Multiple Human Tumor Research Articles

Dissecting the Tumor Myeloid Compartment Reveals Rare Activating Antigen-Presenting Cells Critical for T Cell Immunity

It is well understood that antigen-presenting cells (APCs) within tumors typically do not maintain cytotoxic Tcell (CTL) function, despite engaging them. Across multiple mouse tumor models and human tumor biopsies, we have delineated the intratumoral dendritic cell (DC) populations as distinct from macrophage populations. Within these, CD103(+) DCs are extremely sparse and yet remarkably capable CTL stimulators. These are uniquely dependent on IRF8, Zbtb46, and Batf3 transcription factors and are generated by GM-CSF and FLT3L cytokines. Regressing tumors have higher proportions of these cells, T-cell-dependent immune clearance relies on them, and abundance of their transcripts in human tumors correlates with clinical outcome. This cell type presents opportunities for prognostic and therapeutic approaches across multiple cancer types.

Abstract LB-320: Systemic tumor-stroma interactions are prognostic indicators of breast tumor invasiveness

Abstract Tumor invasiveness dysregulates homeostatic processes within tissues, driving both tumor and stroma cells to locally proliferate while also disrupting tissue boundaries and recruiting cells from distal sites to promote metastatic dissemination. Here, we use an RNA sequencing approach in mouse models of invasive and noninvasive breast cancer to directly test whether the transcriptional changes that occur as invasive tumors disrupt their microenvironment can be used to predict tumor subtype and patient outcomes. We find that tumor and stroma gene expression levels are largely synchronized within the tumor microenvironment, but that the expression levels of a subset of genes with high prognostic potential are strongly negatively correlated and may reflect opposing responses of tumor and normal stroma cells to tissue disruption. We clinically validate these observations in multiple human breast tumor gene expression datasets and demonstrate that stromal transcript levels enable superior classification of breast cancer subtypes relative to those expression changes observed between invasive and noninvasive tumor tissue. We show that local stromal remodelling is partially driven by differential recruitment of macrophages to invasive tumor sites by CCL5, and that invasion-specific gene expression changes that are prognostic for metastasis-free survival in human patients are detectable in stromal tissues far from the primary lesion. Our data indicate that dysregulation of tissue homeostasis by invasive tumors generates transcriptional changes both within the tumor microenvironment and in peripheral tissues that have significant diagnostic and prognostic potential and could facilitate novel patient therapeutic strategies. Citation Format: Russell Bainer, Casey Frankenberger, Daniel Rabe, Sadiq Saleh, Morag Park, Kiran Chada, Yoav Gilad, Marsha Rosner. Systemic tumor-stroma interactions are prognostic indicators of breast tumor invasiveness. [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 LB-320. doi:10.1158/1538-7445.AM2014-LB-320

Abstract 1764: Inhibition of R-spondin (RSPO) signaling reduces the growth of multiple human tumors

Abstract Deregulation of the canonical Wnt/beta-catenin signaling pathway has long been associated with cancer. Intracellular components of this pathway, including Axin, APC, and beta-catenin are frequently mutated in a range of human tumors. The identity of specific extracellular ligands that contribute to human cancer development through this signaling axis has remained unclear. Molecular characterization of the secreted beta-catenin signaling activities produced by minimally passaged human tumor xenograft models identified RSPO family members produced by multiple tumor types including ovarian, pancreatic, colon, breast and non-small cell lung cancer. In human tumor xenograft models that had RSPO overexpression, in some instances due to genomic translocation, anti-RSPO treatment markedly inhibited tumor growth. In addition, striking combination activity with standard of care chemotherapy agents resulted in regression of established tumors. These results highlight the potential for therapeutic intervention with this newly appreciated signaling axis. Citation Format: Austin Gurney, Fumiko Axelrod, Chris Bond, Jennifer Cain, Cecile Chartier, Marcus Fischer, May Ji, Chris Murriel, Janak Raval, Jalpa Shah, Min Wang, Wan-Ching Yen, Ann Kapoun, John Lewicki, Timothy Hoey. Inhibition of R-spondin (RSPO) signaling reduces the growth of multiple human tumors. [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 1764. doi:10.1158/1538-7445.AM2014-1764

Abstract 1713: PDK1 and hexokinase 2 are downstream effectors of PTEN loss and regulate response to targeted therapies in multiple tumor types

Abstract Recent advances in molecular profiling of many human tumor types has enabled the development and clinical use of molecularly-targeted therapies in patients. Many tumors exhibit inactivation of PTEN which is predicted to sensitize tumor cells to PI3 kinase or AKT inhibitor therapy. However, clinical responses to PI3 kinase or AKT inhibitors are variable and not curative. Furthermore, these responses in patients do not correlate uniformly with PTEN inactivation. Therefore, we investigated whether PTEN inactivation could lead to dependence on signaling pathways and components that function independently of PI3K or AKT and that could serve as novel therapeutic targets in PTEN deficient tumors. Using multiple human tumor models including lung adenocarcinoma, lung squamous, renal cell carcinoma, and prostate adenocarcinoma that express or lack PTEN and Gene Set Enrichment Analysis, we identified alterations in numerous metabolic regulatory genes in response to PTEN inactivation, including hexokinase 2 (HK2) and pyruvate dehydrogenase kinase 1 (PDK1). We found that in PTEN-deficient tumor cells HK2 is upregulated and selectively localizes to the mitochondria, where it enhances glycolytic flux and inhibits apoptosis. Furthermore, we found that PDK1 is also upregulated upon PTEN inactivation and suppresses oxidative phosphorylation, upregulating glycolysis and the production of lactate. Thus, we uncovered a novel role for HK2 and PDK1 in the metabolic reprogramming that occurs as a consequence of PTEN inactivation in tumor cells. Furthermore, we found that overexpression of either HK2 or PDK1 confers resistance to targeted therapies against PI3K and other oncogenic drivers more broadly. Conversely, genetic or pharmacological suppression of HK2 or PDK1 enhances response to targeted therapy in multiple tumor cell types. Together, our data identify HK2 and PDK1 as novel molecular biomarkers and promising therapeutic targets in multiple PTEN deficient tumor types. Citation Format: Evangelos Pazarentzos, Trever G. Bivona. PDK1 and hexokinase 2 are downstream effectors of PTEN loss and regulate response to targeted therapies in multiple tumor types. [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 1713. doi:10.1158/1538-7445.AM2014-1713

CMTM1_v17 is a novel potential therapeutic target in breast cancer.

Chemokine-like factor (CKLF)-like MARVEL transmembrane domain-containing1 (CMTM1) consists of at least 23 alternatively spliced isoforms designated CMTM1_v1-v23. In the present study, we detected CMTM1_v17 expression in multiple human normal and tumor tissues and found that CMTM1_v17 was highly expressed in testis and many tumor tissues including breast tumor. The overexpression of CMTM1_v17 in the breast cancer cell line MDA-MB-231 promoted cell proliferation and resistance to tumor necrosis factor-α (TNF-α)-induced apoptosis. Moreover, siRNA-mediated silencing of CMTM1_v17 sensitized MDA-MB-231 cells to TNF-α-induced apoptosis. We propose that CMTM1_v17 may be a novel potential target for therapy in breast cancer patients. The present study provides insight into a novel mechanism by which CMTM1_v17 enhances cellular proliferation and abrogates TNF-α-induced apoptosis. These findings also have implications for clinical practice as they highlight the potential for therapeutic targeting of CMTM1_v17 for the treatment of breast and other cancers in which CMTM1_v17 impacts cellular proliferation and survival.

Conserved oncogenic behavior of the FAM83 family regulates MAPK signaling in human cancer.

FAM83B (family with sequence similarity 83, member B) was recently identified as a novel oncogene involved in activating CRAF/MAPK signaling and driving epithelial cell transformation. FAM83B is one of eight members of a protein family (FAM83) characterized by a highly conserved domain of unknown function (DUF1669), which is necessary and sufficient to drive transformation. Here, it is demonstrated that additional FAM83 members also exhibit oncogenic properties and have significantly elevated levels of expression in multiple human tumor types using a TissueScan Cancer Survey Panel PCR array and database mining. Furthermore, modeling the observed tumor expression of FAM83A, FAM83C, FAM83D, or FAM83E promoted human mammary epithelial cell (HMEC) transformation, which correlated with the ability of each FAM83 member to bind CRAF (RAF1) and promote CRAF membrane localization. Conversely, ablation of FAM83A or FAM83D from breast cancer cells resulted in diminished MAPK signaling with marked suppression of growth in vitro and tumorigenicity in vivo. Importantly, each FAM83 member was determined to be elevated in at least one of 17 distinct tumor types examined, with FAM83A, FAM83B, and FAM83D most frequently overexpressed in several diverse tissue types. Finally, evidence suggests that elevated expression of FAM83 members is associated with elevated tumor grade and decreased overall survival. FAM83 proteins represent a novel family of oncogenes suitable for the development of cancer therapies aimed at suppressing MAPK signaling.

PPM1D silencing by RNA interference inhibits the proliferation of lung cancer cells.

BackgroundPPM1D (protein phosphatase, Mg2+/Mn2+ dependent, 1D) has been reported to be involved in multiple human tumors. This study was designed to investigate the functional role of PPM1D in lung cancer cells.MethodsExpression levels of PPM1D were analyzed in A549 and H1299 cells by real-time PCR and Western blotting. Lentivirus-mediated short hairpin RNA (shRNA) was used to knock down PPM1D expression in both cell lines. The effects of PPM1D on lung cancer cell growth were investigated by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide), colony formation and flow cytometry assays.ResultsKnockdown of PPM1D in lung cancer cells resulted in decreased cell proliferation and impaired colony formation ability. Moreover, flow cytometry analysis showed that knockdown of PPM1D arrested cell cycle at the G0/G1 phase. Furthermore, PPM1D silencing downregulated the expression of cyclin B1 in H1299 cells. Therefore, it is reasonable to speculate that the mechanisms by which PPM1D knockdown alleviates cell growth may be partly via the induction of cell cycle arrest due to the suppression of cyclin B1.ConclusionsThese results suggest that PPM1D silencing by RNA interference (RNAi) may be a potential therapeutic approach for the treatment of lung cancer.

Research progress of negative costimulator B7-H4 in urologic tumors

B7-H4 is a recently-identified member of B7 costimulation superfamily. B7-H4 serves as an inhibitory modulator of T cell response through suppressing the proliferation, activation, and cytokine production of T cells. B7-H4 is found to be abnormally expressed in multiple human tumors. Many studies have shown that B7-H4 expression is closely related to the progression and prognosis of urologic tumors. Investigation of B7-H4 function will provide new strategies for diagnosis and immunotherapy of urologic tumors. This article reviews the recent progress of B7-H4 research in urologic tumors.

The role of TET family proteins and 5-hydroxymethylcytosine in human tumors.

Tumorigenesis correlates with hypermethylation of tumor suppressors and hypomethylation of oncogenes. DNA methyltransferases (DNMTs) catalyze DNA methylation, and mutations and aberrant expression in DNMT genes are found in multiple human tumors. The discovery of the DNA demethylation function of TET proteins has opened up new avenues for the study of DNA methylation regulation. TET proteins regulate the DNA demethylation pathway through oxidizing 5-mC into 5-hmC, 5-fC, and 5-aC. TET genes have been reported to be frequently mutated in hematopoietic malignancies and are associated with the malignant transformation of cells. Loss-of-function mutations in TET genes have not been reported in human solid tumors. However, 5-hmC has been found to be reduced in various solid tumors, indicating that TET genes may contribute to cellular transformation via regulation of DNA demethylation. As a new epigenetic modification, 5-hmC may be a useful biomarker for the diagnosis of cancers. To better understand the roles of TET and 5-hmC in tumors, the biological functions of TET and 5-hmC should be studied further.

CHIP Regulates AKT/FoxO/Bim Signaling in MCF7 and MCF10A Cells

A number of studies have shown that apoptosis resistance can be observed in multiple human tumors; however the detailed mechanism remains unclear. In the present study, we demonstrated that the abnormal overexpression of the C terminus of Hsc70-interacting protein (CHIP) induced apoptosis resistance by regulating the AKT/FoxO/Bim signaling pathway in the breast cancer cell MCF7 and the human non-tumorigenic cell MCF10A. We found that CHIP overexpression in MCF7 and MCF10A cells activated AKT and inhibited the Forkhead box O (FoxO) transcription factors FoxO1, FoxO3, and FoxO4, thereby inhibiting transcription of the target genes bim and pten. Inhibition of PI3K by a chemical reagent revealed that these events may be critical for CHIP-induced apoptosis resistance. We also determined that inhibition of FoxO3 by CHIP led to the decrease in PTEN and further activated the AKT survival pathway. We corroborated our findings in breast cancer tissues. In general, the CHIP-modulated AKT/FoxO/Bim signaling pathway was shown to induce apoptosis resistance by decreasing the protein level of the tumor suppressor PTEN in both transcriptional and post-translational regulations.

Patterns of TPD52 overexpression in multiple human solid tumor types analyzed by quantitative PCR

Tumor protein D52 (TPD52) is located at chromosome 8q21, a region that is frequently gained or amplified in multiple human cancer types. TPD52 has been suggested as a potential target for new anticancer therapies. In order to analyze TPD52 expression in the most prevalent human cancer types, we employed quantitative PCR to measure TPD52 mRNA levels in formalin-fixed tissue samples from more than 900 cancer tissues obtained from 29 different human cancer types. TPD52 was expressed at varying levels in all tested normal tissues, including skin, lymph node, lung, oral mucosa, breast, endometrium, ovary, vulva, myometrium, liver, pancreas, stomach, kidney, prostate, testis, urinary bladder, thyroid gland, brain, muscle and fat tissue. TPD52 was upregulated in 18/29 (62%) tested cancer types. Strongest expression was found in non-seminoma (56-fold overexpression compared to corresponding normal tissue), seminoma (42-fold), ductal (28-fold) and lobular breast cancer (14-fold). In these tumor types, TPD52 upregulation was found in the vast majority (>80%) of tested samples. Downregulation was found in 11 (38%) tumor types, most strongly in papillary renal cell cancer (-8-fold), leiomyosarcoma (-6-fold), clear cell renal cell cancer (-5-fold), liposarcoma (-5-fold) and lung cancer (-4-fold). These results demonstrate that TPD52 is frequently and strongly upregulated in many human cancer types, which may represent candidate tumor types for potential anti-TPD52 therapies.

Ras-Pathway Inhibition With Targeted Therapies Abrogates Self-Renewal In Acute Myelogenous Leukemia

Hyperactivated Ras-pathways serve as oncogenic drivers in multiple human tumors including acute myelogenous leukemia (AML) (Ahearn et al. Nat Rev Mol Cell Biol 2011). The specific functions of these pathways in AML are unclear, thwarting the rational application of targeted therapeutics. Recently, we have shown that NRASG12V–activated signaling pathways are critical to leukemia stem cell maintenance (Sachs et al. submitted). To elucidate which Ras-activated signaling molecules mediate self-renewal in AML, we employed a murine model that harbors Mll-AF9 and a tetracycline repressible, activated NRAS (NRASG12V) and develops AML (Kim et al. Blood 2009). Primary leukemia cells were treated with therapeutic agents targeting Ras-activated signaling pathways. We used PD325901 to inhibit the Mek-Erk pathway, GDC0941 to inhibit the Pi3k pathway, and RAD001 to inhibit the mTor pathway. Using MTS assays, we identified the IC50 dose for each of these agents. Inhibitor-treated leukemia cells were submitted for RNA sequencing in order to investigate the effects of these agents on leukemia gene expression. Previously, we identified a list of NRASG12V responsive genes in our model. In these studies, we identified that PD325901-treatment most closely recapitulates the effect of NRASG12V inhibition on this comprehensive list of RAS-responsive genes. However, when we study the effects of these inhibitors on the subset of RAS-responsive genes that mediate leukemia self-renewal, we find that both PD325901 and RAD001 independently recapitulate the effects of NRASG12V withdrawal on this subset of genes implicating the Mek and mTor pathways in leukemia self renewal. Next, we treated primary leukemia cells with the IC50 dose of each drug and plated them in colony forming assays. We found that Mek or mTor inhibition, but not Pi3k inhibition, abrogated secondary colony formation corroborating our gene expression analyses and showing that, at doses that have equivalent effects on cell growth, only the Mek and mTor pathways are important for leukemia cell stem cell maintenance. These studies provide potential targets for leukemia stem cell-specific therapies. Disclosures:Sachs:Silicon Valley Biosystems: Consultancy. Bendall:DVS Sciences: Consultancy. Nolan:SAB for DVS Sciences and Nodality: Chairman Other; Cell Signalling Technologies and Becton Dickenson, Inc: Consultancy. Largaespada:Discovery Genomics, Inc: Consultancy, Share Holder Other; NeoClone Biotechnology, Inc: Consultancy, Share Holder, Share Holder Other.

Myc — Proteinstabilität als Angriffsziel für zielgerichtete Therapien

The Myc transcription factor family affects the expression of a large group of genes that play an important role during tumourigenesis. Therefore, deregulated expression of the MYC gene family is a hallmark of multiple human tumours. An indirect reduction of Myc-protein levels due to interference with protein-stabilizing mechanisms could be a promising therapeutic approach targeting Myc-dependent tumours.

Abstract A17: ME-344, a novel mitochondrial oxygenase inhibitor: Results from a first-in-human Phase I study.

Abstract Background: ME-344 is a second generation isoflavone-derived drug candidate that causes caspase-independent cell death in multiple human tumor cell lines including CD44+/MyD88+ ovarian cancer stem cells by interfering with mitochondrial energy generation. Treatment with ME-344 results in decreased ATP production, an increase in reactive oxygen species and disruption of tumor cell mitochondrial integrity. Decreased ATP also lead to inhibition of mTOR1 and mTOR2-dependent signaling pathways via the activation of AMP kinase and the appearance of autophagic vacuoles. We report clinical and pharmacokinetic (PK) results from the first-in-human phase I study of ME-344. Methods: A 3+3 dose escalation design enrolled patients with refractory solid tumors in 6 cohorts at 1.25, 2.5, 5, 10, 15, and 20mg/kg IV weekly times 3, followed by 1 week of rest, then continuous weekly dosing. Restaging occurred every 2 cycles and PK was assessed at day 1 and 15 of cycle 1. Results: Safety: Thirty patients were enrolled. The MTD was established at 10 mg/kg. DLT's were observed at 15 mg/kg (2) and 20 mg/kg (2), all grade 3 neuropathy, and one patient at 10 mg/kg with cardiac chest pain. Possibly related grade 1/2 AE's occurring in >1 patient included: dizziness (5), fatigue (5), nausea (5), asthenia (3), diarrhea (3), dyspnea (3), vomiting (3), abdominal pain (2), chest pain (2), dysphonia (2), headache (2), and neuropathy (2). Pharmacokinetics: ME[[Unable to Display Character: ‐]]344 plasma concentrations declined in a multi[[Unable to Display Character: ‐]]exponential fashion, with a mean half-life of 6 hrs. There was a linear relationship between both Cmax and AUC as a function of dose (mg/kg), with no evidence of accumulation between Day 1 and Day 15. At the MTD of 10 mg/kg, Cmax was 25.78μg/mL and AUC∞ was 25.9 hr*μg/mL. Volume of distribution, half-life and clearance parameters appear dose[[Unable to Display Character: ‐]]independent. Efficacy: Median time on treatment is 56 days (range 1 to 372+). To date, 1 confirmed PR (SCLC, duration 52+ weeks) and 7 prolonged SD (carcinoid (1), cervical (1), leiomyosarcoma (2), NSCLC (1), urothelial (1), uterine (1), 8 - 40+ weeks) have been observed in 23 patients evaluable for efficacy. Conclusions: ME-344 demonstrated dose-limiting toxicity of neuropathy, a known effect of mitochondrial respiratory chain inhibitors. PK data at the 10 mg/kg MTD dose level suggests a sufficient therapeutic index. Evidence of clinical activity at the MTD was observed and supports continued clinical development. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):A17. Citation Format: Johanna C. Bendell, Manish Patel, Jeffrey R. Infante, Carla Kurkjian, Suzanne Jones, Shubham Pant, Howard A. Burris, John D. Hainsworth, Ofir Moreno, Kathleen Moore. ME-344, a novel mitochondrial oxygenase inhibitor: Results from a first-in-human Phase I study. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr A17.

Abstract A268: TAS-119, a selective Aurora A inhibitor, enhanced the antitumor efficacy of taxanes in multiple human tumor cell lines including paclitaxel-resistant cells.

Abstract Background: Aurora A is a serine/ threonine kinase that plays a critical role in regulating multiple mitosis processes. Aberrant expression of Aurora A has been associated with spindle checkpoint dysfunction and increased resistance to the microtubule-polymerizing agents, paclitaxel and docetaxel (1). Aurora A has been identified as one of the genes associated with enhancement of paclitaxel cytotoxicity in a siRNA library screen (2). Moreover, Aurora A overexpression/ gene amplification has been reported in various types of human cancers. In this report, we demonstrate that a selective Aurora kinase A inhibitor, TAS-119, synergistically enhances the activity of paclitaxel in multiple cell lines in vitro. Materials and Methods: Evaluation of synergistic effects in combination was conducted by curve-shift analysis and Bliss additivity model, based on cell viability. Cell viability was assayed by using CellTiter-Glo™ which measures cellular ATP. Selectivity of TAS-119 for three isotypes of Aurora kinases was determined via monitoring the auto-phosphorylation of kinases by Western blot. Apoptosis induction was assessed by cleaved-PARP detection. As paclitaxel-resistant cell lines, both A549.T12 (3) and PTX10 (4) were used in combination experiments. A549.T12 and PTX10 were reported to have altered expression of β-tubulin isotypes and β-tubulin mutation, respectively. Results: TAS-119 selectively inhibited cellular auto-phosphorylation of Aurora A without affecting the activity of both Aurora B and Aurora C. TAS-119 showed a synergistic effect with paclitaxel or docetaxel in multiple tumor cell lines from different origins, even in cells insensitive to TAS-119 treatment alone. Furthermore, TAS-119 also showed synergistic effects in paclitaxel-resistant cell lines. Cleaved-PARP analysis revealed that the cytotoxic synergism in combination with paclitaxel was induced by promoting the caspase-dependent apoptotic cell death. Conclusion: The unique characteristic of TAS-119 as a highly selective Aurora A inhibitor contributed to the synergistic enhancement of antiproliferative effects of taxanes in multiple cell lines, possibly mediated by apoptotic pathways. These findings provide a rationale for the clinical development of TAS-119 as a combination therapy with taxanes. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):A268. Citation Format: Akihiro Miura, Hiroshi Sootome, Keiko Ishihara, Norio Masuko, Hiroshi Hirai, Teruhiro Utsugi. TAS-119, a selective Aurora A inhibitor, enhanced the antitumor efficacy of taxanes in multiple human tumor cell lines including paclitaxel-resistant cells. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr A268.

Abstract C275: CEP-40783: A potent and selective AXL/c-Met inhibitor for use in breast, non-small cell lung (NSCLC), and pancreatic cancers.

Abstract The oncogenic receptor tyrosine kinases AXL and c-Met are over-expressed and constitutively activated in a variety of human cancers. Activation of these receptors contributes to multiple steps in tumor progression by promoting cancer cell migration and invasion, enhancing tumor angiogenesis, facilitating cancer cell survival and tumor growth, and contributing to tumor resistance to several chemotherapy and molecular-targeted therapeutic agents. CEP-40783 is an orally active, potent and selective AXL and c-Met kinase inhibitor, with enzyme IC50 values of 7 nM and 12 nM, respectively. In AXL-transfected 293GT cells, CEP-40783 was 27-fold more active compared to recombinant enzyme with an IC50 value of 0.26 nM. Comparably high cellular potency was observed in NCI-H1299 human NSCL cells. CEP-40783 also demonstrated superior activity against c-Met in GTL-16 cells (IC50 = 6 nM). The increased inhibitory activity of CEP-40783 in cells could be attributed to its extended residence time on both AXL and c-Met, consistent with a Type II mechanism. The prolonged residence time of CEP-40783 at the target may provide for improved in vivo efficacy, selectivity and therapeutic index. Additionally, CEP-40783 showed high kinome selectivity against 298 kinases with an S90 of 0.04 (fraction of kinases showing >90% inhibition at 1 µM). In PK/PD studies, CEP-40783 showed dose- and time-dependent inhibition of AXL phosphorylation using NCI-H1299 NSCL xenografts with ∼80% target inhibition at 0.3 mg/kg 6 h post dose and complete target inhibition to >90% inhibition at 1 mg/kg between 6-24 h, while a 10 mg/kg po dose resulted in complete AXL inhibition up to 48 h post dosing. In AXL/NIH3T3 xenografts, 0.3 mg/kg po resulted in complete tumor regressions. CEP-40783 was also efficacious in reducing spontaneous lymph node and pulmonary metastatic tumor burden in the MDA-MB-231-luc and 4T1-luc orthotopic breast cancer models, respectively, at 10 and 30 mg/kg po. PK/PD evaluation of the c-Met activity of CEP-40783 (10, 30, 55 mg/kg po qdX5d) showed significant to complete inhibition of c-Met phosphorylation in GTL-16 gastric carcinoma xenografts. Efficacy studies in GTL-16 xenografts demonstrated significant anti-tumor efficacy (tumor stasis and regressions) at 10 and 30 mg/kg po. In EBC-1 NSCL xenografts, administration of CEP-40783 (3, 10 and 30 mg/kg, po qd) resulted in dose-related efficacy, with tumor stasis at 3 mg/kg, tumor regressions and >96% TGI at 10 mg/kg. In all studies CEP-40783 was well tolerated with no compound-related body weight loss. CEP-40783 demonstrated potent AXL and c-Met pharmacodynamic and anti-tumor efficacy in established tumor xenograft models, having potential therapeutic utility in multiple human tumor types in which c-Met and AXL activity play a critical role in tumor formation, local invasion and metastasis. Studies in primary human tumorgrafts are in progress. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):C275. Citation Format: Sheila Miknyoczki, Mangeng Cheng, Robert Hudkins, Thelma Angeles, Lisa Aimone, Jean Husten, Jie Qian, Seetha Murthy, Thomas Conners, Robert Bendesky, Amy Landis, Jennifer Grobelny, Hong Chang, Bruce Dorsey, Mark Ator, Bruce Ruggeri. CEP-40783: A potent and selective AXL/c-Met inhibitor for use in breast, non-small cell lung (NSCLC), and pancreatic cancers. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr C275.

Harnessing the genome for characterization of G-protein coupled receptors in cancer pathogenesis.

G-protein coupled receptors (GPCRs) mediate numerous physiological processes and represent the targets for a vast array of therapeutics for diseases ranging from depression to hypertension to reflux. Despite the recognition that GPCRs can act as oncogenes and tumour suppressors by regulating oncogenic signalling networks, few drugs targeting GPCRs are utilized in cancer therapy. Recent large-scale genome-wide analyses of multiple human tumours have uncovered novel GPCRs altered in cancer. However, work aiming to determine which GPCRs from these lists are the drivers of tumourigenesis, and hence valid therapeutic targets, comprises a formidable challenge. The present review highlights recent studies providing evidence that GPCRs are relevant targets for cancer therapy through their effects on known cancer signalling pathways, tumour progression, invasion and metastasis, and the microenvironment. Furthermore, the review also explores how genomic analysis is beginning to highlight GPCRs as therapeutic targets in the age of personalized medicine.

Modulation of OATP1B-Type Transporter Function Alters Cellular Uptake and Disposition of Platinum Chemotherapeutics

Expression of the human organic anion transporting polypeptides OATP1B1 and OATP1B3 has been previously believed to be restricted to hepatocytes. Here we show that the gene encoding OATP1B3, but not OATP1B1, is abundantly expressed in multiple human solid tumors that include hepatocellular, lung, and ovarian carcinomas. Surprisingly, OATP1B3 gene expression in a panel of 60 human tumor cell lines was linked with sensitivity to multiple cytotoxic agents, including the platinum anticancer drugs cisplatin, carboplatin, and oxaliplatin. In addition, overexpression of OATP1B3 in mammalian cells increased cellular accumulation of platinum agents and decreased cell survival. In mice with a targeted disruption of the ortholog transporter Oatp1b2, the liver-to-plasma ratio of cisplatin was significantly reduced compared with wild-type mice, without concurrent changes in expression profiles of other transporter genes. Our findings indicate an unexpected role for tumoral and host OATP1B-type carriers in the toxicity and disposition of platinum anticancer drugs, and may provide a foundation for understanding the extensive interindividual pharmacodynamic variability seen with these drugs in patients.

Protective Role of the Inflammatory CCR2/CCL2 Chemokine Pathway through Recruitment of Type 1 Cytotoxic γδ T Lymphocytes to Tumor Beds

Tumor-infiltrating lymphocytes (TILs) are important prognostic factors in cancer progression and key players in cancer immunotherapy. Although γδ T lymphocytes can target a diversity of tumor cell types, their clinical manipulation is hampered by our limited knowledge of the molecular cues that determine γδ T cell migration toward tumors in vivo. In this study we set out to identify the chemotactic signals that orchestrate tumor infiltration by γδ T cells. We have used the preclinical transplantable B16 melanoma model to profile chemokines in tumor lesions and assess their impact on γδ TIL recruitment in vivo. We show that the inflammatory chemokine CCL2 and its receptor CCR2 are necessary for the accumulation of γδ TILs in B16 lesions, where they produce IFN-γ and display potent cytotoxic functions. Moreover, CCL2 directed γδ T cell migration in vitro toward tumor extracts, which was abrogated by anti-CCL2 neutralizing Abs. Strikingly, the lack of γδ TILs in TCRδ-deficient but also in CCR2-deficient mice enhanced tumor growth in vivo, thus revealing an unanticipated protective role for CCR2/CCL2 through the recruitment of γδ T cells. Importantly, we demonstrate that human Vδ1 T cells, but not their Vδ2 counterparts, express CCR2 and migrate to CCL2, whose expression is strongly deregulated in multiple human tumors of diverse origin, such as lung, prostate, liver, or breast cancer. This work identifies a novel protective role for CCL2/CCR2 in the tumor microenvironment, while opening new perspectives for modulation of human Vδ1 T cells in cancer immunotherapy.

Phase I study of the safety and pharmacokinetics of epitinib, an oral EGFR tyrosine kinase inhibitor, in patients with advanced solid tumors.

e19042 Background: Epitinib is an oral small molecule compound selectively inhibits epidermal growth factor receptor (EGFR). It has demonstrated potent inhibitory effects on multiple human tumor xenografts with a large safety window and broad tissue distribution including high drug contribution in brain. This first-in-human study is being conducted to assess the safety and tolerability, pharmacokinetics (PK), and preliminary anti-tumor activity of Epitinib. Methods: This phase I study uses a 3+3 design for dose-escalation of Epitinib given once daily (QD) in 28-day treatment cycles in patients with solid tumors who failed or have no access to standard therapies. Safety and tumor response are assessed according to NCI CTCAE 4.0 and RECIST 1.1, respectively. Results: By Dec 31, 2012, 19 pts aged 40-66 yr old (ECOG PS 1) with NSCLC (n=16) or breast cancer (n=3) were enrolled in 5 dose cohorts of 20-160 mg. Six NSCLC pts had EGFR mutations (3 with T790M). All pts had ≥2 lines of prior systemic treatments (7 pts with gefitinib or elotinib). Most AEs were mild (Gr1). Treatment-related AEs included skin rash (36.8%), abnormal liver function tests (42.1%), diarrhea (10.5%), vomiting (5.3%), paronychia (5.3%), decreased neutrophil count (5.3%) and increased creatinine (5.3%). Three SAEs were reported as possibly unrelated to the study drug. No DLT was observed and MTD has not been reached yet. PK analysis showed that the mean elimination half-life of Epitinib was approximately 40 hours and Tmax was around 2 h. Both Cmaxand AUC exhibited good dose proportionality over the studied dose range with low inter-patient variability following single and multiple doses. Among 15 evaluable pts, 1 EGFR mutation+ NSCLC pt achieved confirmed partial response (66.7% tumor reduction) for more than 6 months and the treatment is ongoing; 8 pts had stable disease, including 1 with T790M for 6 months and 1 with brain metastasis for 12 months who is still on treatment. Conclusions: Epitinib was well tolerated at doses up to 160mg QD to date with manageable adverse events and excellent pharmacokinetic properties. Encouraging clinical activity was observed, including durable SD in NSCLC patients with T790M or brain mets.