Peloton Therapeutics | Date: 2015-05-14
Benzimidazole derivatives of Formula I, that modulate the activity of ACSS2 are disclosed for therapeutic use. The fused imidazole ring of the compounds disclosed has a diarylmethyl or diarylmethanol moiety attached at the 2-position and the as compounds have at least one other substituent at the 5 or 6 position of the benzimidazole. Also disclosed are methods of using the benzimidazole compounds for the treatment of diseases or disorders, such as cancer.
News Article | November 2, 2016
Throughout the manuscript and figures, XP refers to the tumourgraft line; V refers to vehicle; S refers to sunitinib; and P refers to PT2399. Numbers following V, S, or P refer to the mouse identifier (ear tag) of that sample. Drug trials in tumourgraft-bearing mice were done as previously described12, 13. Briefly, ~64-mm3 fragments of tissue from stably growing orthotopic tumourgrafts were implanted subcutaneously in 4–6-week-old female and male non-obese diabetic (NOD)/severe combined immunodeficiency (SCID) mice. When tumour volumes reached ~300–600 mm3, mice were segregated into treatment groups (3–5 mice per group) based on (i) tumour volume, (ii) growth rate, and (iii) mouse weight. A sample size of five mice per treatment arm gave us 80% power to detect a significant tumour volume differential on the 28th day after treatment between the reference arm and a treatment arm using a two-sample t-test, assuming a true 600-mm3 tumour volume difference with a standard deviation of 250 and attrition margin of ~20%. Since the mixed model analysis uses about eight repeated measures from each mouse, even with a few more covariates included in the model, the power will be similar or even higher. Vehicle (10% EtOH, 30% PEG400, 60% MCT (0.5% methyl cellulose, 0.5% Tween 80 (aq))) was administered by gavage every 12 h. Sunitinib (LC Laboratories) was administered by oral gavage every 12 h at 10 mg kg−1 in 0.5% carboxymethylcellulose (CMC) in D5W. PT2399 (Peloton Therapeutics, Inc.) was administered at 100 mg kg−1 by oral gavage in 10% EtOH, 30% PEG400, 60% MCT. Mouse weights were taken weekly and treatment doses were adjusted weekly. Tumours were generally measured twice a week using a digital caliper. While leading to an overestimation in tumour volumes, to minimize bias12, tumour volume was calculated by the formula: tumour volume = l × w × h, where l is the largest dimension of the tumour, w is the largest diameter perpendicular to l, and h is maximal height of the tumour. Trials typically lasted 4 weeks, but this varied depending upon tumour growth rates. Overall, > 14,000 measurements were obtained. Assuming a digital caliper measurement error rate up to 10%, 99.8% of measurements were within protocol limits. Consideration was given to tumour growth rates, curve separation and the foreseeable need for additional mice for repeat experiments. Mice were monitored during treatment and provided appropriate veterinary care. In accordance with UT Southwestern’s Institutional Animal Care and Use Committee (IACUC) policies, animals were euthanized within timeframes specified by the veterinary staff once tumour diameters were greater than 2 cm. Mice were also euthanized if they exhibited signs of adverse clinical health. A total of n = 22 tumourgraft trials were completed with n = 89 vehicle-treated tumours (sensitive: n = 39; intermediate: n = 22; resistant: n = 28), n = 96 PT2399-treated tumours (sensitive: n = 42; intermediate: n = 24; resistant: n = 30), and n = 82 sunitinib-treated tumours (sensitive: n = 32; intermediate: n = 22; resistant: n = 28). Complete blood counts (CBC) (platelets, white blood cells, neutrophils, lymphocytes, and haemoglobin) were measured at the end of ~28-day trials and run on an IDEXX ProCyte Dx analyser. CBCs were available for 17 tumourgraft trials, with 52 vehicle-treated mice (sensitive: n = 10; intermediate: n = 21; resistant: n = 21), 58 PT2399-treated mice (sensitive: n = 13; intermediate: n = 19; resistant: n = 26), and 53 sunitinib-treated mice (sensitive: n = 8; intermediate: n = 22; resistant: n = 23). 3′-[18F]fluoro-3′-deoxythymidine (FLT) was synthesized at 160 °C for 10 min using a GE FXN module through the nucleophilic substitution reaction between 2,3′-anhydro-5′-O-benzoyl-2′-deoxythymidine and [18F]KF (potassium fluoride) in DMSO, followed by deprotecting the benzoyl group in 1N NaOH solution. The product was separated and purified by HPLC. The injection dose of FLT was prepared in saline containing 10% ethanol. Small animal PET/CT imaging studies were performed on a Siemens Inveon PET/CT Multimodality System. PET/CT scans were conducted on mice with both orthotopic and subcutaneous tumours. Orthotopic tumourgrafts were implanted using 2–3 pieces of 2 × 2 × 2-mm tissue underneath the left renal capsule of NOD/SCID mice. Once tumours became palpable, a baseline PET/CT scan was performed, and within 72 h, PT2399 treatment was started. PT2399 was continued for 8–10 days, after which a second PET/CT scan was performed to assess tumour response. After injection with 0.12 mCi of FLT via the tail vein, and a 60-min wait period to allow for the radiotracer’s distribution and uptake, mice were anaesthetized using 3% isoflurane, which was decreased to 2% during imaging. CT imaging was acquired at 80 kV and 500 μA with a focal spot of 58 μm. The PET imaging was acquired for 500 s directly following the acquisition of CT data. CT images were reconstructed with Cobra Reconstruction Software, and PET images were reconstructed using the OSEM3D algorithm. Reconstructed CT and PET images were fused and analysed using the manufacturer’s software. For quantification, regions of interest were drawn aided by CT images and then quantitatively expressed as per cent injected dose per gram of tissue (%ID/g). Immunohistochemistry (IHC) was performed using Dako Autostainer Link 48. The HIF-1α and HIF-2α immunohistochemical procedures and interpretations were standardized based on expression profiles in well-characterized cell lines (786-O, 786-O empty vector, and 786-O VHL-reconstituted cell lines) and human ccRCC tissue with known expression for these two proteins by western blot. Multiple commercially available antibodies were evaluated and the antibodies with most consistent results were selected for further studies. Briefly, for HIF-1α and HIF-2α staining, after hydration, antigen retrieval was accomplished with EnVision FLEX Target Retrieval Solution, Low pH (K800521, Dako) in a Dakocytomation Pascal pressure cooker; Ki67 and CD31 antigen retrieval was done using a Dako PT Link. Slides were incubated in 3% hydrogen peroxide for 10 min. Primary antibodies were added and incubated for 40 min at room temperature. Primary antibodies: HIF-1α (1:500, NB100-105, Novus), HIF-2α (1:200, sc-46691, Santa Cruz), Ki67 (ready-to-use, IR-626, Dako) and CD31 (1:200, LS-B1932, LifeSpan BioSciences). After rinsing with wash buffer, EnVision FLEX mouse/rabbit linker (K802121/K800921, Dako) was applied to the tissue and incubated for 10 min. Secondary antibody, EnVision FLEX/HRP (K800021, Dako), was incubated for 20 min. Sections were then processed using the Envision FLEX Substrate Working Solution for 10 min followed by dehydration in a standard ethanol–xylene series and mounting medium (8310-4, Thermo Scientific). IHC of HIF-1α and HIF-2α was performed on pre-treated tumourgraft tissue for n = 22 tumourgraft lines. Appropriate positive and negative controls were used with each run of immunostaining. The percentage of tumour cells in the entire section examined was recorded by a pathologist blinded to the western blot results. Only a 2 or 3+ nuclear positive reaction was considered as positive expression (staining scale: 0 = no staining, 1 = weak, 2 = moderate, 3 = strong). To assess tumour proliferation index, we performed immunostaining for Ki67, and to assess tumour microvessels, we performed CD31 immunostaining on tumours following treatment with vehicle or PT2399. IHC was completed on n = 10 sensitive tumourgraft lines, with n = 28 vehicle-treated tumours and n = 31 PT2399-treated tumours. Slides were digitally scanned using an Aperio Scanscope AT Turbo and reviewed using the Aperio eSlide Manager (ver. 22.214.171.12440) and Imagescope (ver. 126.96.36.19929) systems (Leica Biosystems). For Ki67, Aperio Genie (ver. 11.2) pattern recognition software was used to identify and select tumour areas for quantitative analysis with the Aperio Nuclear algorithm (version 11.2), yielding a percentage of tumour nuclei positive for Ki67. In a small subset of tumours where Genie inadequately identified tumour cells, representative tumour regions were manually selected (tumour necrosis areas were avoided) and reanalysed. Quantitative measurements of microvessels, including density and average lumen area, were obtained using the Aperio Microvessel algorithm (version 11.2) from manually selected representative tumour regions. RT–PCR data was generated for 16 tumourgraft trials, except for CA9 and LDHA, which were evaluated in 12 tumourgraft trials. Three RT–PCR reactions were run concurrently for each tumour. Total RNA was isolated as described previously31. cDNA was synthesized using iScript Reverse Transcription Supermix for RT–qPCR (170-8841, Bio-Rad). qRT–PCR was performed on a Bio-Rad CFX96 Real-Time PCR system using iTaq Universal SYBR Green SMX (1725124, Bio-Rad). Primers were synthesized by Invitrogen. Primers sequence available upon request. HIF2-I sensitive ccRCC tumourgrafts that had wild-type VHL status (XP164, XP373, XP453, and XP454) were tested for VHL methylation using the Affymetrix Promoter Methylation PCR Kit (MP1100). Tumour tissue was lysed in IP buffer containing 25 mM Tris-HCl pH 7.4, 150 mM NaCl, 1% NP-40, 1 mM EDTA, 5% glycerol, 0.5 mM DTT, with 3–4 freeze–thaw cycles. 10–20% of the lysate was saved for input; 40 μg was mixed with 3× loading buffer (10% SDS, 33.3% glycerol, 300 mM DDT, 0.2% bromophenol blue) for input. After pre-clearing the lysate with 50 μl of a 1:1 solution of recombinant protein G-sepharose 4B (101242, Life Technologies) for 1 h, 1 mg protein was mixed with 20 μl ARNT/HIF-1β antibody (sc-55526, Santa Cruz) and rocked overnight at 4 °C. 30 μl protein G-sepharose 4B equilibrated with IP buffer was then added, rocked for 1 h at 4 °C, and spun at 3,000 rpm for 10 s. The supernatant was removed and the beads washed three times with IP buffer containing DTT. 20 μl of 1× loading buffer was added to the beads and vortexed gently, then boiled for 5 min and spun at max speed for 5 min. The entire sample was loaded for western blot analysis. For western blot analysis, both HIF-1α antibody (A300-286A, Bethyl) and HIF-2α antibody (NB100-122, Novus) were diluted at 1:1,000 in 5% BSA in TBS and incubated overnight at 4 °C. Tubulin antibody (T5168, Sigma) was diluted at 1:5,000. Primary antibodies were detected using horseradish peroxidase-conjugated secondary antibodies (31430, 31460, Pierce) followed by exposure to enhanced chemiluminescence substrate (mixing 1:1 solution 1 (2.5 mM luminol, 0.4 mM pCoumaric acid, 0.1 M Tris-HCl) and solution 2 (0.015% H O , 0.1 M Tris-HCl)). Primer sequences are available upon request. HEK293T cells (ATCC; no perceived need for authentication; negative for mycoplasma) were cotransfected with the indicated expression plasmids using Lipofectamine 2000 (Invitrogen) following the manufacturer’s instructions. After 36 h, cells were treated with PT2399 (10 μM) at 37 °C for 5 h, harvested for immunoprecipitation with anti-FLAG beads (A2220-1ML, Sigma) and then subjected to western blot analysis. Plasmid laboratory database: #930 (pcDNA3.1 Flag-HIF1β), #931 (pcDNA3.1 Flag-HIF1β [F446L]), #932 (pLVX HA-HIF-2α-IRES-zsGreen), and #933 (pLVX-HA-HIF-2α [G323E]-IRES-zsGreen). The G323E and F446L mutations were evaluated using PyMOL and Protein Data Bank 4ZP4 (ref. 28). Mouse VEGF (MMV00), human VEGF (DVE00), and mouse erythropoietin (MEP00B) ELISA kits were from R&D Systems. Briefly, 50 μl assay diluent was added to each well. 50 μl of either standard, control, or sample was then added to a well. For mEPO and mVEGF ELISA, the serum was diluted twofold and fivefold, respectively, with calibrator diluent. For hVEGF, no dilution was performed. Plates were incubated for 2 h at room temperature on a horizontal orbital microplate shaker, then aspirated and washed for a total of five washes. After the last wash, 100 μl of mEPO/mVEGF/hVEGF conjugate was added to each well and incubated for 2 h at room temperature on a shaker. Plates were washed five times with wash buffer and 100 μl of substrate solution was added to each well and incubated for 30 min at room temperature, during which time the plates were covered to protect from the light. Stop solution was then added to each well, with gentle tapping to ensure thorough mixing. The optical density of each well was determined using a microplate reader set to 450 nm. Wavelength correction was set to 540 nm. The final optical density (OD) value was obtained by subtracting readings at 540 nm from the readings at 450 nm. ELISA data were generated for a total of 20 tumourgraft trials. Mouse anti-HIF-1α (NB100-105, Novus), mouse anti-HIF-2α (sc-46691X, Santa Cruz) and rabbit anti-ARNT/HIF-1β (A302-765A, Bethyl) were used. Primary antibodies were concentrated and buffers were exchanged using a Vivaspin 500 Centrifugal Concentrator (VS0131, Fisher Scientific). Antibodies were diluted to 1 mg ml−1 in PBS. Primary antibody conjugation was done with a Duolink In situ Probemaker MINUS/PLUS kit (DUO92010 & DUO92009, Sigma-Aldrich). Briefly, 2 μl of conjugation buffer was added to 20 μl of the antibody (1 mg ml−1), mixed gently, transferred to one vial of lyophilized oligonucleotide (PLUS or MINUS), and incubated at room temperature overnight. 2 μl of stop reagent was then added to the reaction and incubated at room temperature for 30 min. 24 μl of storage solution was added and the conjugation stored at 4 °C. Tumour tissue was blocked with PBS-T (0.1% Triton X-100) + 1% BSA for 30 min after antigen retrieval. Conjugated HIF1-α-MINUS, HIF2-α-MINUS and ARNT-PLUS were diluted in blocking buffer containing 1× assay reagent (20×) at a dilution of 1:50, 1:50, and 1:200, respectively. The mixture was allowed to sit for 20 min at room temperature before diluted primary antibody was added to each sample. Slides were incubated in a humidity chamber overnight at 4 °C. Duolink In situ Detection Reagents Red (DUO92008, Sigma-Aldrich) were used for signal detection. Briefly, slides were washed with wash buffer A, ligation solution containing ligase at a 1:40 dilution was added, and slides were incubated in a pre-heated humidity chamber for 30 min at 37 °C. After washing in 1× wash buffer A with gentle agitation, amplification solution containing polymerase was added at a 1:80 dilution, and slides were then incubated in a pre-heated humidity chamber for 100 min at 37 °C. After washing in 1× wash buffer B and then 0.01× wash buffer B, slides were dried at room temperature in the dark and mounted with a cover slip using a minimal volume of Duolink In situ Mounting Medium with DAPI (DUO82040, Sigma-Aldrich). After approximately 15 min, slides were analysed by fluorescence microscopy (Olympus) using a 40× objective. Image analysis was done with the ImageJ 1.48V program. Pictures of three fields for each sample were used. At least 100 cells of each sample were counted. 23 vehicle- and 23 PT2399-treated tumour RNA samples, including 5 sensitive XPs (XP144, XP164, XP373, XP374, and XP453) and 4 resistant XPs (XP169, XP296, XP490, and XP506), underwent RNA sequencing at the New York Genome Center. RNA sequencing libraries were prepared using the Illumina TruSeq Stranded mRNA Sample Preparation Kit. Briefly, 500 ng total RNA was purified by oligo-dT beads selecting for polyadenylated RNA species and fragmented by divalent cations under elevated temperature. The fragmented RNA underwent first strand synthesis using reverse transcriptase and random primers. Second strand synthesis created the cDNA fragments using DNA polymerase I. Following RNaseH treatment, the cDNA fragments went through end repair, adenylation of the 3′ ends, and ligation of adapters. The cDNA library was enriched using eight cycles of PCR and purified. Quality control consisted of assaying the final library size using the Agilent Bioanalyzer and quantifying the final library by RT–PCR and PicoGreen (fluorescence) methods. A single peak between 250 and 350 bp indicated a properly constructed and amplified library ready for sequencing. Sequencing was performed on a HiSeq 2500 using v4 SBS chemistry according to the Illumina protocol, as described32. Sequencing libraries were loaded onto the HiSeq 2500 flowcell for clustering on the cBot using the instrument-specific clustering protocol. Given HiSeq 2500 capabilities (200–250M passed filter 2 × 50-bp sequencing reads per flow cell lane), we sequenced 5 samples per lane in order to obtain a minimum of 50M PF reads per sample. With one exception, > 100 million reads were obtained per sample (median 146,644,355; 95% distribution-free CI: 142,380,928–151,324,826; Extended Data Table 1). Any gene with more than 50 reads in any sample was kept; only genes that had low reads in all of the samples were removed. This left 20,667 genes after removal of pseudogenes. cDNA sequences were aligned to a combined index of mouse and human reference sequences with STAR v 2.4.0c. Mouse reads were filtered out and the remaining reads were re-mapped to the NCBI hg37 using STAR aligner (v2.3.1z)33. Quantification of genes annotated in Gencode v19 was performed using HTSeq34. Picard and RSeQC35 were used to collect QC metrics (http://broadinstitute.github.io/picard/). Differential gene expression analysis was measured using edgeR36. A false discovery rate (FDR) cutoff of 0.05 was applied to identify the statistically significant genes between comparison groups. FDR was calculated using the Benjamini and Hochberg method37 for adjusting for multiple hypothesis testing. RNA-seq data were deposited into the Sequence Read Archive (SRP073253). For RNA-seq data, the tumourgraft number is preceded by an S for sensitive or R for resistant followed by treatment and ear tag. SRS1395449 (S144-P4340), SRS1395526 (S144-P4342), SRS1397028 (S164-P3281), SRS1397038 (S164-P3287), SRS1397048 (S164-P3297), SRS1397056 (S373-P4241), SRS1397057 (S373-P4244), SRS1397060 (S373-P4250), SRS1397059 (S374-P5172), SRS1397058 (S453-P5103), SRS1396986 (S453-P5104), SRS1396988 (S453-P5109), SRS1396987 (S144-V4352), SRS1396989 (S144-V4377), SRS1396991 (S164-V3290), SRS1396993 (S164-V3294), SRS1397021 (S164-V3298), SRS1397024 (S373-V4232), SRS1397025 (S373-V4236), SRS1397026 (S373-V4237), SRS1397027 (S374-V5170), SRS1397029 (S453-V5105), SRS1397031 (S453-V5107), SRS1397030 (S453-V5108), SRS1397032 (R169-P5231), SRS1397033 (R169-P5240), SRS1397034 (R169-P5241), SRS1397035 (R296-P4512), SRS1397036 (R296-P4531), SRS1397037 (R490-P3207), SRS1397039 (R490-P3210), SRS1397040 (R490-P3214), SRS1397042 (R506-P4734), SRS1397041 (R506-P4735), SRS1397043 (R506-P4736), SRS1397044 (R169-V5230), SRS1397045 (R169-V5235), SRS1397046 (R169-V5239), SRS1397047 (R296-V4519), SRS1397049 (R296-V4524), SRS1397050 (R490-V3211), SRS1397052 (R490-V3218), SRS1397051 (R490-V3224), SRS1397053 (R506-V4743), SRS1397054 (R506-V4745), SRS1397055 (R506-V4777). Apart from the RNA-seq analysis, all reported P values were obtained from two-tailed tests at the 0.05 significance level. All bar charts depict the mean with the error bar representing s.e.m., while all boxplots have median centre values with fences extending to the greatest value inside the upper and lower fences (1.5[IQR] away from the 75th and 25th percentiles, respectively). Transformations were used where indicated to meet normality assumptions for analysis. These tests were completed using SAS 9.4 (SAS Institute Inc.). Except where indicated, the experiments were not randomized and the investigators were not blinded to allocation during experiments and outcome assessment. Written informed consent was obtained from the patient participating in the phase 1 clinical trial ‘A Phase 1, Dose-Escalation Trial of PT2385 Tablets In Patients With Advanced Clear Cell Renal Cell Carcinoma’ (NCT02293980). UT Southwestern IACUC-approved animal protocol, APN 2015-100932, includes all live vertebrate experimental procedures.
News Article | February 23, 2017
Like many living things, a cancer cell cannot survive without oxygen. When young and tiny, a malignancy nestles inside a bed of blood vessels that keep it fed. As the mass grows, however, its demand for oxygen outpaces supply. Pockets within the tumor become deprived and send emergency signals for new vessel growth, a process called angiogenesis. In the 1990s, a popular cancer-fighting theory proposed interfering with angiogenesis to starve tumors to death. One magazine writer in 2000 called the strategy “the most important single insight about cancer of the past 50 years.” It made such intuitive sense. Rakesh Jain viewed angiogenesis through a different lens. Trained as an engineer, not a biologist, Jain was studying tumor vasculature during the height of excitement about drugs that could impede vessel growth. He was bothered by the fact that capillaries that arise in the tumor aren’t normal; they’re gnarled and porous, incapable of effective blood flow in the same way a leaky pipe is lousy at delivering water. The expanding tumor squeezes smaller vessels, making them even less able to transport blood. “The mantra was, ‘Let’s starve tumors,’ ” recalls Jain, director of the Edwin L. Steele Laboratories for Tumor Biology at Harvard Medical School. “I said, ‘No, we need to do the opposite.’ ” In 2001, he published a commentary in Nature Medicine predicting that angiogenesis inhibitors would not entirely shrivel the tumor. Instead, he argued, starving tumors might make them harder to treat. “I was sticking my neck out and saying this is not a good thing to do,” he says. “I had tremendous resistance.” Time has proved him right. Once they came on the market, anti-angiogenesis drugs were not the boon doctors had hoped for. Most disturbing, some patients saw their tumors shrink, only to have the disease return with renewed vengeance. Today, more than a decade after the introduction of the first tumor-starving drug, researchers have a far greater understanding of the role of oxygen deprivation in cancer. Instead of slowing tumors, hypoxia appears to trigger a metabolic panic that can drive growth, drug resistance and metastasis. Rescue proteins called hypoxia-inducible factors, or HIFs, open a bag of tricks so tumors can adapt and outrun the body’s defenses. But there’s now reason for hope: Recent insights into the effects of oxygen deprivation in cancer are sparking new ideas and providing the blueprint for treatments that could short-circuit a cancer’s ability to survive and spread, and help make existing drugs more effective. While the idea of starving cancer made sense, the approach may have underestimated the strength and complexity of a tumor’s resilience. Since oxygen is essential for so much of life, nature equips cells with elaborate safeguards that kick in when the oxygen-rich blood supply dwindles — whether the cells are part of a tumor or part of a muscle straining for one last push of strength. When oxygen levels drop, newly minted proteins stampede throughout the cell, turning on a frenzy of chemical reactions that offer protection from the crisis. Cancer cells distort this natural coping mechanism for their own means. Growing new vessels is just one move in an elaborate strategy. Many changes accompany hypoxia, including: The malignant cells loosen from each other and become less adhesive, ready to break free; tendrils of collagen, a natural binding substance, form and start to reach out to nearby vessels; and proteins appear on the cell surface to pump out lactic acid, a product of the tumor’s switch from primarily aerobic to anaerobic respiration. Researchers now think stopping enough of these and other changes could cripple the cancer. Much of the research focuses on the proteins that are among the first to deploy when a cell senses a danger of asphyxiation. “At zero oxygen, the cell can’t survive,” says Daniele Gilkes of Johns Hopkins University School of Medicine. “Inside a tumor you will see these regions of necrosis,” or dead cells. But those cells that are low on oxygen but still alive will produce new proteins: Key among them are HIF-1 and HIF-2. Both are transcription factors — they help transcribe DNA instructions into RNA. Under normal conditions, the genes that make HIF proteins are mostly silent. Once HIF proteins are made, they turn on genes — Gilkes estimates there are hundreds — that enable cells to live when oxygen concentrations are low. Gilkes’ target of choice is HIF-1. It is not only a first responder, but the protein also appears to be key to cancer’s spread. Tumors with high levels of HIF-1, particularly when concentrated at the invasive outer edge of the mass, are more likely to become metastatic, invading other parts of the body. The reverse is also true: Human tumors transplanted into mice that genetically can’t produce HIF-1 are less likely to spread. The reasons are complicated, Gilkes says, but she considers one thing really interesting: HIF-1 is involved with a lot of enzymes in collagen formation. The collagen appears to provide a means of escape. Last year, in a review in the International Journal of Molecular Sciences, Gilkes described genes, found by her lab group and others, that breast tumors activate to degrade the surrounding environment. In turn, the tumor wraps itself in a stringy web of collagen. As the collagen forms, the strands stretch outward from the tumor and latch onto nearby vessels. “We think cancer cells will find this collagen and use it to migrate and glide.” She calls them “collagen highways.” Her laboratory captured video of human tumor cells migrating along a fibrous strand. “To see them move is really scary.” Once they’ve broken from their home tumor, many types of cancer, including prostate and breast cancers, commonly move into bones. This is no coincidence, Gilkes says. Bones lack the dense thickets of blood vessels that run through soft tissues. That means cancer cells migrating from a hypoxic environment, and therefore already trained for low oxygen, would find hospitable surroundings in the bone. Her lab group is now looking for ways to block collagen formation to close the travel lanes and perhaps keep the cancer from spreading. She and others are also working to find a way to inhibit HIF-1 directly, but so far those efforts have proved challenging. HIF-1’s accomplice, HIF-2, may be a more available target. HIF-2 is a molecule made of two parts that clamp onto DNA to trigger production of other proteins that make tumors tougher to kill. In 2009, structural biologists at University of Texas Southwestern Medical Center in Dallas discovered that the HIF-2 protein had a large cavity. “Usually proteins don’t have holes inside them,” says James Brugarolas, leader of UT Southwestern’s kidney cancer program. With the discovery, researchers began working on a way to use the gap as a foothold for drugs. Now in development, the experimental drug PT2399 slips inside HIF-2 and effectively breaks the molecule in two. Brugarolas and colleagues from six other institutions and the biotech company Peloton Therapeutics Inc. in Dallas published results of the first animal tests of the compound in Nature in November. In mice with implanted grafts of human kidney tumors, PT2399 split HIF-2 and slowed growth in 56 percent of tumors — better than a standard treatment. Brugarolas hypothesizes that the drug worked only about half the time because the other half of tumors relied more heavily on HIF-1. A similar HIF-2–busting drug is now in Phase I safety testing in humans, described in June in Chicago at the annual meeting of the American Society of Clinical Oncology. While Phase I studies are not designed to test whether the treatment works, the drug showed few side effects among 51 people with advanced kidney cancer who took the drug at ever-increasing doses. The patients had already been through multiple types of treatments, one as many as seven. After taking the drug, 16 patients experienced a slowing in disease progression, three more had a partial response and one a complete reversal. Given the dearth of treatments for advanced kidney cancer, Brugarolas says, “this is a big deal.” Still more molecules throw a lifeline to hypoxic tumors in ways that scientists are just beginning to understand. In 2008, pathologist David Cheresh and colleagues at the University of California, San Diego announced a curious discovery in Nature: Depriving cells of vascular endothelial growth factor, or VEGF — the key protein responsible for new vessel growth in a tumor and the main target of drugs that block angiogenesis — could actually make tumors more aggressive. His team went on to discover the same was true for another popular class of drugs, which work by depriving a tumor cell of nutrients in the same way anti-angiogenesis drugs limit oxygen. The drugs, called EGFR inhibitors, were capable of doing the opposite of what was expected: They could make tumors stronger. Cheresh believes that hypoxia — and other stresses of low blood supply, like nutrient deprivation — inflict a wound on the tumor. When normal tissues sustain an injury (like a cut), they immediately enter a period of healing and regeneration. The bleeding stops and the skin grows back. Low oxygen delivers a blow to tumor cells, sending them into a similar state of rejuvenation, he says. “They’re now prepared to survive not only the hypoxia, but everything else thrown at it.” In 2014, Cheresh published his take on why this occurs, at least in some cases, in Nature Cell Biology. He and his team described a molecule called avb3 found on the surface of drug-resistant tumors that appears to reprogram tumor cells into a stem cell–like state. As embers of the original tumor that are often impermeable to treatment, these stem cell–like cells can lie quietly for a time and then reignite. The discovery of avb3 has redefined how Cheresh thinks about resistance. He no longer believes that tumors defy chemotherapy in the way bacteria overcome antibiotics, with only the strongest cells surviving and then roaring back to become dominant. “The tumor cells are adapting, changing in real time,” Cheresh says. In short, his data suggest that when EGFR inhibitors deprive a cell of nutrients, some cells survive not because they are naturally tougher, but because the appearance of avb3 transforms them into drug-resistant stem cells. The good news is that laboratory tests suggest an experimental drug might block this reprogramming, and it may even prevent chemotherapy resistance. A clinical trial will soon begin that combines usual cancer treatment with this avb3-disabling drug, in a one-two punch aimed at reversing or delaying resistance so the treatment can do its job. There are still more ways tumors withstand low oxygen. They start eating leftovers. HIF-1 triggers a switch from oxygen-based aerobic respiration to anaerobic respiration using pyruvate, a product of glucose breaking down. The strategy works in the short term; it’s the reason your muscles keep pumping for a time, even when you’re gasping for air on the last few yards of a sprint. Problem is, anaerobic respiration leaves a trail of lactic acid. A lot of it. “Lactic acid buildup leads to a precipitous drop in pH inside of the tumor,” says Shoukat Dedhar of the BC Cancer Research Centre in Vancouver. To compensate, HIF-1 deploys a fleet of proteins that remove the acid so it won’t accumulate and burn up the cell. Dedhar’s laboratory didn’t start out studying hypoxia. “We had tumors that were readily metastatic and genetically related tumors that couldn’t metastasize,” he says. Those tumors that easily spread were producing HIF-1, along with products from other genes. Searching for the functions of those genes, his group and others found two proteins important in pH balance. The first, MCT-4, acts like a molecular sump pump, bailing out lactic acid. But it’s not enough to normalize the pH, Dedhar says. That job goes to the second protein, carbonic anhydrase 9, or CAIX. “Its job is simply to convert carbon dioxide to bicarbonate, which then neutralizes the acid,” he says. In March 2016, in a review in Frontiers in Cell and Developmental Biology, Dedhar and colleagues described how to improve cancer treatment by taking away some of the tools for hypoxia survival — that is, keeping the cell from neutralizing acid — while simultaneously giving drugs that boost the immune system. His team has developed new compounds that specifically block CAIX. Since CAIX is almost exclusively produced in tumor cells, CAIX inhibitors should theoretically have few side effects. A Phase I safety trial is testing possible drugs now. Harvard’s Jain is still making the case for bathing the tumors in oxygen, giving them more blood, not less. This could keep the tumor from becoming hypoxic and throwing up a new series of defenses, including a flood of angiogenesis-promoting proteins, which produce tormented circulation. When he proposed that concept in 2001, “I thought abnormal vessels were bad,” he says. “I now think they are worse.” His idea is to make tumor vasculature more normal, using the very drugs that he was concerned about almost two decades ago. His research suggests that giving anti-angiogenesis drugs in modest doses will keep the vessels from becoming abnormal, making them less tortured and more capable of normal blood flow (SN: 10/5/13, p. 20). He believes the restored oxygen not only shuts down the hypoxic response that gives the cancer a survival advantage, but also serves as a conduit for chemotherapy drugs and immune cells to penetrate deeper into the tumor. Oxygen is also necessary for radiation to work. His latest experiments take the concept of more oxygen, not less, even further. He combined two chemotherapy drugs with losartan, a generic medicine used to control blood pressure. The result, reported in Nature Communications in 2013, was a delay in pancreatic and breast tumor growth in mice. Another experiment from Jain and colleagues, published in 2016 in Translational Oncology, had similar results. “We are finding every therapy works better when we do this,” he says. A clinical trial is now under way at Massachusetts General Hospital testing whether giving losartan during radiation and chemotherapy will improve results for pancreatic cancer patients. The concept still remains unproven, but Jain has reason for optimism. And he is no longer in the scientific minority. Last May, he received the National Medal of Science from President Barack Obama, who commended Jain for “groundbreaking discoveries of principles leading to the development and novel use of drugs for treatment of cancer.” Jain hopes to see the day, not long in the future, when hypoxic tumors are defeated by giving them the very thing they need the most. This article appears in the March 4, 2017, issue of Science News with the headline, "Deflating cancer: New approaches to low oxygen may thwart tumors."
News Article | December 20, 2016
REDWOOD CITY, Calif.--(BUSINESS WIRE)--REVOLUTION Medicines, Inc., a company focused on frontier cancer targets and drug discovery inspired by nature’s lessons, today announced that it has raised $25 million in a Series A extension financing. The Column Group, a leading life sciences investment firm focused on science-driven companies, joined founding investor, Third Rock Ventures, in the ongoing funding of the company. Proceeds from this expanded round will support the company as it continues advancing its discovery programs toward clinical development. Larry Lasky, Ph.D., partner at The Column Group, has joined the company’s Board of Directors. “This financing brings a second top-tier group to REVOLUTION Medicines and reflects the excellent progress the company has made in advancing multiple oncology drug discovery programs,” said Mark A. Goldsmith, M.D., Ph.D., president and chief executive officer of REVOLUTION Medicines. “I am excited that Dr. Lasky has joined our board of directors and look forward to working with him. His extensive background and track record in oncology drug discovery, coupled with the recent expansion of our founder and senior advisor groups that contributes additional oncology expertise, mark our deepening commitment to bringing innovative drugs forward for cancer patients.” Dr. Lasky brings over 30 years of experience in the biotechnology industry to REVOLUTION Medicines. From 2008-2014, Dr. Lasky was a partner at US Venture Partners, and from 2002-2008, he served as general partner at Latterell Venture Partners. Prior to that, Dr. Lasky was a scientist at Genentech for 20 years, where he worked in various disciplines including vaccinology, immunology, stem cell biology, cellular signaling mechanisms and monoclonal antibody therapy of tumors, and was also named Genentech Fellow, the company’s highest scientific position. Dr. Lasky has been a lecturer at the University of California, Berkeley’s Haas School of Business and currently sits on the boards of Carmot Therapeutics, eFFECTOR Therapeutics, Ribon Therapeutics, ORIC Pharmaceuticals, Peloton Therapeutics and OncoMed Pharmaceuticals, Inc. (NASDAQ:OMED). “There remain many unserved needs in oncology, including continued demand for new drugs directed to emerging targets in cancer cells and the immune system,” said Dr. Lasky. “REVOLUTION Medicines is taking a truly innovative approach to drug discovery through a special combination of biology and chemistry seen through the lens of nature. I look forward to helping the company realize the full potential of its product engine and pipeline.” The mission of REVOLUTION Medicines is to discover and develop new drugs directed toward frontier oncology targets for cancer patients. The company draws inspiration from nature’s lessons including natural products that are inherently rich with biological function. REVOLUTION Medicines deploys an innovative toolkit including REVBLOCKS™, an integrated suite of modular synthesis methodologies applied to simple chemical “building blocks,” and the REVEAL™ computational platform, which uses evolution’s lessons to inform selection of chemical scaffolds and guide drug design for non-classical drug targets. Headquartered in Redwood City, Calif. at the intersection of Silicon Valley and the birthplace of biotechnology, REVOLUTION Medicines is a private company financed by top-tier investors Third Rock Ventures and The Column Group. For more information, please visit www.revolutionmedicines.com.
Kwon I.,University of Texas Southwestern Medical Center |
Kato M.,University of Texas Southwestern Medical Center |
Xiang S.,University of Texas Southwestern Medical Center |
Wu L.,University of Texas Southwestern Medical Center |
And 8 more authors.
Cell | Year: 2013
The low-complexity (LC) domains of the products of the fused in sarcoma (FUS), Ewings sarcoma (EWS), and TAF15 genes are translocated onto a variety of different DNA-binding domains and thereby assist in driving the formation of cancerous cells. In the context of the translocated fusion proteins, these LC sequences function as transcriptional activation domains. Here, we show that polymeric fibers formed from these LC domains directly bind the C-terminal domain (CTD) of RNA polymerase II in a manner reversible by phosphorylation of the iterated, heptad repeats of the CTD. Mutational analysis indicates that the degree of binding between the CTD and the LC domain polymers correlates with the strength of transcriptional activation. These studies offer a simple means of conceptualizing how RNA polymerase II is recruited to active genes in its unphosphorylated state and released for elongation following phosphorylation of the CTD. PaperFlick © 2013 Elsevier Inc.
Peloton Therapeutics | Date: 2016-06-08
Compounds comprising a fused tricylic core that modulate HIF-2 activity, pharmaceutical compositions containing these chemical entities, and methods of using these chemical entities for treating diseases associated with HIF-2 activity are described herein.
Peloton Therapeutics | Date: 2014-09-05
The present disclosure relates to HIF-2 inhibitors and methods of making and using them for treating cancer. Certain compounds were potent in HIF-2 scintillation proximity assay, luciferase assay, and VEGF ELISA assay, and led to tumor size reduction and regression in 786-O xenograft bearing mice in vivo.
News Article | April 2, 2015
Life science industry leaders and Dotmatics' innovators to introduce the future of scientific informatics at two UGMs in Japan Dotmatics, the leading provider of scientific informatics solutions and services to the pharmaceutical, biotechnology and chemical industries, announced today that after the success of previous User Group Meetings across North America and Europe, the event is to be expanded in Japan in May 2015. The objective of the User Group Meetings is to bring the scientific informatics community together to exchange ideas and information. Both events are free to attend, open to all and will include the discussion of a wide range of technical topics including upgrading from obsolete research and development informatics platforms, implementing a secure collaborative infrastructure, and managing and analysing chemicals and biologicals. The UGMs will take place on 13th May at the Tokyo Conference Center Shinagawa and on the 15th May at the Hyatt Regency Osaka. Expert users of Dotmatics' solutions will showcase their data and knowledge management practices and implementation as well as their use of the Dotmatics Platform, including Studies Notebook, Vortex, Browser, Register and BioRegister, among other modules. Presenters include scientists from the life science industry including Cancer Research UK - Beatson Institute, Peloton Therapeutics and IRBM as well as scientific leaders from Dotmatics. Dotmatics' own scientists will share the platform technicalities, the company strategy and provide tactics for upgrading outdated informatics infrastructures. "We would like to take this opportunity to invite the esteemed Japanese scientific community to our User Group Meetings which will formally introduce our Informatics Platform in Japan," said Richard Lingard, Senior Vice President of Global Commercial Operations at Dotmatics. "We deliver an established, modern and scalable informatics solution that is simple to adopt and easy to migrate to from old technologies." The event presents a unique opportunity for attending scientists to discuss various approaches to working in collaborative and dynamic environments. Dotmatics invites researchers interested in scientific informatics data management and analysis to register today at: http://www.dotmatics.com/events-list/ Dotmatics is a leading global scientific informatics software and services provider, delivering solutions tailored to the modern, highly collaborative and mobile scientific environments. The company provides solutions to several vertical markets, including the pharmaceutical, biotechnology, academia, food and beverage, oil and gas and agrochemical industries. Dotmatics' enterprise solutions are flexible, scalable and configurable, providing effective scientific information management across entire organisations, from discovery research to development and early manufacturing. Dotmatics has significant expertise in scientific informatics, including database management for chemistry and biologics, electronic laboratory notebooks, chemical and biological registration, screening data management, SAR analysis, reporting, and visualisation. Dotmatics solutions are available for local or cloud deployment and they are supported on Microsoft Windows, Mac OS X and Linux. A privately owned company, Dotmatics was founded in 2005 and their headquarters is based south of Cambridge in the UK.
News Article | April 19, 2015
DALLAS & PHILADELPHIA--(BUSINESS WIRE)--Peloton Therapeutics, Inc., a drug discovery and development company focused on advancing first-in-class, small molecule cancer therapies targeting unexploited molecular vulnerabilities, presented preclinical data on its lead investigational candidate, PT2385, at the American Association for Cancer Research Annual Meeting in Philadelphia, PA. PT2385 is the first clinical stage antagonist of hypoxia inducible factor-2α (HIF-2α), a transcription factor implicated in the development and progression of renal cancer. “HIF-2α can act as a tumorigenic driver in cancer. As a transcription factor, HIF-2α has historically been seen by the scientific community as impossible to directly target,” said Eli Wallace, Ph.D., Vice President of Chemistry for Peloton. “Our preclinical evidence indicates that PT2385 is potent, selective, and readily absorbed. We believe this program has the potential to become a significant therapy for renal cancer.” PT2385 is currently being investigated in a Phase 1 clinical trial for the treatment of advanced or metastatic clear cell renal cell carcinoma (ccRCC). Loss of the von Hippel-Lindau tumor suppressor (VHL) is the key oncogenic event in up to 95% of patients with ccRCC. With the loss of the VHL protein (pVHL), the transcription factor HIF-2α accumulates and drives the unbalanced expression of numerous gene products. Preclinical data indicate that orally bioavailable PT2385 disrupts HIF-2α activity in ccRCC and thereby blocks the expression of multiple tumorigenic factors responsible for unrestrained cancer cell growth and proliferation, tumor angiogenesis, and suppression of anti-tumor immune responses characteristic of ccRCC. “Loss of VHL, and resulting activation of HIF-2α, is the signature driving event in clear cell renal cell carcinoma but HIF-2α had been largely dismissed as ‘undruggable,’ which is one reason the potential of PT2385 is so exciting,” remarked William G. Kaelin, Jr., M.D., Professor in the Department of Medicine at the Dana-Farber Cancer Institute, Harvard Medical School, a scientific advisor to Peloton, and a noted expert on VHL and hypoxia inducible factors. “PT2385 is the first molecule to advance to the clinic that binds directly and specifically to HIF-2α and potently inhibits its transcriptional activity.” The American Cancer Society estimates that more than 61,000 new cases of kidney cancer will be diagnosed and about 14,000 people will die from this disease this year. The National Cancer Institute reports that the prognosis for any treated renal cell cancer patient with progressing, recurring, or relapsing disease is poor, regardless of cell type or stage. Peloton Therapeutics is a drug discovery and development company focused on advancing first-in-class small molecule therapies for cancer and other life-threatening diseases. Initial efforts are focused on hypoxia inducible factor-2α (HIF-2α), which has been implicated in the development and progression of several types of cancers. Peloton’s lead candidate, PT2385, is the first HIF-2α inhibitor to enter clinical development and is being evaluated in a Phase 1 clinical trial for the treatment of advanced or metastatic clear cell renal cell carcinoma (ccRCC). Peloton is headquartered in Dallas, TX and was founded in 2011 by Professor Steven McKnight and The Column Group. The company raised more than $30 million in a Series C financing in October 2014 to fund its development activities for PT2385 as well as advance its research pipeline. Peloton is backed by The Column Group, Remeditex Ventures, Tichenor Ventures, Topspin Fund, Nextech Invest, and other investors. For more information, please visit www.pelotontherapeutics.com.