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H3B-6527 response in 40 HCC cell lines and 625 cell lines.

Supplement for Statistical Methods from Synergistic Antitumor Effects of Combined Epidermal Growth Factor Receptor and Vascular Endothelial Growth Factor Receptor-2 Targeted Therapy

Legends describing each of the supplementary figures and tables

RNA-seq analysis of 24 CCLE HCC cell lines.

Purpose: Combination therapies that target the epidermal growth factor receptor (EGFR) and vascular endothelial growth factor receptor (VEGFR) pathways, are being actively tested for the treatment of cancer. In evaluating combination strategies, the ideal combination would be one in which the treatments interact in a way that is synergistic with regard to antitumor effects. Here, we have evaluated the interaction between anti-EGFR antibody Erbitux (cetuximab) and anti-VEGFR2 antibody, DC101, in preclinical models of pancreatic (BxPC-3) and colon (GEO) cancer.Experimental Design: Analysis of the interaction between cetuximab and DC101 in vivo used a novel method for establishing the upper 95% confidence limits for the combination index (CI) of isobologram analyses, where CI < 1 indicates synergy. Assessment of tumor cell proliferation, apoptosis, VEGF production, and hypoxia, as well as tumor vascularization, was performed to gain insights into the mechanistic basis for synergy between agents targeting different tumor compartments.Results: Monotherapy ED50 values for tumor growth inhibition ranged from 1.8 to 2.3 mg/kg and 10.5 to 16.6 mg/kg for cetuximab and DC101, respectively. From the dose response of the combination treatment, it was determined that cetuximab and DC101 are synergistic in the BxPC-3 (CI = 0.1, P < 0.01) and GEO (CI = 0.1, P < 0.01) models. Overlapping effects on the tumor cell and vascular compartments form a basis for the interaction, with VEGF production and hypoxia-inducible factor 1α potentially acting as molecular links between EGFR and VEGFR2 inhibition.Conclusions: Results show antitumor synergy for combined EGFR and VEGFR2 targeted therapy, supporting the significant therapeutic potential of this combination strategy.

Activation of the fibroblast growth factor receptor FGFR4 by FGF19 drives hepatocellular carcinoma (HCC), a disease with few, if any, effective treatment options. While a number of pan-FGFR inhibitors are being clinically evaluated, their application to FGF19-driven HCC may be limited by dose-limiting toxicities mediated by FGFR1–3 receptors. To evade the potential limitations of pan-FGFR inhibitors, we generated H3B-6527, a highly selective covalent FGFR4 inhibitor, through structure-guided drug design. Studies in a panel of 40 HCC cell lines and 30 HCC PDX models showed that FGF19 expression is a predictive biomarker for H3B-6527 response. Moreover, coadministration of the CDK4/6 inhibitor palbociclib in combination with H3B-6527 could effectively trigger tumor regression in a xenograft model of HCC. Overall, our results offer preclinical proof of concept for H3B-6527 as a candidate therapeutic agent for HCC cases that exhibit increased expression of FGF19. Cancer Res; 77(24); 6999–7013. ©2017 AACR.

Cancer patients receiving epidermal growth factor receptor (EGFR) antibody therapy often experience an acneiform rash of uncertain etiology in skin regions rich in pilosebaceous units. Currently, this condition is treated symptomatically with very limited, often anecdotal success. Here, we show that a monoclonal antibody targeting murine EGFR, ME1, caused a neutrophil-rich hair follicle inflammation in mice, similar to that reported in patients. This effect was preceded by the appearance of lipid-filled hair follicle distensions adjacent to enlarged sebaceous glands. The cytokine tumor necrosis factor-α (TNFα), localized immunohistochemically to this affected region of the pilosebaceous unit, was specifically up-regulated by ME1 in skin but not in other tissues examined. Moreover, skin inflammation was reduced by cotreatment with the TNFα signaling inhibitor, etanercept, indicating the involvement of TNFα in this inflammatory process. Interleukin-1, a cytokine that frequently acts in concert with TNFα, is also involved in this process given the efficacy of the interleukin-1 antagonist Kineret. Our results provide a mechanistic framework to develop evidence-based trials for EGFR antibody–induced skin rash in patients with cancer. [Cancer Res 2009;69(14):5643–7]

Supplementary Figures 1-9 from Tumor Necrosis Factor-α and Interleukin-1 Antagonists Alleviate Inflammatory Skin Changes Associated with Epidermal Growth Factor Receptor Antibody Therapy in Mice

Activation of the fibroblast growth factor receptor FGFR4 by FGF19 drives hepatocellular carcinoma (HCC), a disease with few, if any, effective treatment options. While a number of pan-FGFR inhibitors are being clinically evaluated, their application to FGF19-driven HCC may be limited by dose-limiting toxicities mediated by FGFR1–3 receptors. To evade the potential limitations of pan-FGFR inhibitors, we generated H3B-6527, a highly selective covalent FGFR4 inhibitor, through structure-guided drug design. Studies in a panel of 40 HCC cell lines and 30 HCC PDX models showed that FGF19 expression is a predictive biomarker for H3B-6527 response. Moreover, coadministration of the CDK4/6 inhibitor palbociclib in combination with H3B-6527 could effectively trigger tumor regression in a xenograft model of HCC. Overall, our results offer preclinical proof of concept for H3B-6527 as a candidate therapeutic agent for HCC cases that exhibit increased expression of FGF19. Cancer Res; 77(24); 6999–7013. ©2017 AACR.

Cancer patients receiving epidermal growth factor receptor (EGFR) antibody therapy often experience an acneiform rash of uncertain etiology in skin regions rich in pilosebaceous units. Currently, this condition is treated symptomatically with very limited, often anecdotal success. Here, we show that a monoclonal antibody targeting murine EGFR, ME1, caused a neutrophil-rich hair follicle inflammation in mice, similar to that reported in patients. This effect was preceded by the appearance of lipid-filled hair follicle distensions adjacent to enlarged sebaceous glands. The cytokine tumor necrosis factor-α (TNFα), localized immunohistochemically to this affected region of the pilosebaceous unit, was specifically up-regulated by ME1 in skin but not in other tissues examined. Moreover, skin inflammation was reduced by cotreatment with the TNFα signaling inhibitor, etanercept, indicating the involvement of TNFα in this inflammatory process. Interleukin-1, a cytokine that frequently acts in concert with TNFα, is also involved in this process given the efficacy of the interleukin-1 antagonist Kineret. Our results provide a mechanistic framework to develop evidence-based trials for EGFR antibody–induced skin rash in patients with cancer. [Cancer Res 2009;69(14):5643–7]

Purpose: Vascular endothelial growth factor receptor-1 (VEGFR-1) plays important roles in promotion of tumor growth by mediating cellular functions in tumor vascular endothelium and cancer cells. Blockade of VEGFR-1 activation has been shown to inhibit pathologic angiogenesis and tumor growth, implicating VEGFR-1 as a potential therapeutic target for the treatment of cancer. We have thus developed a VEGFR-1 antagonist human monoclonal antibody designated as IMC-18F1 and evaluated its antitumor activity in preclinical experimental models to show the therapeutic potential of the antibody for cancer treatment in clinic. Experimental Design: Human IgG transgenic mice were used for generation of anti-VEGFR-1 antibodies. Anti-VEGFR-1-specific blocking antibodies were identified using solid-phase binding and blocking assays. Inhibitory antitumor cell activity of IMC-18F1 was assessed in cell-based kinase and growth assays. Pharmacokinetic/pharmacodynamic studies were done to determine the association of antibody blood level with antitumor efficacy of the antibody in vivo. Antitumor efficacy of the anti-VEGFR-1 antibodies as monotherapy and in combination with cytotoxic agents was evaluated in human breast cancer xenograft models. Results: A fully human neutralizing antibody, IMC-18F1, was shown to be a high-affinity (KD = 54 pmol) inhibitor of VEGFR-1 ligand binding (VEGF-A, VEGF-B, and placental growth factor). IMC-18F1 inhibited ligand-induced intracellular activation of VEGFR-1 and mitogen-activated protein kinase signaling and prevented ligand-stimulated in vitro growth of breast cancer cells. In vivo, IMC-18F1 suppressed the growth of human breast tumor xenografts in association with reduced mitogen-activated protein kinase and Akt activation, reduced tumor cell proliferation, and increased tumor cell apoptosis. Pharmacokinetic/pharmacodynamic studies established a plasma elimination half-life of 5 days for IMC-18F1 and a steady-state trough plasma therapeutic threshold of 88 μg/mL. Importantly, inhibition of mouse and human VEGFR-1 with MF1 and IMC-18F1, respectively, enhanced the antitumor efficacy of cytotoxic agents commonly used to treat breast cancer. Conclusions: Based on preclinical validation studies, IMC-18F1 anti-VEGFR-1 has potential to provide clinical benefit to cancer patients.

Purpose: Combination therapies that target the epidermal growth factor receptor (EGFR) and vascular endothelial growth factor receptor (VEGFR) pathways, are being actively tested for the treatment of cancer. In evaluating combination strategies, the ideal combination would be one in which the treatments interact in a way that is synergistic with regard to antitumor effects. Here, we have evaluated the interaction between anti-EGFR antibody Erbitux (cetuximab) and anti-VEGFR2 antibody, DC101, in preclinical models of pancreatic (BxPC-3) and colon (GEO) cancer. Experimental Design: Analysis of the interaction between cetuximab and DC101 in vivo used a novel method for establishing the upper 95% confidence limits for the combination index (CI) of isobologram analyses, where CI < 1 indicates synergy. Assessment of tumor cell proliferation, apoptosis, VEGF production, and hypoxia, as well as tumor vascularization, was performed to gain insights into the mechanistic basis for synergy between agents targeting different tumor compartments. Results: Monotherapy ED50 values for tumor growth inhibition ranged from 1.8 to 2.3 mg/kg and 10.5 to 16.6 mg/kg for cetuximab and DC101, respectively. From the dose response of the combination treatment, it was determined that cetuximab and DC101 are synergistic in the BxPC-3 (CI = 0.1, P < 0.01) and GEO (CI = 0.1, P < 0.01) models. Overlapping effects on the tumor cell and vascular compartments form a basis for the interaction, with VEGF production and hypoxia-inducible factor 1α potentially acting as molecular links between EGFR and VEGFR2 inhibition. Conclusions: Results show antitumor synergy for combined EGFR and VEGFR2 targeted therapy, supporting the significant therapeutic potential of this combination strategy.

Hepatocellular carcinoma (HCC) has limited treatment options and generally poor prognosis. Recent genomic studies have identified FGF19 as a driver oncogene in HCC. FGF19 is a gut secreted hormone that acts in the liver through FGFR4 to regulate bile acid synthesis. Consistent with the notion that FGF19 is a driver oncogene in HCC, transgenic mice overexpressing FGF19 form liver tumors and genetic ablation of FGFR4 prevented tumor formation. These data suggest targeting FGFR4 would have therapeutic benefit in HCC with altered FGF19 signaling. While a number of Pan-FGFR inhibitors are being clinically evaluated, their application to FGF19-driven HCC may be limited by their FGFR1-3 related dose limiting toxicities. Using structure guided drug design, we have generated a highly selective covalent FGFR4 inhibitor, H3B-6527. Biochemical and cellular selectivity assays showed that H3B-6527 is >300 fold selective towards FGFR4 compared to other FGFR isoforms. Addition of H3B-6527 to FGF19 amplified HCC cell lines led to dose dependent inhibition of FGF19/FGFR4 signaling and concomitant reduction in cell viability. In a panel of 40 HCC cell lines, H3B-6527 selectively reduced the viability of cells that harbor FGF19 amplification and showed no effect in FGF19 non-amplified HCC cell line models. Oral dosing of H3B-6527 to mice led to dose-dependent pharmacodynamic modulation of FGFR4 signaling and tumor regression in FGF19 altered HCC cell line derived xenograft models. H3B-6527 demonstrated inhibition of tumor growth in an orthotopic liver xenograft model of FGF19 altered HCC grown in nude mice. Importantly, the inhibition of tumor growth occurred at doses that were well tolerated in mice and no evidence of FGFR1-3 related toxicities were observed at efficacious doses. In a panel of 30 HCC patient-derived xenograft (PDX) models, H3B-6527 demonstrated tumor regressions in the context of FGF19-amplified tumors. In addition, H3B-6527 showed antitumor activity and tumor regressions in PDX models with high FGF19 expression but no FGF19 amplification. The mechanism for FGF19 overexpression in the absence of gene amplification is under investigation. In conclusion, our preclinical studies demonstrate that FGF19 expression is a predictive biomarker for response to FGFR4 inhibitor therapy. Genomic analysis of public and proprietary data sets indicates that at least approximately 30% of HCC patients exhibit altered FGF19 expression and could potentially benefit from H3B-6527 monotherapy treatment. Citation Format: Anand Selvaraj, Erik Corcoran, Heather Coffey, Sudeep Prajapati, Ming-Hong Hao, Nicholas Larsen, Jennifer Tsai, Takashi Satoh, Kana Ichikawa, Julie Jaya Joshi, Raelene Hurley, Jeremy Wu, Chia-Ling Huang, Suzanna Bailey, Craig Karr, Pavan Kumar, Victoria Rimkunas, Crystal Mackenzie, Nathalie Rioux, Amy Kim, Sandeep Akare, George Lai, Lihua Yu, Peter Fekkes, John Wang, Markus Warmuth, Peter Smith, Dominic Reynolds. H3B6527, a selective and potent FGFR4 inhibitor for FGF19-driven hepatocellular carcinoma [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 3126. doi:10.1158/1538-7445.AM2017-3126

The benefits of inhibiting vascular endothelial growth factor (VEGF) signaling in cancer patients are predominantly attributed to effects on tumor endothelial cells. Targeting non–endothelial stromal cells to further impact tumor cell growth and survival is being pursued through the inhibition of additional growth factor pathways important for the survival and/or proliferation of these cells. However, recent data suggest that VEGF receptor (VEGFR)–specific inhibitors may target lymphatic vessels and pericytes in addition to blood vessels. Here, in fact, we demonstrate that DC101 (40 mg/kg, thrice a week), an antibody specific to murine VEGFR2, significantly reduces all three of these stromal components in subcutaneous (SKRC-29) and orthotopic (786-O-LP) models of renal cell carcinoma (RCC) established in nu/nu athymic mice. Sunitinib (40 mg/kg, once daily), a receptor tyrosine kinase inhibitor of VEGFR2 and other growth factor receptors, also caused significant loss of tumor blood vessels in RCC models but had weaker effects than DC101 on pericytes and lymphatic vessels. In combination, sunitinib did not significantly add to the effects of DC101 on tumor blood vessels, lymphatic vessels, or pericytes. Nevertheless, sunitinib increased the effect of DC101 on tumor burden in the SKRC-29 model, perhaps related to its broader specificity. Our data have important implications for combination therapy design, supporting the conclusion that targeting VEGFR2 alone in RCC has the potential to have pleiotropic effects on tumor stroma.

Rational strategies utilizing anticancer efficacy and biological principles are needed for the prioritization of specific combination targeted therapy approaches for clinical development, from among the many with experimental support.Antibodies targeting epidermal growth factor receptor (EGFR) (cetuximab), insulin-like growth factor-1 receptor (IGF-IR) (IMC-A12) or vascular endothelial growth factor receptor 2 (VEGFR2) (DC101), were dosed alone or in combination, in 11 human tumor xenograft models established in mice. Efficacy readouts included the tumor burden and incidence of metastasis, as well as tumor active hypoxia inducible factor-1 (HIF-1), human VEGF and blood vessel density.Cetuximab and DC101 contributed potent and non-overlapping benefits to the combination approach. Moreover, DC101 prevented escape from IMC-A12 + cetuximab in a colorectal cancer model and cetuximab prevented escape from DC101 therapy in a pancreatic cancer model.Targeting VEGFR2 + EGFR was prioritized over other treatment strategies utilizing EGFR, IGF-IR and VEGFR2 antibodies. The criteria that proved to be valuable were a non-overlapping spectrum of anticancer activity and the prevention of resistance to another therapy in the combination.

Targeted therapy for cancer is shifting towards an approach of inhibiting multiple pathways, justified in part by the ability of cancer cells to overcome the inhibition of a single pathway. However the literature is replete with preclinical data supporting the anticancer potential of numerous combinations of targeted agents, making it difficult to select the combination strategies to invest in through clinical development. One characteristic of a combination strategy that can be utilized for prioritization is synergy. Synergy indicates that the effect of the combination is greater than that predicted from the monotherapy potencies. Here we describe a detailed method for establishing synergy between two treatments in vivo. We utilized this method to establish that antibodies targeting the epidermal growth factor receptor and vascular endothelial growth factor receptor-2 are synergistic with regard to antitumor effects, in a BxPC-3 subcutaneous xenograft model for pancreatic cancer.

Targeted therapy for cancer is shifting towards an approach of inhibiting multiple pathways, justified in part by the ability of cancer cells to overcome the inhibition of a single pathway. However the literature is replete with preclinical data supporting the anticancer potential of numerous combinations of targeted agents, making it difficult to select the combination strategies to invest in through clinical development. One characteristic of a combination strategy that can be utilized for prioritization is synergy. Synergy indicates that the effect of the combination is greater than that predicted from the monotherapy potencies. Here we describe a detailed method for establishing synergy between two treatments in vivo. We utilized this method to establish that antibodies targeting the epidermal growth factor receptor and vascular endothelial growth factor receptor-2 are synergistic with regard to antitumor effects, in a BxPC-3 subcutaneous xenograft model for pancreatic cancer.

RWJ-54428 (also known as MC-02,479) is a new cephalosporin with promising activity against gram-positive bacteria. The pharmacodynamics (PDs) of RWJ-54428 against Staphylococcus aureus , Streptococcus pneumoniae , and Enterococcus faecalis were studied in a neutropenic mouse thigh infection model. The RWJ-54428 MICs ranged from 0.25 to 1 mg/liter. Mice with ca. 10 6 CFU/thigh at the initiation of therapy were treated intraperitoneally with RWJ-54428 at doses that ranged from 3 to 1,200 mg/kg of body weight/day (in 2, 3, 4, 6, or 12 divided doses) for 24 h. The maximal reductions in bacterial counts in thigh tissues at 24 h for the methicillin-resistant S. aureus , penicillin-resistant S. pneumoniae , and E. faecalis strains were −2.8, −3.8, and −1.7 log 10 CFU/thigh, respectively. The percentage of a 24-h dosing interval that the unbound serum RWJ-54428 concentrations exceeded the MIC ( fT > MIC ) was the pharmacokinetic (PK)-PD parameter that best described the efficacy of RWJ-54428. The fT > MIC s for a bacteriostatic effect (no net change in the numbers of CFU/thigh over 24 h) ranged from 14 to 20% for staphylococci and streptococci; for maximal reductions in the numbers of CFU/thigh, the fT > MIC s ranged from 22 to 36% for these strains. For E. faecalis , the ranges of fT > MIC s for static and maximal effects were 30 to 46% and 55 to 60%, respectively. These data show that treatment with RWJ-54428 results in marked antibacterial effects in vivo, with the PK-PD parameters for efficacy being comparable to those for the efficacy of penicillins and carbapenems active against staphylococci and pneumococci.

A novel class of 1-[4-(1H-benzoimidazol-2-yl)-phenyl]-3-[4-(1H-benzoimidazol-2-yl)-phenyl]-ureas are described as potent inhibitors of heparanase. Among them are 1,3-bis-[4-(1H-benzoimidazol-2-yl)-phenyl]-urea (7a) and 1,3-bis-[4-(5,6-dimethyl-1H-benzoimidazol-2-yl)-phenyl]-urea (7d), which displayed good heparanase inhibitory activity (IC(50) 0.075-0.27 microM). Compound 7a showed good efficacy in a B16 metastasis model.

Recurrent heterozygous mutations of the spliceosome protein SF3B1 have been identified in myelodysplastic syndromes, chronic lymphocytic leukemia (CLL), breast, pancreatic and skin cancers. SF3B1 is a component of the U2 snRNP complex which binds to the pre-mRNA branch point site and is involved in recognition and stabilization of the spliceosome at the 3′ splice site. To understand the impact of SF3B1 mutations, we compared RNAseq profiles from tumor samples with SF3B1 hotspot mutations (SF3B1-MUT) or wild-type SF3B1 (SF3B1-WT) in breast cancer, melanoma and CLL. This analysis revealed significant increases in the usage of novel alternative splice junctions in SF3B1-MUT samples including selection of alternative 3′ splice sites and less frequently exon skipping. These events induce expression of alternative mRNAs that are translated into novel proteins or aberrant mRNAs that are decayed by cells. A common alternative splicing profile was shared across different hotspot mutations and lineages (e.g. ZDHHC16 and COASY); however, unique alternative splicing profiles were also observed suggesting lineage specific effects. RNAseq analysis of several cell lines with endogenous SF3B1 hotspot mutations confirmed the presence of the same spliced isoforms as observed in tumor samples. To prove that SF3B1-MUT were inducing alternative splicing, transient transfection of several SF3B1 hotspot mutations in 293FT cells induced the expression of the common alternatively spliced genes suggesting functional similarity. Selective shRNA depletion of mutant SF3B1 allele in SF3B1-MUT cells resulted in downregulation of the same splice isoforms. Furthermore, isogenic B-cell lines (NALM-6) expressing the most frequent SF3B1 mutation (K700E) were generated and profiled by RNAseq. As expected, similar alternatively spliced genes were observed in NALM-6 SF3B1-K700E cells exclusively. To investigate the role of nonsense-mediated mRNA decay (NMD) in eliminating aberrant mRNAs induced by SF3B1-MUT, we treated NALM-6 SF3B1-K700E cells with cycloheximide, a translation inhibitor known to inhibit NMD. In the treated samples, expression of several aberrant mRNAs was revealed and some of these transcripts were shown to be downregulated in patient samples. Taken together, these results confirm the association between different SF3B1 hotspot mutations and the presence of novel splice isoforms. We demonstrated that E7107, a potent and selective inhibitor of wild-type SF3B1, also binds and inhibits SF3B1-MUT protein. In addition, E7107 represses the expression of several common aberrant splice mRNA products in SF3B1-MUT cells in vitro and in vivo. When tested in a NALM-6 mouse model, E7107 induced tumor regression and increased the overall survival of animals implanted with NALM-6 SF3B1-K700E cells. These data suggest splicing inhibitors as a promising therapeutic approach for cancer patients carrying SF3B1 mutations. Citation Format: Silvia Buonamici, Kian Huat Lim, Jacob Feala, Eunice Park, Laura Corson, Michelle Aicher, Daniel Aird, Betty Chan, Erik Corcoran, Rachel Darman, Peter Fekkes, Gregg Keaney, Pavan Kumar, Kaiko Kunii, Linda Lee, Xiaoling Puyang, Jose Rodrigues, Anand Selvaraj, Michael Thomas, John Wang, Markus Warmuth, Lihua Yu, Ping Zhu, Peter Smith, Yoshiharu Mizui. SF3B1 mutations induce aberrant mRNA splicing in cancer and confer sensitivity to spliceosome inhibition. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2932. doi:10.1158/1538-7445.AM2014-2932

SCH 56592 (posaconazole), a new triazole antifungal agent, was tested in vitro, and its activity was compared to that of itraconazole against 39 Aspergillus strains and to that of fluconazole against 275 Candida and 9 Cryptococcus strains. The SCH 56592 MICs for Aspergillus ranged from ≤0.002 to 0.5 μg/ml, and those of itraconazole ranged from ≤0.008 to 1 μg/ml. The SCH 56592 MICs for Candida and Cryptococcus strains ranged from ≤0.004 to 16 μg/ml, and those of fluconazole ranged from ≤0.062 to >64 μg/ml. SCH 56592 showed excellent activity against Aspergillus fumigatus and Aspergillus flavus in a pulmonary mouse infection model. When administered therapeutically, the 50% protective doses (PD 50 s) of SCH 56592 ranged from 3.6 to 29.9 mg/kg of body weight, while the PD 50 s of SCH 56592 administered prophylactically ranged from 0.9 to 9.0 mg/kg; itraconazole administered prophylactically was ineffective (PD 50 s, >75 mg/kg). SCH 56592 was also very efficacious against fluconazole-susceptible, -susceptible dose-dependent, or -resistant Candida albicans strains in immunocompetent or immunocompromised mouse models of systemic infection. The PD 50 s of SCH 56592 administered therapeutically ranged from 0.04 to 15.6 mg/kg, while the PD 50 s of SCH 56592 administered prophylactically ranged from 1.5 to 19.4 mg/kg. SCH 56592 has excellent potential for therapy against serious Aspergillus or Candida infections.

Cetuximab has significant clinical benefits alone or in combination with irinotecan in metastatic colorectal cancer (mCRC) patients with KRAS wild type tumors, but efficacy in patients with KRAS mutant tumors has thus far been elusive. Mutant BRAF in mCRC tumors may also predict for reduced benefit with EGFR antibodies. Interestingly, in preclinical CRC models, cetuximab, alone or in combination with irinotecan, has significant efficacy in tumor models with mutant KRAS or BRAF. Here we examine if this preclinical efficacy can be significantly increased in a screen for add-on therapies that may provide clinically meaningful efficacy in mCRC patients requiring an EGFR antibody. HT-29 (BRAF mutant-V600E) and HCT-116 (KRAS mutant-G38D) cell lines were utilized as models of CRC. We screened 14 compounds that could potentially add to the effects of cetuximab+irinotecan (C+I) treatment, including inhibitors of inflammation, enzymes, receptor tyrosine kinases, HSP90, cyclins, mTOR, an ATP mimetic, activators of apoptosis and PPARγ. Of the compounds evaluated only 17-AAG (HSP90 inhibitor), everolimus(mTOR inhibitor) and DC101 (antibody targeting murine VEGFR2) significantly broke the ceiling of tumor stasis established with C+I treatment alone in both models. 17-AAG+C+I resulted in T/C% values of 14 and 32% in HCT-116 and HT-29, respectively, compared to 25 and 62% in the C+I treatment group. Follow up studies showed this benefit to be due to an additive interaction between 17-AAG and irinotecan, which was also associated with significant weight loss. Everolimus+C+I treatment resulted in a T/C% of 15 and 22% in HCT-116 and HT-29, respectively, compared to 22 and 35% in the C+I treatment group. The benefits of everolimus+C+I on the other hand required all three agents. The combination of DC101+C+I was significantly more efficacious than C+I in both models, with a T/C% of 11 and 33% in HCT-116 and HT-29, respectively, compared to 19 and 53% in C+I treatment group. In contrast to these three therapies, all other compounds evaluated did not improve the antitumor effects of C+I alone. Results demonstrate a ceiling effect in CRC models with KRAS or BRAF gene mutations that may be associated with the limited clinical benefits observed with cetuximab in patients. The fact that this ceiling can be broken with everolimus in a manner that requires cetuximab, supports the potential utility of cetuximab in these patients in a combination approach. 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 3641. doi:10.1158/1538-7445.AM2011-3641

FLT3 is a class III receptor tyrosine kinase overexpressed by blast cells in approximately 90% of acute myeloid leukemia and acute lymphoid leukemia (ALL) patients. Recent findings testing a small molecule FLT3 inhibitor (PKC412) in combination with eight conventional antileukemic agents, reported that methotrexate in particular did not combine well with PKC412 against leukemia cells (Furukawa, Y. et al., Leukemia 2007, 21: 1005–1014). IMC-EB10 is a fully human neutralizing antibody that binds FLT3 with high affinity (Kd 158 pM) and blocks FLT3 ligand binding to FLT3 (IC50 ~10 nM), leading to inhibition of MAPK, STAT5, and PI3K/Akt signaling in leukemia cells. Here we test for benefits of adding methotrexate to IMC-EB10. In an SEMK2 model of ALL we evaluated survival effects of treatments administered intraperitoneally, beginning 1 day after after intravenous injection of 5 × 10e5 cells/mouse. In this model the maximum tolerated dose (MTD) of methotrexate administered once per week for 3 weeks, was determined to be 100 mg/kg. At this MTD, Methotrexate increased median survival to 44 days, compared to 30 days in the control group (p

Platelet-derived growth factor receptor α (PDGFRα) is a type III receptor tyrosine kinase that is expressed on a variety of tumor types. A neutralizing monoclonal antibody to human PDGFRα, which did not cross-react with the β form of the receptor, was generated. The fully human antibody, termed 3G3, has a Kd of 40 pmol/L and blocks both PDGF-AA and PDGF-BB ligands from binding to PDGFRα. In addition to blocking ligand-induced cell mitogenesis and receptor autophosphorylation, 3G3 inhibited phosphorylation of the downstream signaling molecules Akt and mitogen-activated protein kinase. This inhibition was seen in both transfected and tumor cell lines expressing PDGFRα. The in vivo antitumor activity of 3G3 was tested in human glioblastoma (U118) and leiomyosarcoma (SKLMS-1) xenograft tumor models in athymic nude mice. Antibody 3G3 significantly inhibited the growth of U118 (P = 0.0004) and SKLMS-1 (P < 0.0001) tumors relative to control. These data suggest that 3G3 may be useful for the treatment of tumors that express PDGFRα.