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Supplementary Table 1 from A Fully Human Insulin-Like Growth Factor-I Receptor Antibody SCH 717454 (Robatumumab) Has Antitumor Activity as a Single Agent and in Combination with Cytotoxics in Pediatric Tumor Xenografts

Supplementary Figure Legends 1-3, Table Legend 1 from A Fully Human Insulin-Like Growth Factor-I Receptor Antibody SCH 717454 (Robatumumab) Has Antitumor Activity as a Single Agent and in Combination with Cytotoxics in Pediatric Tumor Xenografts

The insulin-like growth factor-I receptor (IGF-IR) and its ligands (IGF-I and IGF-II) have been implicated in the growth, survival, and metastasis of a broad range of malignancies including pediatric tumors. Blocking the IGF-IR action is a potential cancer treatment. A fully human neutralizing monoclonal antibody, SCH 717454 (19D12, robatumumab), specific to IGF-IR, has shown potent antitumor effects in ovarian cancer in vitro and in vivo. In this study, SCH 717454 was evaluated in several pediatric solid tumors including neuroblastoma, osteosarcoma, and rhabdomyosarcoma. SCH 717454 is shown here to downregulate IGF-IR as well as inhibit IGF-IR and insulin receptor substrate-1 phosphorylation in pediatric tumor cells. IGF-IR and insulin receptor substrate-1 phosphorylation in the tumor cells. In vivo, SCH 717454 exhibits activity as a single agent and significantly inhibited growth of neuroblastoma, osteosarcoma, and rhabdomyosarcoma tumor xenografts. Combination of SCH 717454 with cisplatin or cyclophosphamide enhanced both the degree and the duration of the in vivo antitumor activity compared with single-agent treatments. Furthermore, SCH 717454 treatment markedly reduced Ki-67 expression and blood vessel formation in tumor xenografts, showing that the in vivo activity is derived from its inhibition of tumor cell proliferation and angiogenesis activity. Mol Cancer Ther; 9(2); 410–8

Supplementary Figure 1 from A Fully Human Insulin-Like Growth Factor-I Receptor Antibody SCH 717454 (Robatumumab) Has Antitumor Activity as a Single Agent and in Combination with Cytotoxics in Pediatric Tumor Xenografts

Supplementary Tables 1, 2, and 3 and Supplementary Figures 1 and 2

Supplementary Figure 3 from A Fully Human Insulin-Like Growth Factor-I Receptor Antibody SCH 717454 (Robatumumab) Has Antitumor Activity as a Single Agent and in Combination with Cytotoxics in Pediatric Tumor Xenografts

Supplementary Figure 2 from A Fully Human Insulin-Like Growth Factor-I Receptor Antibody SCH 717454 (Robatumumab) Has Antitumor Activity as a Single Agent and in Combination with Cytotoxics in Pediatric Tumor Xenografts

Kinesin spindle protein (KSP) is a mitotic kinesin required for the formation of the bipolar mitotic spindle, and inhibition of this motor protein results in mitotic arrest and cell death. KSP inhibitors show preclinical antitumor activity and are currently undergoing testing in clinical trials. These agents have been dosed intravenously using various dosing schedules. We sought to identify a KSP inhibitor that could be delivered orally and thus provide convenience of dosing as well as the ability to achieve more continuous exposure via the use of dose-dense administration. We discovered SCH 2047069, a potent KSP inhibitor with oral bioavailability across species and the ability to cross the blood-brain barrier. The compound induces mitotic arrest characterized by a monaster spindle and is associated with an increase in histone H3 and mitotic protein monoclonal 2 phosphorylation both in vitro and in vivo. SCH 2047069 showed antitumor activity in a variety of preclinical models as a single agent and in combination with paclitaxel, gemcitabine, or vincristine. Mol Cancer Ther; 9(11); 2993–3002. ©2010 AACR.

Tedizolid phosphate, the prodrug of the active antibiotic tedizolid, is an oxazolidinone for the treatment of acute bacterial skin and skin structure infections. Studies in a mouse thigh infection model demonstrated that tedizolid has improved potency and pharmacokinetics/pharmacodynamics (PK/PD) compared with those of linezolid. Subsequent studies showed that the efficacy of tedizolid was enhanced in immunocompetent (IC) mice compared with neutropenic (immunosuppressed [IS]) mice, with stasis at clinically relevant doses being achieved only in the presence of granulocytes. The tedizolid label therefore contains a warning about its use in neutropenic patients. This study reevaluated the PK/PD of tedizolid and linezolid in the mouse thigh infection model in IC and IS mice using a methicillin-resistant Staphylococcus aureus (MRSA) strain (ATCC 33591) and a methicillin-susceptible S. aureus (MSSA) strain (ATCC 29213). The antistaphylococcal effect of doses ranging from 1 to 150 mg/kg of body weight tedizolid (once daily) or linezolid (twice daily) was determined at 24, 48, and 72 h after initiating treatment. In IC mice, stasis was achieved in the absence of antibiotics, and both tedizolid and linezolid reduced the burden further beyond a static effect. In IS mice, tedizolid achieved stasis against MRSA ATCC 33591 and MSSA ATCC 29213 at 72 h at a human clinical dose of 200 mg, severalfold lower than that in earlier studies. Linezolid achieved a static effect against MRSA ATCC 33591 in IS mice at a dose lower than that used clinically. This study demonstrates that, with time, both tedizolid and linezolid at clinically relevant exposures achieve stasis in neutropenic mice with an MRSA or MSSA thigh infection.

The imidazo-[1,2-a]-pyrazine (1) is a dual inhibitor of Aurora kinases A and B with modest cell potency (IC50 = 250 nM) and low solubility (5 μM). Lead optimization guided by the binding mode led to the acyclic amino alcohol 12k (SCH 1473759), which is a picomolar inhibitor of Aurora kinases (TdF K d Aur A = 0.02 nM and Aur B = 0.03 nM) with improved cell potency (phos-HH3 inhibition IC50 = 25 nM) and intrinsic aqueous solubility (11.4 mM). It also demonstrated efficacy and target engagement in human tumor xenograft mouse models.

Abrogation of p53 function occurs in almost all human cancers, with more than 50% of cancers harboring inactivating mutations in p53 itself. Mutation of p53 is indicative of highly aggressive cancers and poor prognosis. The vast majority of mutations in p53 occur in its core DNA binding domain (DBD) and result in inactivation of p53 by reducing its thermodynamic stability at physiological temperature. Here, we report a small molecule, SCH529074, that binds specifically to the p53 DBD in a saturable manner with an affinity of 1-2 microm. Binding restores wild type function to many oncogenic mutant forms of p53. This small molecule reactivates mutant p53 by acting as a chaperone, in a manner similar to that previously reported for the peptide CDB3. Binding of SCH529074 to the p53 DBD is specifically displaced by an oligonucleotide with a sequence derived from the p53-response element. In addition to reactivating mutant p53, SCH529074 binding inhibits ubiquitination of p53 by HDM2. We have also developed a novel variant of p53 by changing a single amino acid in the core domain of p53 (N268R), which abolishes binding of SCH529074. This amino acid change also inhibits HDM2-mediated ubiquitination of p53. Our novel findings indicate that through its interaction with p53 DBD, SCH529074 restores DNA binding activity to mutant p53 and inhibits HDM2-mediated ubiquitination.

Clostridium difficile is the causative agent of C. difficile -associated diarrhea (CDAD), with increased risk in elderly populations. Kibdelomycin, a novel natural-product inhibitor of type II topoisomerase enzymes, was evaluated for activity against C. difficile and gastrointestinal anaerobic organisms. Toxigenic C. difficile isolates ( n = 168) from U.S. hospitals and anaerobic Gram-positive and Gram-negative organisms ( n = 598) from Chicago-area hospitals were tested. Kibdelomycin showed potent activity against toxigenic C. difficile (MIC 90 = 0.25 μg/ml) and most Gram-positive aerobic organisms but had little activity against Bacteroides species (MIC 50 > 32 μg/ml; n = 270). Potent anti- C. difficile activity was also observed in the hamster model of C. difficile colitis. Dosing at 1.6 mg/kg (twice-daily oral dose) resulted in protection from a lethal infection and a 2-log reduction in C. difficile cecal counts. A 6.25-mg/kg twice-daily oral dose completely eliminated detectable C. difficile counts in cecal contents. A single 6.25-mg/kg oral dose showed that cecal contents were exposed to the drug at >2 μM (eightfold higher than the MIC), with no significant plasma exposure. These findings support further exploration of kibdelomycin for development of an anti- C. difficile agent.

CD45 is a receptor-type protein-tyrosine phosphatase (PTP) that is required for antigen-specific stimulation and proliferation in lymphocytes. This study was designed to determine the nature of specific kinases in lymphocytes that phosphorylate CD45 and to determine the effect of phosphorylation on CD45 PTP activity. A major cytoplasmic lymphocyte kinase that phosphorylated CD45 was identified as casein kinase 2 (CK2) by use of an in-gel kinase assay in combination with immunoprecipitation, immunodepletion, and specific inhibition. Mutational analysis of CK2 consensus sites showed that the target for CK2 was in an acidic insert of 19 amino acids in the D2 domain, and Ser to Ala mutations at amino acids 965, 968, 969, and 973 abrogated CK2 phosphorylation of CD45. CK2 phosphorylation increased CD45 activity 3-fold toward phosphorylated myelin basic protein, and this increase was reversible by PP2A treatment. Mutation of Ser to Glu at the CK2 sites had the same effect as phosphorylation and also tripled the Vmax of CD45. CD45 isolatedin vivo was highly phosphorylated and could not be phosphorylated by CK2 without prior dephosphorylation with phosphatase PP2A. We conclude that CK2 is a major lymphocyte kinase that is responsible for in vivo phosphorylation of CD45, and phosphorylation at specific CK2 sites regulates CD45 PTP activity.

Summary Innovative strategies are needed to combat drug resistance associated with methicillin-resistant Staphylococcus aureus (MRSA). Here, we investigate the potential of wall teichoic acid (WTA) biosynthesis inhibitors as combination agents to restore β-lactam efficacy against MRSA. Performing a whole-cell pathway-based screen, we identified a series of WTA inhibitors (WTAIs) targeting the WTA transporter protein, TarG. Whole-genome sequencing of WTAI-resistant isolates across two methicillin-resistant Staphylococci spp. revealed TarG as their common target, as well as a broad assortment of drug-resistant bypass mutants mapping to earlier steps of WTA biosynthesis. Extensive in vitro microbiological analysis and animal infection studies provide strong genetic and pharmacological evidence of the potential effectiveness of WTAIs as anti-MRSA β-lactam combination agents. This work also highlights the emerging role of whole-genome sequencing in antibiotic mode-of-action and resistance studies.

Kinesin spindle protein (KSP) is a mitotic kinesin required for the formation of the bipolar mitotic spindle, and inhibition of this motor protein results in mitotic arrest and cell death. KSP inhibitors show preclinical antitumor activity and are currently undergoing testing in clinical trials. These agents have been dosed intravenously using various dosing schedules. We sought to identify a KSP inhibitor that could be delivered orally and thus provide convenience of dosing as well as the ability to achieve more continuous exposure via the use of dose-dense administration. We discovered SCH 2047069, a potent KSP inhibitor with oral bioavailability across species and the ability to cross the blood-brain barrier. The compound induces mitotic arrest characterized by a monaster spindle and is associated with an increase in histone H3 and mitotic protein monoclonal 2 phosphorylation both in vitro and in vivo. SCH 2047069 showed antitumor activity in a variety of preclinical models as a single agent and in combination with paclitaxel, gemcitabine, or vincristine. Mol Cancer Ther; 9(11); 2993–3002. ©2010 AACR.

The insulin-like growth factor-I receptor (IGF-IR) and its ligands (IGF-I and IGF-II) have been implicated in the growth, survival, and metastasis of a broad range of malignancies including pediatric tumors. Blocking the IGF-IR action is a potential cancer treatment. A fully human neutralizing monoclonal antibody, SCH 717454 (19D12, robatumumab), specific to IGF-IR, has shown potent antitumor effects in ovarian cancer in vitro and in vivo. In this study, SCH 717454 was evaluated in several pediatric solid tumors including neuroblastoma, osteosarcoma, and rhabdomyosarcoma. SCH 717454 is shown here to downregulate IGF-IR as well as inhibit IGF-IR and insulin receptor substrate-1 phosphorylation in pediatric tumor cells. IGF-IR and insulin receptor substrate-1 phosphorylation in the tumor cells. In vivo, SCH 717454 exhibits activity as a single agent and significantly inhibited growth of neuroblastoma, osteosarcoma, and rhabdomyosarcoma tumor xenografts. Combination of SCH 717454 with cisplatin or cyclophosphamide enhanced both the degree and the duration of the in vivo antitumor activity compared with single-agent treatments. Furthermore, SCH 717454 treatment markedly reduced Ki-67 expression and blood vessel formation in tumor xenografts, showing that the in vivo activity is derived from its inhibition of tumor cell proliferation and angiogenesis activity. Mol Cancer Ther; 9(2); 410–8

CD45 contains tandem repeated protein tyrosine phosphatase (PTP) domains and is essential for the initiation of the earliest activation events resulting from Ag ligation of the TCR. The second PTP domain (D2) contains four CK2 phosphorylation sites in a unique 19-aa insert, which are targets of CK2 phosphorylation. This study was designed to evaluate the roles of these Ser residues in T cell activation. Transient transfection of the CD45− T cell line, J45.01, with CD45 cDNA incorporating four Ser to Ala (S/A) mutations in the 19-aa insert did not affect the magnitude of NF-AT activation resulting from TCR ligation. However, the basal level of NF-AT activity in unstimulated cells expressing the CD45 S/A mutation was elevated 9- to 10-fold. Increased basal NF-AT was dependent on extracellular Ca2+ stores as judged by EGTA treatment. In additional experiments, isolation of stable clones derived from transfection of the CD45 S/A mutant into CD45− H45.01 cells showed sustained calcium flux after TCR engagement. The sustained calcium flux returned to baseline levels after addition of EGTA, suggesting that the expression of the CD45 S/A mutant may have prevented deactivation of plasma membrane calcium channels. Consideration of both transient and stable transfection systems suggests that in addition to being essential for initial events in T cell triggering, the intact CD45 D2, 19-aa insert is necessary for regulation of TCR-mediated calcium signaling pathways.