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Purpose: Insulin-like growth factor 1 receptor (IGF-IR) has been implicated in the pathogenesis of ovarian cancer. Ganitumab is an investigational, fully human monoclonal antibody against IGF-IR. Here, we explore the therapeutic potential of ganitumab for the treatment of ovarian cancer.Experimental Design: The effects of ganitumab were tested in vitro against a panel of 23 established ovarian cancer cell lines. The ability of ganitumab to inhibit IGF-I–, IGF-II–, and insulin-mediated signaling was examined in vitro and in tumor xenografts using ovarian cancer models displaying IGF-IR/PI3K/AKT pathway activation by two distinct mechanisms, PTEN loss and IGF-II overexpression. Drug interactions between ganitumab and cisplatin, carboplatin, or paclitaxel were studied in vitro and in vivo.Results:In vitro, growth inhibition varied significantly among individual ovarian cancer cell lines. IGF-II mRNA and phospho–IGF-IR protein expression were quantitatively correlated with response to ganitumab, and PTEN mutations conferred resistance to ganitumab. Ganitumab potently inhibited baseline and IGF-I–, IGF-II–, and insulin-induced IGF-IR and IGF-IR/insulin hybrid receptor signaling in vitro and in vivo. Synergistic and additive drug interactions were seen for ganitumab and carboplatin or paclitaxel in vitro. Furthermore, ganitumab significantly increased the efficacy of cisplatin in ovarian cancer xenograft models in vivo.Conclusions: These observations provide a biologic rationale to test ganitumab as a single agent or in combination with carboplatin/cisplatin and paclitaxel in patients with ovarian cancer. Moreover, assessment of tumor expression of IGF-II, phospho–IGF-IR, or PTEN status may help select patients with ovarian cancer who are most likely to benefit from ganitumab. Clin Cancer Res; 20(11); 2947–58. ©2014 AACR.

PDF file - 114KB, Expression of Akt and PTEN in OV-90 and TOV-21G cells.

PDF file - 56KB, Inhibition of cell growth by ganitumab.

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PDF file - 37KB, Primer sequences.

PDF file - 55KB, Description of ovarian cancer cell lines.

Supplementary Table S1 from AMG 479, a fully human anti–insulin-like growth factor receptor type I monoclonal antibody, inhibits the growth and survival of pancreatic carcinoma cells

Pancreatic carcinoma is a leading cause of cancer deaths, and recent clinical trials of a number of oncology therapeutics have not substantially improved clinical outcomes. We have evaluated the therapeutic potential of AMG 479, a fully human monoclonal antibody against insulin-like growth factor (IGF) type I receptor (IGF-IR), in two IGF-IR–expressing pancreatic carcinoma cell lines, BxPC-3 and MiaPaCa2, which also differentially express insulin receptor (INSR). AMG 479 bound to IGF-IR (KD 0.33 nmol/L) and blocked IGF-I and IGF-II binding (IC50 < 0.6 nmol/L) without cross-reacting to INSR. AMG 479 completely inhibited ligand-induced (IGF-I, IGF-II, and insulin) activation of IGF-IR homodimers and IGF-IR/INSR hybrids (but not INSR homodimers) leading to reduced cellular viability in serum-deprived cultures. AMG 479 inhibited >80% of basal IGF-IR activity in BxPC-3 and MiaPaCa2 xenografts and prevented IGF-IR and IGF-IR/INSR hybrid activation following challenge with supraphysiologic concentrations of IGF-I. As a single agent, AMG 479 inhibited (∼80%) the growth of pancreatic carcinoma xenografts, and long-term treatment was associated with reduced IGF-IR signaling activity and expression. Efficacy seemed to be the result of two distinct biological effects: proapoptotic in BxPC-3 and antimitogenic in MiaPaCa2. The combination of AMG 479 with gemcitabine resulted in additive inhibitory activity both in vitro and in vivo. These results indicate that AMG 479 is a clinical candidate, both as a single agent and in combination with gemcitabine, for the treatment of patients with pancreatic carcinoma.[Mol Cancer Ther 2009;8(5):1095–105]

PDF file - 47KB, Expression analysis in ovarian cancer cell lines.

Supplementary Fig. S1 from AMG 479, a fully human anti–insulin-like growth factor receptor type I monoclonal antibody, inhibits the growth and survival of pancreatic carcinoma cells

PDF file - 53KB, IGF1R and INSR signaling in OV-90 and TOV-21G cell lines.

The prosurvival BCL2 family member MCL1 is frequently dysregulated in cancer. To overcome the significant challenges associated with inhibition of MCL1 protein–protein interactions, we rigorously applied small-molecule conformational restriction, which culminated in the discovery of AMG 176, the first selective MCL1 inhibitor to be studied in humans. We demonstrate that MCL1 inhibition induces a rapid and committed step toward apoptosis in subsets of hematologic cancer cell lines, tumor xenograft models, and primary patient samples. With the use of a human MCL1 knock-in mouse, we demonstrate that MCL1 inhibition at active doses of AMG 176 is tolerated and correlates with clear pharmacodynamic effects, demonstrated by reductions in B cells, monocytes, and neutrophils. Furthermore, the combination of AMG 176 and venetoclax is synergistic in acute myeloid leukemia (AML) tumor models and in primary patient samples at tolerated doses. These results highlight the therapeutic promise of AMG 176 and the potential for combinations with other BH3 mimetics. Significance: AMG 176 is a potent, selective, and orally bioavailable MCL1 inhibitor that induces a rapid commitment to apoptosis in models of hematologic malignancies. The synergistic combination of AMG 176 and venetoclax demonstrates robust activity in models of AML at tolerated doses, highlighting the promise of BH3-mimetic combinations in hematologic cancers. See related commentary by Leber et al., p. 1511. This article is highlighted in the In This Issue feature, p. 1494

Therapeutic resistance is a major obstacle to achieving durable clinical responses with targeted therapies, highlighting a need to elucidate the underlying mechanisms responsible for resistance and identify strategies to overcome this challenge. An emerging body of data implicates the tyrosine kinase MET in mediating resistance to BRAF inhibitors in BRAFV600E mutant melanoma. In this study we observed a dominant role for the HGF/MET axis in mediating resistance to BRAF and MEK inhibitors in models of BRAFV600E and NRAS mutant melanoma. In addition, we showed that MAPK pathway inhibition induced rapid increases in MET and GAB1 levels, providing novel mechanistic insight into how BRAFV600E mutant melanoma is primed for HGF-mediated rescue. We also determined that tumor-derived HGF, not systemic HGF, may be required to convey resistance to BRAF inhibition in vivo and that resistance could be reversed following treatment with AMG 337, a selective MET inhibitor. In summary, these findings support the clinical evaluation of MET-directed targeted therapy to circumvent resistance to BRAF and MEK inhibitors in BRAFV600E mutant melanoma. In addition, the induction of MET following treatment with BRAF and MEK inhibitors has the potential to serve as a predictive biomarker for identifying patients best suited for MET inhibitor combination therapy.

Ganitumab is a fully human MAB to the human type 1 IGF receptor (IGF1R). Binding assays showed that ganitumab recognized murine IGF1R with sub-nanomolar affinity (KD=0.22 nM) and inhibited the interaction of murine IGF1R with IGF1 and IGF2. Ganitumab inhibited IGF1-induced activation of IGF1R in murine lungs and CT26 murine colon carcinoma cells and tumors. Addition of ganitumab to 5-fluorouracil resulted in enhanced inhibition of tumor growth in the CT26 model. Pharmacological intervention with ganitumab in naïve nude mice resulted in a number of physiological changes described previously in animals with targeted deletions ofIgf1andIgf1r, including inhibition of weight gain, reduced glucose tolerance and significant increase in serum levels of GH, IGF1 and IGFBP3. Flow cytometric analysis identified GR1/CD11b-positive cells as the highest IGF1R-expressing cells in murine peripheral blood. Administration of ganitumab led to a dose-dependent, reversible decrease in the number of peripheral neutrophils with no effect on erythrocytes or platelets. These findings indicate that acute IGF availability for its receptor plays a critical role in physiological growth, glucose metabolism and neutrophil physiology and support the presence of a pituitary IGF1R-driven negative feedback loop that tightly regulates serum IGF1 levels through Gh signaling.

Acute myeloid leukemia (AML) is a heterogeneous malignancy involving the clonal expansion of abnormal myeloid progenitor cells. The anti-apoptotic BCL-2 protein family member MCL1 has been implicated in AML pathogenesis. AMG 176 is a potent and selective MCL1 inhibitor currently being evaluated in an AML Phase I clinical trial. Standard of care (SOC) treatment for AML involves “7+3” induction therapy with cytarabine and an anthracycline (i.e. doxorubicin). In patient’s ineligible for conventional induction therapy, the hypomethylating agent decitabine is a well-tolerated alternative. Here we investigate the activity of AMG 176 and AM-8621, a closely related analog of AMG 176 with similar potency and selectivity, in combination with SOC therapies in preclinical models of AML. We profiled a panel of AML cell lines (MOLM-13, MV-4-11, GDM-1 and EOL-1), testing AM-8621 in combination with cytarabine, doxorubicin and decitabine in 3-day viability assays. Evidence for synergistic activity was detected with each of the combinations across subsets of the cell lines. A time course of AM-8621 treatment revealed a rapid induction of caspase 3/7 activity, with significant increases observed within the first 2-4 hours of treatment while longer duration treatment (>8 hours) was required to activate caspase 3/7 with SOC agents. Combined treatment with AM-8621 and SOC agents exhibited significant improvements in caspase 3/7 activity beyond either single agent alone. To provide mechanistic insight into the ability of SOC agents to sensitize AML cell lines to AM-8621 treatment, signaling experiments were performed to characterize changes in BCL-2 protein family members following treatment with SOC agents. Clear changes in the levels of anti-apoptotic BCL-2 family members (MCL1, BCL-2 and BCL-XL), BH3-only domain containing proteins (BIM, NOXA, PUMA) and the executioner protein BAK were observed, providing insight into potential mechanisms of priming cells to MCL1 inhibition. Combinations of AMG 176 and each of the SOC therapies were also tested in a MOLM-13 orthotopic xenograft model of AML. Mice were treated with combinations of AMG 176 and cytarabine, decitabine or doxorubicin and compared against the single agent therapies. Significant reductions in tumor burden compared to single agent treatments were observed with the AMG 176 + doxorubicin (30% regression) and AMG 176 + decitabine (98% tumor growth inhibition) combinations. These data highlight the promise of combining AMG 176 with SOC therapies in the setting of AML. Citation Format: Sean Caenepeel, Brian Belmontes, Tao Osgood, Elaina Cajulis, Angela Coxon, Jude Canon, Paul E. Hughes. AMG 176 exhibits robust antitumor activity in combination with standard of care agents in models of acute myeloid leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2180.

Acute myeloid leukemia (AML) is a heterogeneous hematological malignancy involving the clonal expansion of immature myeloid cells. Dysregulated expression of the BCL-2 family of proteins has been implicated in AML pathogenesis. Specifically, the anti-apoptotic family members MCL1 and BCL-2 have been reported to play key roles in AML survival. The selective inhibition of these two proteins represents an emerging strategy in AML treatment. AMG 176 is a potent and selective MCL1 inhibitor currently being tested in an AML Phase I clinical trial. Here we describe the activity of AMG 176 and AM-8621, a structural analog, as single agents and in combination with the BCL-2 inhibitor venetoclax, in models of AML. AM-8621 and venetoclax were profiled as single agents against a panel of AML cell lines to characterize their dependency on MCL1 and BCL-2 for survival. A wide range of sensitivities to both compounds was observed, with several lines exhibiting dependency on both MCL1 and BCL-2 for survival, suggesting functional redundancy and a requirement for combined inhibition to maximize response. To test this hypothesis, we profiled a subset of cell lines with the combination of AM-8621 and venetoclax. A synergistic interaction was detected in each cell line, highlighting their codependence on MCL1 and BCL-2. We also evaluated the synergistic potential of this combination on primary AML patient samples. Here, freshly purified bone marrow aspirates were treated with equimolar concentrations of AM-8621 and venetoclax and compared against single agents in a flow cytometry based viability assay. Marked improvements in activity and potency were observed with the combination over either agent alone. We also tested the combination of AMG 176 and venetoclax in a MOLM-13 orthotopic xenograft model of AML. Mice were treated twice weekly with AMG 176 (60 mg/kg) and daily with venetoclax (100 mg/kg). While both single agents achieved significant reductions in MOLM-13 tumor burden (69% and 33% reduction in BLI respectively), the combination exhibited complete inhibition of tumor growth (100% reduction in BLI) and achieved tumor regression relative to the first day of dosing. We next characterized the effects of this combination on subsets of hematopoietic cells in vivo. The reduced affinity of AMG 176 for murine MCL1 (200-fold) required the use of a human MCL1 knock-in mouse for these studies. Terminal analysis of mice treated with the combination or AMG 176 alone showed significant decreases in peripheral blood B-cells and monocytes, whereas venetoclax alone exhibited significant reductions in B-cells only. Analysis of spleens revealed greater reductions in both cell types following treatment with the combination compared with either single agent. These data highlight the promise of combined MCL1 and BCL2 inhibition as a novel therapeutic strategy for the treatment of AML. Citation Format: Sean R. Caenepeel, Tao Osgood, Brian Belmontes, Jan Sun, Elaina Cajulis, Andrew Wei, Angela Coxon, Jude Canon, Paul Hughes. Combined inhibition of MCL1 and BCL-2 with AMG 176 and venetoclax induces anti-tumor effects in primary patient samples and models of acute myeloid leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3972.

Rheumatoid arthritis involves psychoneuroendocrine-immunopathological comorbidities. In the stoma, patients with rheumatoid arthritis frequently show signs of periondontal disease consequent to elevated levels of crevicular proinflammatory cytokines. It is not clear whether rheumatoid arthritis may manifest in association with immunopathological manifestations of the oral soft mucosa. Oral lichen planus (OLP), first described by E. Wilson in 1859, is a T-cell-mediated inflammatory disease whose lesions characteristically lack B cells, plasma cells, immunoglobulin. or complement. It is increasingly well characterized and recognized as a model for psychoneuroimmunology research in oral biology and medicine. To date, we have shown an association between changes in hypothalamic-pituitary-adrenal (HPA) regulation, systemic markers of cellular immunity and mood states, with clinical stages of OLP (i.e., atrophic vs. erosive vs. bullous lesions). We report significant associations (p < 0.05) between the stage of OLP, HPA deregulation, and altered distribution and functional responses of naive CD4(+) cells. We emphasize the need to study in greater details the psychoneuroendocrine-immune inter-relationships in OLP, and we propose a novel neuroimmune hypothesis for OLP.

BACKGROUND: Therapeutic antibodies targeting the IGF1R have shown diverse efficacy and safety signals in oncology clinical trials. The success of these agents as future human therapeutics depends on understanding the specific mechanisms by which these antibodies target IGF1R signaling. METHODOLOGY/PRINCIPAL FINDINGS: A panel of well-characterized assays was used to investigate the mechanisms by which ganitumab, a fully human anti-IGF1R antibody undergoing clinical testing, inhibits IGF1R activity. Epitope mapping using IGF1R subdomains localized the ganitumab binding site to the L2 domain. Binding of ganitumab inhibited the high-affinity interaction of IGF-1 and IGF-2 required to activate IGF1R in cells engineered for IGF1R hypersensitivity and in human cancer cell lines, resulting in complete blockade of ligand-induced cellular proliferation. Inhibition of IGF1R activity by ganitumab did not depend on endosomal sequestration, since efficient ligand blockade was obtained without evidence of receptor internalization and degradation. Clinically relevant concentrations of ganitumab also inhibited the activation of hybrid receptors by IGF-1 and IGF-2. Ganitumab was not an agonist of homodimeric IGF1R or hybrid receptors in MCF-7 and COLO 205 cells, but low-level IGF1R activation was detected in cells engineered for IGF1R hypersensitivity. This activation seems biologically irrelevant since ganitumab completely inhibited ligand-driven proliferation. The in vivo efficacy profile of ganitumab was equivalent or better than CR and FnIII-1 domain-specific antibodies, alone or in combination with irinotecan. CR domain-specific antibodies only blocked IGF-1 binding to IGF1R but were more potent than ganitumab at inducing homodimer and hybrid receptor downregulation in vitro, however this difference was less obvious in vivo. No inhibition of hybrid receptors was observed with the FnIII-1 domain antibodies, which were relatively strong homodimer and hybrid agonists. CONCLUSIONS/SIGNIFICANCE: The safety and efficacy profile of ganitumab and other anti-IGF1R antibodies may be explained by the distinct molecular mechanisms by which they inhibit receptor signaling.

Ewing's and osteogenic sarcoma are two of the leading causes of cancer deaths in children and adolescents. Recent data suggest that sarcomas may depend on the insulin-like growth factor type 1 (IGF-1) receptor (IGF1R) and/or the insulin receptor (INSR) to drive tumor growth, survival, and resistance to mammalian target of rapamycin complex 1 (mTORC1) inhibitors. We evaluated the therapeutic value of ganitumab (AMG 479; C(6472)H(10028)N(1728)O(2020)S(42)), an anti-IGF1R, fully human monoclonal antibody, alone and in combination with rapamycin (mTORC1 inhibitor) in Ewing's (SK-ES-1 and A673) and osteogenic (SJSA-1) sarcoma models. IGF1R was activated by IGF-1 but not by insulin in each sarcoma model. INSR was also activated by IGF-1 in the SJSA-1 and SK-ES-1 models, but not in the A673 model where insulin was the preferred INSR ligand. Ganitumab significantly inhibited the growth of SJSA-1 and SK-ES-1 xenografts; inhibition was associated with decreased IGF1R and Akt phosphorylation, reduced total IGF1R and bromodeoxyuridine detection, and increased caspase-3 expression. Ganitumab inhibited rapamycin-induced IGF1R, Akt, and glycogen synthase kinase-3β hyperphosphorylation in each sarcoma model. However, ganitumab in combination with rapamycin also resulted in a marked increase in INSR expression and activity in the SJSA-1 and A673 models. The in vivo efficacy of ganitumab in the two ganitumab-sensitive models (SJSA-1 and SK-ES-1) was significantly enhanced in combination with rapamycin. Our results support studying ganitumab in combination with mTORC1 inhibitors for the treatment of sarcomas and suggest that INSR signaling is an important mechanism of resistance to IGF1R blockade.

Brequinar sodium (BQR) is a novel immunosuppressive drug that inhibits cell proliferation by virtue of its disruption of the de novo pyrimidine biosynthesis. The basis of the immunosuppressive activity of BQR is distinctively different from that of cyclosporine (CsA), and we have recently evaluated in vivo and in vitro the efficacy of the two drugs when used in combination. Subtherapeutic doses of BQR and CsA were tested for their ability to prolong heterotopic cardiac allograft survival in the MHC- and non-MHC-mismatched ACI--LEW rat strain combination. The graft survival data derived from these experiments were analyzed using the median-effect analysis to establish the immunosuppressive interaction of both drugs. The administration of BQR 3 mg/kg three times weekly or CsA 2.5 mg/kg daily moderately prolonged cardiac allograft survival, with a mean survival of 10 +/- 0.5 and 16 +/- 5.3 days, respectively. The use of the two drugs in combination with the same dose schedule exerted a synergistic effect on graft survival, prolonging the graft function to a mean of 31 +/- 5.7 days. Sera from animals treated with the two drugs displayed, when compared with single treatment groups (BQR 3 mg/kg and CsA 2.5 mg/kg), significantly (P0.01) increased in vitro inhibition of lymphocyte proliferation following stimulation with PHA. Finally, a clear correlation between the mean survival time and BQR plasma levels of animals treated with BQR alone or in combination with CsA was seen. Those treatment groups with BQR levels below 2 micrograms/ml (1.5 and 3.0 mg/kg/3x/week) had a mean graft survival of less than 10 days). In contrast, recipients treated with a combination of low doses of BQR and CsA displayed higher drug plasma levels (2 micrograms/ml) and longer mean graft survival times. These observations suggest that BQR and CsA may be highly effective when used in combination to prevent organ allograft rejection for clinical transplantation.

Pancreatic carcinoma is a leading cause of cancer deaths, and recent clinical trials of a number of oncology therapeutics have not substantially improved clinical outcomes. We have evaluated the therapeutic potential of AMG 479, a fully human monoclonal antibody against insulin-like growth factor (IGF) type I receptor (IGF-IR), in two IGF-IR–expressing pancreatic carcinoma cell lines, BxPC-3 and MiaPaCa2, which also differentially express insulin receptor (INSR). AMG 479 bound to IGF-IR (KD 0.33 nmol/L) and blocked IGF-I and IGF-II binding (IC50 < 0.6 nmol/L) without cross-reacting to INSR. AMG 479 completely inhibited ligand-induced (IGF-I, IGF-II, and insulin) activation of IGF-IR homodimers and IGF-IR/INSR hybrids (but not INSR homodimers) leading to reduced cellular viability in serum-deprived cultures. AMG 479 inhibited >80% of basal IGF-IR activity in BxPC-3 and MiaPaCa2 xenografts and prevented IGF-IR and IGF-IR/INSR hybrid activation following challenge with supraphysiologic concentrations of IGF-I. As a single agent, AMG 479 inhibited (∼80%) the growth of pancreatic carcinoma xenografts, and long-term treatment was associated with reduced IGF-IR signaling activity and expression. Efficacy seemed to be the result of two distinct biological effects: proapoptotic in BxPC-3 and antimitogenic in MiaPaCa2. The combination of AMG 479 with gemcitabine resulted in additive inhibitory activity both in vitro and in vivo. These results indicate that AMG 479 is a clinical candidate, both as a single agent and in combination with gemcitabine, for the treatment of patients with pancreatic carcinoma.[Mol Cancer Ther 2009;8(5):1095–105]

Introduction: Amplification of the HER-2 receptor tyrosine kinase has been implicated in the pathogenesis and aggressive behavior of approximately 25% of invasive human breast cancers. Clinical and experimental evidence suggest that aberrant HER-2 signaling contributes to tumor initiation and disease progression. Transforming growth factor beta (TGF-β) is the dominant factor opposing growth stimulatory factors and early oncogene activation in many tissues, including the mammary gland. Thus, to better understand the mechanisms by which HER-2 overexpression promotes the early stages of breast cancer, we directly assayed the cellular and molecular effects of TGF-β1 on breast cancer cells in the presence or absence of overexpressed HER-2. Methods: Cell proliferation assays were used to determine the effect of TGF-β on the growth of breast cancer cells with normal or high level expression of HER-2. Affymetrix microarrays combined with Northern and western blot analysis were used to monitor the transcriptional responses to exogenous TGF-β1 in luminal and mesenchymal-like breast cancer cells. The activity of the core TGF-β signaling pathway was assessed using TGF-β1 binding assays, phospho-specific Smad antibodies, immunofluorescent staining of Smad and Smad DNA binding assays. Results: We demonstrate that cells engineered to over-express HER-2 are resistant to the anti-proliferative effect of TGF-β1. HER-2 overexpression profoundly diminishes the transcriptional responses induced by TGF-β in the luminal MCF-7 breast cancer cell line and prevents target gene induction by a novel mechanism that does not involve the abrogation of Smad nuclear accumulation, DNA binding or changes in c-myc repression. Conversely, HER-2 overexpression in the context of the mesenchymal MDA-MB-231 breast cell line potentiated the TGF-β induced pro-invasive and pro-metastatic gene signature. Conclusion: HER-2 overexpression promotes the growth and malignancy of mammary epithelial cells, in part, by conferring resistance to the growth inhibitory effects of TGF-β. In contrast, HER-2 and TGF-β signaling pathways can cooperate to promote especially aggressive disease behavior in the context of a highly invasive breast tumor model.

The mechanism of BRCA1 tumor suppression in human breast and ovarian cells is the focus of intense investigation. In this report, full length BRCA1 (230 kDa) introduced into cells with CMV promoter constructs was nuclear when transgene expression was low whereas high expression resulted in cytoplasmic accumulation, aberrant nuclear and cell morphology. A nuclear localization signal (NLS) was mapped to BRCA1 amino acid positions 262-570. We describe a splice variant, BRCA1-delta11b, missing the majority of exon 11 including the NLS. Exogenous BRCA1-delta11b (110 kDa) was cytoplasmic and, unlike the full-length protein, overexpression of the protein encoded by the variant did not appear to be toxic. RNA probe titrations and RT-PCR demonstrated that BRCA1 and delta11b transcripts are coexpressed in a wide variety of cells and tissues. Interestingly, BRCA1-delta11b message was greatly reduced or absent in several breast and ovarian tumor lines relative to exon 11 transcripts and a delta9,10 splice variant. Taken together our results suggest that full-length BRCA1 and BRCA1-delta11b may have distinct roles in cell growth regulation and tumorigenesis.

Background: Resistance to targeted therapies represents a key hurdle in the treatment of cancer. Preclinical data suggest that resistance is frequently driven by redundant growth factor signaling. For example, HGF has been reported to mediate resistance to BRAF inhibitors in V600E BRAF mutant melanoma. Furthermore, clinical data support these findings as circulating and tumor HGF levels have been shown to correlate with outcome in BRAF mutant melanoma patients treated with vemurafenib. In this study, we evaluated the ability of HGF / MET signaling to attenuate the effects of BRAF and MEK inhibitors in the context of V600E BRAF and NRAS mutant cell lines. Our data suggest that combined blockade of MET and MAPK pathway signaling may be required for a robust and durable clinical response in melanoma. Methods: To confirm the ability of HGF to rescue V600E BRAF mutant melanoma cell lines from the effects of vemurafenib, G361 and SKMEL5 cells were treated with vemurafenib alone or in combination with HGF. Time lapse imaging, viability assays and cell cycle analysis were used to measure treatment effects. Analogous experiments were performed with the selective MEK inhibitor PD0325901. Combinations of a potent and selective MET inhibitor (Compound A) with vemurafenib or PD0325901 were tested to evaluate their ability to attenuate the HGF-mediated resistance. To identify other cell lines rescued by HGF, thirteen V600E BRAF mutant melanoma cell lines were treated with a dose response matrix of vemurafenib and HGF. The ability of six additional growth factors to rescue cells from the effects of vemurafenib was also tested. Three NRAS mutant melanoma cell lines were also screened for HGF-mediated resistance to PD0325901. Results: Nine of thirteen V600E BRAF mutant melanoma cell lines were rescued from the effects of vemurafenib by co-treatment with HGF. Detailed analysis of G361 and SKMEL5 cells revealed that HGF was also capable of rescuing the cells from the effects of PD0325901. This HGF-mediated rescue was attenuated by treatment with combinations of vemurafenib + compound A or PD0325901 + compound A. Mechanistic cell cycle analysis of the HGF-mediated rescue of vemurafenib in G361 cells revealed an increase in proliferating cells and a reduction in growth arrested cells, whereas rescue of PD0325901 treatment occurred through a reduction in the percentage of dead or dying cells. Of the seven growth factors tested, HGF was the predominate growth factor capable of conferring resistance to vemurafenib. Furthermore, HGF rescued one of three NRAS mutant melanoma cell lines from the effects of PD0325901, highlighting the breadth of HGF-mediated resistance in melanoma cell lines. Conclusion: These findings demonstrate the potential role for HGF / MET signaling in mediating resistance to BRAF and MEK inhibitors in melanoma, and support the clinical evaluation of MET kinase inhibitors and neutralizing antibodies to HGF in this setting. Citation Format: Sean Caenepeel, Elaina Cajulis, Rick Kendall, Angela Coxon, Paul Hughes. Targeting HGF-mediated resistance to vemurafenib in V600E BRAF mutant melanoma cell lines. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3405. doi:10.1158/1538-7445.AM2013-3405

We have previously demonstrated that the immune response to cardiac allografts in the ACI-to-LEW rat strain combination involves a limited use of the TCR V beta gene repertoire. In the present study we analyzed the expression of V beta genes by T cells infiltrating kidney and liver allografts to test whether a limited use of the T cell receptor (TCR) repertoire is a common denominator for immune responses to allografts. Graft-infiltrating lymphocytes (GIL) were isolated from allografts on different days after transplantation and analyzed for the expression of V beta genes using a semi-quantitative polymerase chain reaction (PCR) without manipulations in tissue culture. We detected a limited expression of the V beta gene repertoire in fresh GIL harvested from both kidney and liver allografts early in graft rejection. The level of TCR repertoire usage, however, was influenced by the type of graft. The rejection of heart and kidney allografts was associated with more limited use of the V beta gene repertoire when compared with that seen for the rejection of liver allografts. The limited use of the V beta gene repertoire was only apparent when analyzed early in graft rejection; as the rejection reaction progressed T cells using a more diverse V beta repertoire infiltrated the graft. The limited use of TCR repertoire of the early T cell response to allografts may provide the opportunity to therapeutically disrupt the rejection reaction by targeting selected T cell populations for elimination at the time of organ transplantation.

AMG 479, a fully human monoclonal antibody raised to the type I insulin-like growth factor receptor (IGF1R), is currently being studied in the treatment of patients with ovarian carcinoma. We have previously shown that AMG 479 inhibits the growth of pancreatic and sarcoma tumor models by inhibiting IGF1R signaling through ligand blockade and receptor internalization and degradation. In the present study, we tested the ability of AMG 479 to inhibit the growth of four human ovarian carcinoma xenografts (OV-90, SK-OV3, TOV-21G, and OV-CAR3) in nude mice. Recent evidence has shown that multiple players in the IGF-axis are differentially regulated in human ovarian serous carcinomas, suggesting that the IGF1R pathway may play an important role in the growth of these tumors. The OV-90 model showed exquisite sensitivity to single-agent AMG 479, reaching significant (p Citation Information: Mol Cancer Ther 2009;8(12 Suppl):C172.