Supplementary Figures 1-6 from Antitumor and Antiangiogenic Activities of BMS-690514, an Inhibitor of Human EGF and VEGF Receptor Kinase Families

Figure S1. Potency of I-BET762 in inhibition of proliferation of lung tumor cell lines. Lineage of lung cell lines is as indicated. Figure S2. Dose response curves of JQ1 in cell proliferation assays. Shown are examples of lung tumor cell lines with different sensitivity to JQ1. Figure S3. Induction of apoptosis in four SCLC cell lines was monitored by caspase 3 and 7 cleavage after 72 h treatment with JQ1. Data shown are fold changes over DMSO control. Figure S4. Probe sets induced by JQ1 in SCLC cell lines. Shown are microarray data for three probe sets that were induced by JQ1. Figure S5. Examples of probe sets that show dose-dependent changes, but were expressed only at background levels (RNF183) in all four cell lines, or were expressed at low levels in the sensitive cell lines (NR0B2). Figure S6. Dose response in expression of the three ASCL1 probe sets upon JQ1 treatment. Figure S7. (A) Enrichment of BRD4 binding at the ASCL1 gene enhancer compared to a gene desert on the same chromosome. Primers specific for ASCL1 enhancer or a gene desert on chromosome 12 were used to detect BRD4 binding. (B) JQ1 has no effect on non-specific binding of BRD4 to a gene desert in SCLC cell lines. Figure S8. (A) ASCL1 mRNA in SCLC and non-small-cell lung neuroendocrine (UMC-11 and NCI-H1155) cell lines. JQ1 sensitivity cut-off is arbitrarily defined as an IC50 of 0.5 µM. (B) ASCL1 protein abundance in SCLC cell lines that are sensitive or resistant to JQ1. (C) UMC-11 cells were treated with JQ1 for 24h and ASCL1 protein was analyzed by western blotting.

Microarray data for the 5 probe sets that show differential responses to JQ1 treatment. Shown are the Affymetrix RMA anti-log values of probe intensity with each treatment, together with the fold change in signal from JQ1 treatment and the JQ1 concentrations needed to achieve 50% increase or decrease in signal (EC50).

Supplementary Tables 1-3, Figure Legends 1-6 from Antitumor and Antiangiogenic Activities of BMS-690514, an Inhibitor of Human EGF and VEGF Receptor Kinase Families

Legend for Supplementary Tables 1 and 2 and Supplementary Figures 1 through 8

Supplementary Information and Figures 1-3 from Identification of Candidate Molecular Markers Predicting Sensitivity in Solid Tumors to Dasatinib: Rationale for Patient Selection

Supplementary Figures S1-S5. Supplementary Figure S1. Similarity of CNV profiles are seen from both CRC cell lines and CRC primary tumor samples with highlight on chromosome 13 copy number gain. Supplementary Figure S2: The RNA expression levels comparison between the sensitive and resistant groups in all cell lines, KRAS mutants, KRAS/BRAF-WT/WT subpopulations for IR-B, IGF1, IGF2 and IRS1. Supplementary Figure S3: Differential expression patterns of IRS2, IGF-1R, and activation of MAPK between CRC cell lines with KRAS G13D mutation and other KRAS mutations. Supplementary Figure S4: Cell lines with IRS2 CNG or sensitive to BMS-754807 had lower basal level MAPK activation. Supplementary Figure S5: IGF-1R/IR pathway signaling in LS513 and SW-403 CRC cell lines.

JQ1 sensitivity and oncogene mutation or DNA copy number status of small cell lung cancer cell lines. Genomic information was obtained from the COSMIC database (23).

PDF - 32KB, No significant change in body weight was observed throughout the experimental duration.

Supplementary Tables S1-S4. Supplementary Table S1: Association of gene mutations, IRS2 copy number gain, and gene expression level with in vitro sensitivity to BMS-754807 in 60 CRC cell lines. Supplementary Table S2: The relationship between KRAS, BRAF and PIK3CA mutation status and the sensitivity of BMS-754807 in 60 CRC cell lines. Supplementary Table S3: Statistical analyses identified CNV of 197 genes located on chromosome 13 significantly associated with in vitro sensitivity to BMS-754807 in 60 CRC cell lines. Supplementary Table S4: Data mining indicate IRS2 CNG is more prevalent in CRC than in other tumor types.

The hypoxia-inducible factor 2α (HIF-2α) is a key oncogenic driver in clear cell renal cell carcinoma (ccRCC). Our first HIF-2α inhibitor PT2385 demonstrated promising proof of concept clinical activity in heavily pretreated advanced ccRCC patients. However, PT2385 was restricted by variable and dose-limited pharmacokinetics resulting from extensive metabolism of PT2385 to its glucuronide metabolite. Herein we describe the discovery of second-generation HIF-2α inhibitor PT2977 with increased potency and improved pharmacokinetic profile achieved by reduction of phase 2 metabolism. Structural modification by changing the geminal difluoro group in PT2385 to a vicinal difluoro group resulted in enhanced potency, decreased lipophilicity, and significantly improved pharmacokinetic properties. In a phase 1 dose-escalation study, the clinical pharmacokinetics for PT2977 supports the hypothesis that attenuating the rate of glucuronidation would improve exposure and reduce variability in patients. Early evidence of clinical activity shows promise for PT2977 in the treatment of ccRCC.

HIF-2 alpha, a member of the HIF family of transcription factors, is a key oncogenic driver in cancers such as clear cell renal cell carcinoma (ccRCC). A signature feature of these cancers is the overaccumulation of HIF-2 alpha protein, often by inactivation of the E3 ligase VHL (von Hippel Lindau). Herein we disclose our structure based drug design (SBDD) approach that culminated in the identification of PT2385, the first HIF-2 alpha antagonist to enter clinical trials. Highlights include the use of a putative n -> pi*(Ar) interaction to guide early analog design, the conformational restriction of an essential hydroxyl moiety, and the remarkable impact of fluorination near the hydroxyl group. Evaluation of select compounds from two structural classes in a sequence of PK/PD, efficacy, PK, and metabolite profiling identified 10i (PT2385, luciferase EC50 = 27 nM) as the clinical candidate. Finally, a retrospective crystallographic analysis describes the structural perturbations necessary for efficient antagonism.

e13534 Background: Hypoxia, a driver of malignancy, is fundamental to glioblastoma (GBM). Of the hypoxia inducible transcription factors (HIFs), HIF-2a is expressed in GBM tumor cells, responds to prolonged hypoxia, and is a prime candidate for HIF therapeutic targeting. PT2385 is a novel, orally available, first-in-class, HIF-2a inhibitor with a brain:plasma ratio of 0.9 in rats. To establish HIF-2a as a therapeutic target in GBM, in situ expression needs to be described. Methods: Twenty-two paraffin-embedded gliomas (grade II-IV) underwent immunohistochemistry for HIF-2a. After rehydration, a hot Tris EDTA pH 9.0 solution retrieved epitopes, non-specific sites were blocked, and HIF-2a antibody (Santa Cruz, sc-13596) was added. A horseradish peroxidase detection system identified staining. Representative samples were digitally scanned. Localization and quantification of HIF-2a was independently verified by a neuropathologist. Patient survival and molecular genetics were retrospectively collected with Institutional Review Board approval. Results: HIF-2a expression was not detected in the 4 Grade II and 2 Grade III gliomas.Of the 16 GBMs, HIF-2a was expressed in 13 specimens (81%) - highly in 7 ( > 10% cells positive), intermediate in 6 ( < 10% cells positive), and minimally in 3. Recurrent disease stained highly for HIF-2a. Staining was specific to tumor cells and occasionally monocytic cells, clustering in perivascular and perinecrotic regions. Amongst the 16 GBMs, median patient age and survival was 58 and 13.9 months. The two longest survivors harbored positive prognostic genetics (six-year survival with a 1p/19q co-deletion and ten-year survival with MGMT gene methylation). Twelve of 16 patients received chemoradiation. Survival was not correlated with HIF-2a. Conclusions: This first in situ description of HIF-2a demonstrates expression is absent in low-grade gliomas, present in the majority of GBMs, and high in recurrent GBMs. HIF-2a abundance varies with clustering in perivascular and perinecrotic niches. Survival did not appear to be correlated with HIF-2a in this small cohort though well-known prognostic genetic mutations and treatment heterogeneity limit firm conclusions. Preclinical studies with PT2385 are in progress.

Clear cell renal cell carcinoma (ccRCC) is characterized by inactivation of the von Hippel-Lindau tumour suppressor gene (VHL). Because no other gene is mutated as frequently in ccRCC and VHL mutations are truncal, VHL inactivation is regarded as the governing event. VHL loss activates the HIF-2 transcription factor, and constitutive HIF-2 activity restores tumorigenesis in VHL-reconstituted ccRCC cells. HIF-2 has been implicated in angiogenesis and multiple other processes, but angiogenesis is the main target of drugs such as the tyrosine kinase inhibitor sunitinib. HIF-2 has been regarded as undruggable. Here we use a tumourgraft/patient-derived xenograft platform to evaluate PT2399, a selective HIF-2 antagonist that was identified using a structure-based design approach. PT2399 dissociated HIF-2 (an obligatory heterodimer of HIF-2α-HIF-1β) in human ccRCC cells and suppressed tumorigenesis in 56% (10 out of 18) of such lines. PT2399 had greater activity than sunitinib, was active in sunitinib-progressing tumours, and was better tolerated. Unexpectedly, some VHL-mutant ccRCCs were resistant to PT2399. Resistance occurred despite HIF-2 dissociation in tumours and evidence of Hif-2 inhibition in the mouse, as determined by suppression of circulating erythropoietin, a HIF-2 target and possible pharmacodynamic marker. We identified a HIF-2-dependent gene signature in sensitive tumours. Gene expression was largely unaffected by PT2399 in resistant tumours, illustrating the specificity of the drug. Sensitive tumours exhibited a distinguishing gene expression signature and generally higher levels of HIF-2α. Prolonged PT2399 treatment led to resistance. We identified binding site and second site suppressor mutations in HIF-2α and HIF-1β, respectively. Both mutations preserved HIF-2 dimers despite treatment with PT2399. Finally, an extensively pretreated patient whose tumour had given rise to a sensitive tumourgraft showed disease control for more than 11 months when treated with a close analogue of PT2399, PT2385. We validate HIF-2 as a target in ccRCC, show that some ccRCCs are HIF-2 independent, and set the stage for biomarker-driven clinical trials.

The CK2 protein kinases are a small family of two highly related serine/threonine kinases composed of two catalytic subunits, α and α’, and a single β subunit. Numerous substrates have been reported for CK2 and these proteins are known to participate in diverse cellular processes, including cell signaling, transcription, DNA repair, apoptosis regulation and tumor suppression. Elevated CK2 expression and kinase activity has been observed in many cancer types. Further, mRNA knockdown and enzyme inhibition studies have demonstrated that many cancer cell lines are dependent on CK2 for growth and survival. To further evaluate CK2 kinases as targets for therapeutic intervention in cancer, we identified BMS-595, a potent and selective, ATP-competitive CK2 inhibitor. BMS-595 inhibits the in vitro proliferation of human colorectal and lung cancer cell lines with IC50s ranging from less than 10 nM to greater than 1 μM. In sensitive cell lines, anti-proliferative effects of BMS-595 and structurally related analogs strongly correlated with cellular CK2 kinase inhibition. Oral administration of BMS-595 to mice bearing colorectal cancer and lung cancer xenografts demonstrated pharmacodynamic effects and robust efficacy at tolerated doses. These studies confirm the dependence of a subset of human colon and lung cancer cell lines on CK2 activity for growth and demonstrate that pharmacologic inhibition of CK2 can produce anti-tumor efficacy at tolerated doses. Citation Format: Brent A. Rupnow, Chiang Yu, Jonathan G. Pabalan, Urvashi V. Roongta, Jonathan S. Lippy, Ashok R. Dongre, Mary T. Obermeier, Aberra Fura, Paul A. Elzinga, Benjamin J. Henley, Joseph Fargnoli, Francis Y. Lee, William R. Foster, Christine M. Tarby, Brian E. Fink, John S. Tokarski, Ashvinikumar V. Gavai, Tai W. Wong, John T. Hunt, Gregory D. Vite, Ashok V. Purandare. Anti-tumor activity of BMS-595, a novel CK2 kinase inhibitor. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5395. doi:10.1158/1538-7445.AM2015-5395

Abstract CK2 is a highly conserved, and constitutively active family of serine/threonine kinases abnormally elevated in a wide variety of cancers and linked to poor prognosis and disease progression. The enzymes form as hetero-tetrameric complexes comprised of two highly related catalytic subunits (α or α´) with two regulatory β subunits in various combinations and distributions, depending on cell type. While CK2 plays a role in normal growth and development, deregulation of the enzymes has been shown to promote and maintain a malignant phenotype through mechanisms in both the anti-apoptotic and the pro-proliferative signaling pathways. CK2 has been reported to modulate the activity of several oncogenic transcription factors including CREB, Myc, Jun and Fos. Studies with RNAi and small molecule compounds have demonstrated tumor cell dependence on CK2. We sought to identify potent CK2 inhibitors to probe the function of CK2 in cancer-linked pathways and for evaluation in CK2 dependent tumor xenograft models. Herein we report SAR studies in the imidazo[1,2-b]pyridazine chemotype leading to the discovery of BMS-595, a highly potent and selective ATP-competitive CK2 inhibitor with a commensurate level of cellular potency. BMS-595 demonstrates strong PK/PD correlations and robust, oral anti-tumor efficacy in CK2-driven xenograft models at tolerated doses. Citation Format: Christine M. Tarby, Liqi He, Brian E. Fink, Andrew Nation, Yufen Zhao, Soong-Hoon Kim, Libing Chen, John S. Tokarski, Chiang Yu, Jonathan G. Pabalan, Urvashi V. Roongta, Jonathan Lippy, Mary Obermeier, Paul A. Elzinga, Aberra Fura, Benjamin Henley, Joseph J. Fargnoli, William R. Foster, Ashvinikumar V. Gavai, Tai W. Wong, John T. Hunt, Gregory D. Vite, Ashok V. Purandare, Brent A. Rupnow. The identification of BMS-595, an orally active imidazo[1,2-b]pyridazine CK2 inhibitor with in vivo anti-tumor activity. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5417. doi:10.1158/1538-7445.AM2015-5417