martin, Eva Lin, John Chan, Yihong Yu, Dhara Amin, Amy Gustafson, Scott Foster, Joachim Rudolph, Shiva Malek. Pharmacologically induced RAS-GTP levels and CRAF-BRAF hetero-dimerization drive sensitization to Type II pan-RAF inhibitors in KRAS mutant cancer [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 874.

Activating mutations in KRAS and BRAF are found in more than 30% of all human tumours and 40% of melanoma, respectively, thus targeting this pathway could have broad therapeutic effects. Small molecule ATP-competitive RAF kinase inhibitors have potent antitumour effects on mutant BRAF(V600E) tumours but, in contrast to mitogen-activated protein kinase kinase (MEK) inhibitors, are not potent against RAS mutant tumour models, despite RAF functioning as a key effector downstream of RAS and upstream of MEK. Here we show that ATP-competitive RAF inhibitors have two opposing mechanisms of action depending on the cellular context. In BRAF(V600E) tumours, RAF inhibitors effectively block the mitogen-activated protein kinase (MAPK) signalling pathway and decrease tumour growth. Notably, in KRAS mutant and RAS/RAF wild-type tumours, RAF inhibitors activate the RAF-MEK-ERK pathway in a RAS-dependent manner, thus enhancing tumour growth in some xenograft models. Inhibitor binding activates wild-type RAF isoforms by inducing dimerization, membrane localization and interaction with RAS-GTP. These events occur independently of kinase inhibition and are, instead, linked to direct conformational effects of inhibitors on the RAF kinase domain. On the basis of these findings, we demonstrate that ATP-competitive kinase inhibitors can have opposing functions as inhibitors or activators of signalling pathways, depending on the cellular context. Furthermore, this work provides new insights into the therapeutic use of ATP-competitive RAF inhibitors.

The treatment of epidermal growth factor receptor (EGFR)-driven non-small cell lung cancers with the T790M resistance mutation remains a significant unmet medical need. We report the identification of 4-aminoindazolyl-dihydrofuro[3,4-d]pyrimidines as non-covalent inhibitors of EGFR, with excellent activity against the T790M resistance double mutants and initial single activating mutants. Using an optimization strategy focused on structure-based design and improving PK properties through metabolite identification, we obtained advanced leads with high oral exposure.

Because of their increased activity against activating mutants, first-generation epidermal growth factor receptor (EGFR) kinase inhibitors have had remarkable success in treating non-small-cell lung cancer (NSCLC) patients, but acquired resistance, through a secondary mutation of the gatekeeper residue, means that clinical responses only last for 8-14 months. Addressing this unmet medical need requires agents that can target both of the most common double mutants: T790M/L858R (TMLR) and T790M/del(746-750) (TMdel). Herein we describe how a noncovalent double mutant selective lead compound was optimized using a strategy focused on the structure-guided increase in potency without added lipophilicity or reduction of three-dimensional character. Following successive rounds of design and synthesis it was discovered that cis-fluoro substitution on 4-hydroxy- and 4-methoxypiperidinyl groups provided synergistic, substantial, and specific potency gain through direct interaction with the enzyme and/or effects on the proximal ligand oxygen atom. Further development of the fluorohydroxypiperidine series resulted in the identification of a pair of diastereomers that showed 50-fold enzyme and cell based selectivity for T790M mutants over wild-type EGFR (wtEGFR) in vitro and pathway knock-down in an in vivo xenograft model.

Metabolic reprogramming in tumors represents a potential therapeutic target. Herein we used shRNA depletion and a novel lactate dehydrogenase (LDHA) inhibitor, GNE-140, to probe the role of LDHA in tumor growth in vitro and in vivo. In MIA PaCa-2 human pancreatic cells, LDHA inhibition rapidly affected global metabolism, although cell death only occurred after 2 d of continuous LDHA inhibition. Pancreatic cell lines that utilize oxidative phosphorylation (OXPHOS) rather than glycolysis were inherently resistant to GNE-140, but could be resensitized to GNE-140 with the OXPHOS inhibitor phenformin. Acquired resistance to GNE-140 was driven by activation of the AMPK-mTOR-S6K signaling pathway, which led to increased OXPHOS, and inhibitors targeting this pathway could prevent resistance. Thus, combining an LDHA inhibitor with compounds targeting the mitochondrial or AMPK-S6K signaling axis may not only broaden the clinical utility of LDHA inhibitors beyond glycolytically dependent tumors but also reduce the emergence of resistance to LDHA inhibition.

Checkpoint kinase 1 (ChK1) plays a key role in the DNA damage response, facilitating cell-cycle arrest to provide sufficient time for lesion repair. This leads to the hypothesis that inhibition of ChK1 might enhance the effectiveness of DNA-damaging therapies in the treatment of cancer. Lead compound 1 (GNE-783), the prototype of the 1,7-diazacarbazole class of ChK1 inhibitors, was found to be a highly potent inhibitor of acetylcholine esterase (AChE) and unsuitable for development. A campaign of analogue synthesis established SAR delineating ChK1 and AChE activities and allowing identification of new leads with improved profiles. In silico docking using a model of AChE permitted rationalization of the observed SAR. Compounds 19 (GNE-900) and 30 (GNE-145) were identified as selective, orally bioavailable ChK1 inhibitors offering excellent in vitro potency with significantly reduced AChE activity. In combination with gemcitabine, these compounds demonstrate an in vivo pharmacodynamic effect and are efficacious in a mouse p53 mutant xenograft model.

While activating mutations in the KRAS oncogene frequently drive tumorigenesis in human cancers (40% CRC, 20% NSCLC) through constitutive activation of the MAPK pathway, there are currently no targeted treatments available for KRAS mutant cancers. Inhibitors of the individual nodes of the MAPK pathway have been developed, but these molecules have been largely ineffective against KRAS mutant tumors in the clinic. Multiple studies have shown rational combinations of MAPK inhibitors may have anti-tumor activity in KRAS mutant models. In order to understand the versatility of combining RAF inhibitors in this context, we conducted a library screen consisting of 430 small molecule tool compounds in combination with RAF inhibitor AZ-628. Here we show: RAF inhibitors combine especially well with other MAPK pathway inhibitors in KRAS mutant tumor cells. In particular, Type II RAF inhibitors are synergistic with the MEK inhibitor Cobimetinib in vitro and exhibit tumor regressing efficacy in xenograft mouse model studies in vivo. Mechanistically, we have found the MEK inhibitor disables ERK induced negative feedback on the MAPK pathway resulting in activation of CRAF in a KRAS dependent manner. The combination of RAF with MEK inhibition blunts KRAS-dependent activation of CRAF kinase activity and robustly inhibits MAPK signaling, thereby driving efficacy in KRAS mutant tumors. Broad cell line profiling with the combination of RAF and MEK inhibitors demonstrates that a majority of KRAS mutant lung and colorectal tumor lines exhibit synergy with the combination. Therefore, combining a Type II pan-RAF inhibitor with a MEK inhibitor has the potential to improve the therapeutic outcome in KRAS mutant cancers. Citation Format: Ivana Yen, Christiaan Klijn, Frances Shanahan, Mark Merchant, Christine Orr, Thomas Hunsaker, Matthew Durk, Hank La, Xiaolin Zhang, Scott Martin, Eva Lin, John Chan, Yihong Yu, Amy Gustafson, Joachim Rudolph, Shiva Malek. RAF kinase inhibition synergizes with MEK inhibitors in KRAS mutant tumors [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 4967. doi:10.1158/1538-7445.AM2017-4967

A series of 3,6-disubstituted dihydropyrones were identified as inhibitors of human lactate dehydrogenase (LDH)-A. Structure activity relationships were explored and a series of 6,6-spiro analogs led to improvements in LDHA potency (IC50

Kinase domain mutations are frequent drivers of many different types of cancer. While the effect of hotspot point mutations (such as BRAF V600E or EGFR L858R) is well described, the mechanism of deletion mutations, such as the recurrent EGFR exon 19 deletions is not fully understood. In this work, we have discovered and characterized analogous deletions in BRAF mutant patient samples of varying tumor types, with the highest frequency (15 patients) in pancreatic cancer representing ∼5% of KRAS wildtype patients in this data set. In addition, we have identified additional patients (3) with similar deletions in HER2 that have been previously reported at low frequency in breast cancer. The crystal structure of the most frequent BRAF deletion, molecular modeling of lower frequency BRAF deletions, and extensive molecular modeling in conjunction with molecular dynamic simulations of the most frequent EGFR deletion highlight the commonality of the mechanism of activation of this class of oncogenic alterations. These deletions truncate the loop between the β3 strand and αC-helix (β3-αC loop) of the kinase domain, forcing αC into the active (“in”) conformation, and constitutively activating these kinases. Similar to BRAF V600E mutations, constitutive activity of these BRAF β3-αC deletions is CRAF-independent and dimer-independent. Because these deletions genetically restrict the flexibility of this region of the kinase domain, they render these kinases resistant to inhibitors such as vemurafenib (for BRAF) or lapatinib (for EGFR and HER2) that bind in the αC “out” conformation. Functional characterization of the full spectrum of deletion lengths explains the high prevalence of 5 amino acid deletions in BRAF, EGFR, and HER2 in various cancers, with deletions of this length resulting in the strongest activation of kinase activity likely due to the optimal positioning of αC. This work exploits the selective power of oncogenic mutations to highlight a conserved mechanism of kinase activation and underscores the importance of conformation-specific kinase inhibitors to target mutationally activated kinases in cancer. Citation Format: Scott Foster, Dan Whalen, Aysegul Ozen, Matt Wongchenko, JianPing Yin, Ivana Yen, Gabriele Schaefer, John Mayfield, Juliann Chmielecki, Phil Stephens, Lee Albacker, Yibing Yan, Kyung Song, Georgia Hatzivassiliou, Charles Eigenbrot, Christine Yu, Andrey Shaw, Gerard Manning, Nicholas Skelton, Sarah Hymowitz, Shiva Malek. Activation mechanism of oncogenic C-helix shifting mutations in BRAF, EGFR, and HER2. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4374.

The Ras/RAF/MEK/ERK signaling pathway (MAPK pathway) plays a major role in growth factor-mediated cell proliferation and is frequently activated by mutation in human cancers. The precise mechanism of pathway activation is dependent on the underlying mutation. Thus, the recurrent B-RAFV600E mutation, which is found in more than 60% of melanomas, promotes MAPK pathway signaling independent of RAS activation whereas activating K-RAS mutations drive MAPK signaling through C-RAF activation. The hallmarks of C-RAF activation include B and C-RAF heterodimerization, membrane localization and C-RAF phosphorylation. In this work, we have discovered that in addition to its ability to activate the pathway through B-RAF/C-RAF dimerization, B-RAF suppresses the MAPK pathway through formation of a stable B-RAF/MEK complex in the cytoplasm of B-RAFWT and K-RAS mutant (K-RASMT) tumor lines. To understand the molecular mechanism of the B-RAF/MEK interaction, we have determined the crystal structure of a heterotetrameric B-RAF:MEK1 complex in which B-RAF and MEK1 form a face-to-face dimer with B-RAF side-to-side dimers at the heart of the complex. Structural and biochemical evidence illustrate that the molecular determinants for B-RAF enzymatic activity is distinct from those required for interaction with MEK. Our work also reveals the molecular basis for the paradoxical ability of both BRAF-activating and -inactivating oncogenic mutations to stimulate the MAPK pathway. Taken together, these findings reveal a novel regulatory role for B-RAF in the MAPK pathway that is independent of its catalytic activity but dependent on the conformation of the B-RAF kinase domain. Citation Format: Malek Shiva, Jacob R. Haling, Jawahar Sudhamsu, Tony Morales, Ariana Peck, Ivana Yen, Barbara Brandhuber, Sarah Hymowitz. A B-RAF-MEK complex reveals a kinase-independent role for BRAF in MAPK pathway suppression. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2346. doi:10.1158/1538-7445.AM2013-2346

Constitutive activation of the RAS, RAF, mitogen-activated protein kinase (MEK), and extracellular signal regulated kinase (ERK) pathway drives tumor cell proliferation and survival in many cancer types, and activating mutations in KRAS or BRAF occur in over 30% of human tumors. It has been demonstrated that BRAF inhibitors are effective selectively against BRAFV600E tumors and not RAS mutant tumors, despite BRAF functioning as a key effector downstream of RAS and upstream of MEK. Recent Phase I clinical data with RAF inhibitors has demonstrated a significant response rate (81%) in metastatic melanoma patients with BRAFV600E-positive tumors (N Engl J Med. 2010;363:809–19). Interestingly, in contrast to this remarkable response in melanoma, the response rates in BRAFV600E-positive colon tumors have been low in comparison (5.2%) (Kopetz et al., ASCO 2010). It has recently been reported that elevated EGFR levels in a subset of BRAFV600E lines render them resistant to BRAFV600E selective inhibitors (Prahallad et al., Nature. 2012; Corcoran et al., Cancer Discovery. 2012). However, the kinetics of EGFR pathway activation in response to inhibitors and underlying mechanisms of resistance are not entirely clear. In this study, we demonstrate that BRAFV600E/EGFR high tumors show a biphasic response to BRAF inhibitors, whereby the MAPK pathway is effectively blocked upon acute treatment, but subsequently rebounds and becomes resistant to inhibition. Components of the negative feedback loop downstream of the MAPK pathway including Sprouty, MKP3, and CDC25C are downregulated during acute treatment but rebound upon prolonged treatment, coincident with phospho-ERK suppression and subsequent reactivation. We show that this effect is not only dependent on high EGFR levels but also requires EGFR ligand stimulation that can be mediated through autocrine signaling. Resistance to this pathway is associated with increased RAS-GTP levels driving wild-type RAF activation upon extended treatment with B-RAF inhibitors. Importantly, immunohistochemical staining of tissue microarrays representative of both BRAFWT and BRAFV600E mutant CRC tumors with an EGFR antibody illustrates that the BRAFV600E mutation in colorectal cancer is associated with a significantly higher percentage of tumors carrying membrane EGFR compared to BRAFWT CRC tumors, with the median of membrane EGFR positive cells in BRAFV600E tumors 56.5% vs. 15% of cells in BRAFWT tumors, suggesting that activation of both pathways may be required for tumorigenesis in this tissue type. Finally, using both in vitro and in vivo models, we demonstrate that, in BRAFV600E-positive colon tumors with high EGFR pathway activity, inherent resistance to BRAF-selective inhibitors can be overcome by combination treatment with clinically relevant inhibitors of EGFR signaling, providing a potential treatment strategy for BRAFV600E/ EGFR high tumors. In summary, these results provide mechanistic insight into the crosstalk between BRAFV600E and EGFR signaling in CRC, shed light into the low response rate of BRAFV600E mutant colorectal cancer to BRAF inhibitors as single agents, and provide a rationale for the use of BRAF and EGFR inhibitors as combination therapy in this indication.

Constitutive activation of the RAS, RAF, mitogen-activated protein kinase (MEK), and extracellular signal regulated kinase (ERK) pathway is a common finding in many human cancers. Activating mutations in K-RAS or B-RAF constitute over 30% of all mutations in human tumors. It has been demonstrated that RAF inhibitors selectively inhibit B-RAFV600E tumors and not RAS mutant tumors, despite functioning as one of the key effector enzymes downstream of RAS and upstream of MEK. Recent Phase I clinical data with RAF inhibitors has demonstrated a significant response rate (81% response rate) in metastatic melanoma patients with B-RAFV600E-positive tumors (N Engl J Med. 2010 363:809-19). In contrast, the response rates in B-RAFV600E-positive colon tumors have been relatively low. In this work we examine the molecular mechanism of EGFR pathway activation as a mechanism of resistance by comparing the effects of RAF inhibitors against a panel of B-RAFV600E, mutant K-RAS, and WT RAS/RAF tumor lines in the presence and absence of EGF stimulation. We demonstrate that in B-RAF WT lines, pathway induction is observed for RAF inhibitors as evidenced by increases in phospho-MEK and phospho-ERK levels. RAF-inhibitor induced pathway activation in this setting mimics the kinetics and mechanism of EGF-induced pathway stimulation where RAS-GTP levels, C-RAF kinase activity and pERK levels are elevated. Upon stimulation with EGF or serum in B-RAF WT or K-RAS mutant lines, RAF inhibitor induced pathway activation is attenuated. In contrast in B-RAFV600E tumor lines, RAF inhibitors effectively block the MAPK pathway under basal conditions but become ineffective in a subset of B-RAFV600E lines when cells are stimulated with EGF. Additional studies across a broader panel of colon and melanoma mutant B-RAF lines demonstrate that B-RAFV600E lines with high basal levels of EGFR tend to be more resistant to selective B-RAFV600E inhibitors. Immunohistochemistry of colorectal tumor patient samples with either wildtype B-RAF or B-RAFV600E illustrates that colorectal B-RAFV600E tumors are associated with a higher percentage of membrane EGFR. Taken together, these results provide insight into the therapeutic utility of MAPK pathway inhibitors, and emphasize not only the importance of targeting defined genetic backgrounds in cancer treatment but the value in assessing other key pathway markers (such as EGFR) in B-RAFV600E-positive tumor subsets as a factor in sensitivity or resistance to therapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 258. doi:10.1158/1538-7445.AM2011-258

Activating mutations in KRAS or B-RAF are found in over 30% of all human tumors and targeting this pathway could have broad therapeutic impact. Small molecule ATP-competitive RAF kinase inhibitors have potent anti-tumor effects on B-RAFV600E tumors but, in contrast to MEK inhibitors, are not potent against RAS mutant tumor models, despite RAF functioning as a key effector downstream of RAS and upstream of MEK. In this study we show that ATP-competitive RAF inhibitors have two opposing mechanisms of action depending on cellular context. In B-RAFV600E tumors, RAF inhibitors effectively block the MAPK signaling pathway and decrease tumor growth. Surprisingly, in KRAS mutant and RAS/RAF wildtype tumors, RAF inhibitors activate the RAF/MEK/ERK pathway in a RAS-dependent manner, thus enhancing tumor growth in some xenograft models. Inhibitor binding activates wildtype RAF isoforms by inducing dimerization, membrane localization and interaction with RAS-GTP. These events occur independently of kinase inhibition and are, rather, linked to direct conformational effects of inhibitors on the RAF kinase domain. Based on these findings, we demonstrate that ATP-competitive kinase inhibitors can have opposing functions as inhibitors or activators of signaling pathways, depending upon cellular context. In addition, this work provides new insights into the therapeutic use of ATP-competitive RAF inhibitors. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5753.

The phosphatidylinositol-3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) signaling pathway plays a key role in the regulation of cellular growth, survival, and proliferation. Inappropriate activation of the pathway as a result of mutations and amplifications has been implicated in a variety of human cancers. Pathway dysregulation drives tumorigenesis through phosphorylation of proteins that directly regulate protein synthesis, cell cycle, and metabolism. The complexity of the mTOR and PI3K pathways, which include two functionally distinct mTOR complexes, feedback loops, and parallel regulatory pathways, clearly invokes a need for functional assays that can clarify the mechanism of action for drugs targeting mTOR and PI3K either as single agents or in combination. In this work, we used three different types of cell based assays to investigate the effects of PI3K versus mTORc1 inhibition on signaling pathways, cell cycle, and autophagy. The first set of assays differentiates an inhibitor of the mTorc1 complex versus the mTorc2 complex by utilizing two different cell lines and stimulation with either nutrient or growth factors. We show that the mTorc1 inhibitor, rapamycin, has potent activity in the nutrient stimulated mTorc1 assay, while PI3K inhibitors have very weak activity in such an assay. In the second type of assay, we used high content image analysis to produce cell cycle distribution profiles in PC3 cells, clearly showing G1 arrest for both mTOR and PI3K compounds as both single agents and in combination. However, the profiles showed no apoptotic population for either class of compounds in the cell lines tested. Therefore, as a means to investigate an alternative route of cell death to apoptosis, we also developed an autophagy assay using high content image analysis. Our results show that treatment of cells with a PI3K inhibitor or rapamycin as single agents or in combination can have different effects on autophagy and may potentially help differentiate the mechanism of action between the two classes of inhibitors. Taken together, we have generated a panel of mechanistic cellular assays which can help differentiate and characterize the effects of mTor and PI3K inhibition as both single agents and in combination. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):C66.