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EGFR exon 20 insertion driver mutations (Exon20ins) in non–small cell lung cancer (NSCLC) are insensitive to EGFR tyrosine kinase inhibitors (TKI). Amivantamab (JNJ-61186372), a bispecific antibody targeting EGFR–MET, has shown preclinical activity in TKI-sensitive EGFR-mutated NSCLC models and in an ongoing first-in-human study in patients with advanced NSCLC. However, the activity of amivantamab in Exon20ins-driven tumors has not yet been described. Ba/F3 cells and patient-derived cells/organoids/xenograft models harboring diverse Exon20ins were used to characterize the antitumor mechanism of amivantamab. Amivantamab inhibited proliferation by effectively downmodulating EGFR–MET levels and inducing immune-directed antitumor activity with increased IFNγ secretion in various models. Importantly, in vivo efficacy of amivantamab was superior to cetuximab or poziotinib, an experimental Exon20ins-targeted TKI. Amivantamab produced robust tumor responses in two Exon20ins patients, highlighting the important translational nature of this preclinical work. These findings provide mechanistic insight into the activity of amivantamab and support its continued clinical development in Exon20ins patients, an area of high unmet medical need. Significance: Currently, there are no approved targeted therapies for EGFR Exon20ins–driven NSCLC. Preclinical data shown here, together with promising clinical activity in an ongoing phase I study, strongly support further clinical investigation of amivantamab in EGFR Exon20ins–driven NSCLC. This article is highlighted in the In This Issue feature, p. 1079

Purpose Preclinical characterization of cetrelimab (JNJ-63723283), a fully humanized immunoglobulin G4 kappa monoclonal antibody targeting programmed cell death protein-1 (PD-1), in human cancer models. Methods Cetrelimab was generated by phage panning against human and cynomolgus monkey (cyno) PD-1 extracellular domains (ECDs) and affinity maturation. Binding to primate and rodent PD-1 ECDs, transfected and endogenous cell-surface PD-1, and inhibition of ligand binding were measured. In vitro activity was evaluated using cytomegalovirus recall, mixed lymphocyte reaction, staphylococcal enterotoxin B stimulation, and Jurkat-PD-1 nuclear factor of activated T cell reporter assays. In vivo activity was assessed using human PD-1 knock-in mice implanted with MC38 tumors and a lung patient-derived xenograft (PDX) model (LG1306) using CD34 cord-blood-humanized NSG mice. Pharmacodynamics, toxicokinetics, and safety were assessed in cynos following single and/or repeat intravenous dosing. Results Cetrelimab showed high affinity binding to human (1.72 nM) and cyno (0.90 nM) PD-1 and blocked binding of programmed death-ligand 1 (PD-L1; inhibitory concentration [IC] 111.7 ng/mL) and PD-L2 (IC 138.6 ng/mL). Cetrelimab dose-dependently increased T cell-mediated cytokine production and stimulated cytokine expression. Cetrelimab 10 mg/kg reduced mean MC38 tumor volume in PD-1 knock-in mice at Day 21 (P P Conclusion Cetrelimab potently inhibits PD-1 in vitro and in vivo, supporting its clinical evaluation.

The success of targeted or immune therapies is often hampered by the emergence of resistance and/or clinical benefit in only a subset of patients. We hypothesized that combining targeted therapy with immune modulation would show enhanced antitumor responses. Here, we explored the combination potential of erdafitinib, a fibroblast growth factor receptor (FGFR) inhibitor under clinical development, with PD-1 blockade in an autochthonous FGFR2K660N/p53mut lung cancer mouse model. Erdafitinib monotherapy treatment resulted in substantial tumor control but no significant survival benefit. Although anti–PD-1 alone was ineffective, the erdafitinib and anti–PD-1 combination induced significant tumor regression and improved survival. For both erdafitinib monotherapy and combination treatments, tumor control was accompanied by tumor-intrinsic, FGFR pathway inhibition, increased T-cell infiltration, decreased regulatory T cells, and downregulation of PD-L1 expression on tumor cells. These effects were not observed in a KRASG12C-mutant genetically engineered mouse model, which is insensitive to FGFR inhibition, indicating that the immune changes mediated by erdafitinib may be initiated as a consequence of tumor cell killing. A decreased fraction of tumor-associated macrophages also occurred but only in combination-treated tumors. Treatment with erdafitinib decreased T-cell receptor (TCR) clonality, reflecting a broadening of the TCR repertoire induced by tumor cell death, whereas combination with anti–PD-1 led to increased TCR clonality, suggesting a more focused antitumor T-cell response. Our results showed that the combination of erdafitinib and anti–PD-1 drives expansion of T-cell clones and immunologic changes in the tumor microenvironment to support enhanced antitumor immunity and survival.

The protein arginine methyltransferase 5 (PRMT5) methylates a variety of proteins involved in splicing, multiple signal transduction pathways, epigenetic control of gene expression, and mechanisms leading to protein expression required for cellular proliferation. Dysregulation of PRMT5 is associated with clinical features of several cancers, including lymphomas, lung cancer, and breast cancer. Here, we describe the characterization of JNJ-64619178, a novel, selective, and potent PRMT5 inhibitor, currently in clinical trials for patients with advanced solid tumors, non-Hodgkin's lymphoma, and lower-risk myelodysplastic syndrome. JNJ-64619178 demonstrated a prolonged inhibition of PRMT5 and potent antiproliferative activity in subsets of cancer cell lines derived from various histologies, including lung, breast, pancreatic, and hematological malignancies. In primary acute myelogenous leukemia samples, the presence of splicing factor mutations correlated with a higher ex vivo sensitivity to JNJ-64619178. Furthermore, the potent and unique mechanism of inhibition of JNJ-64619178, combined with highly optimized pharmacological properties, led to efficient tumor growth inhibition and regression in several xenograft models in vivo, with once-daily or intermittent oral-dosing schedules. An increase in splicing burden was observed upon JNJ-64619178 treatment. Overall, these observations support the continued clinical evaluation of JNJ-64619178 in patients with aberrant PRMT5 activity-driven tumors. ispartof: MOLECULAR CANCER THERAPEUTICS vol:20 issue:12 pages:2317-2328 ispartof: location:United States status: published

JNJ-78306358 is a first-in-class bispecific antibody (bsAb), engineered using the Zymeworks Azymetric™ platform, to treat advanced stage solid tumors. Human leukocyte antigen G (HLA-G) is a non-classical major histocompatibility class I molecule with an immune tolerance role at the maternal-fetal interface. HLA-G has limited normal tissue expression, mainly detected in placenta and pituitary gland. However, HLA-G is expressed in multiple human cancers, with a potential role in cancer immune evasion. Comprehensive immunohistochemistry analysis of patient-derived tumors revealed high prevalence of HLA-G expression in renal cell carcinoma (RCC, 75%), ovarian (61%), colon (64%) and rectal cancers (40%), and moderate HLA-G expression prevalence in lung adenocarcinoma, endometrial, and pancreatic cancer. JNJ-78306358 induces HLA-G-expressing tumor cell killing via T cell redirection. This bsAb features an anti-HLA-G single-chain fragment variable (scFv) domain that binds with high affinity (KD ~ 13 pM) to HLA-G on tumor cells and a Fab domain that binds with weaker affinity (KD ~22 nM) to the epsilon subunit of the cluster of differentiation 3 (CD3ε). The immunoglobulin (Ig)G1 heavy chains feature Fc region mutations that disrupt interaction with Fcγ receptors. JNJ-78306358 demonstrated potent PBMC- and T cell-mediated in vitro cytotoxicity (EC50 10.4 - 442.3 pM) against endogenous membrane HLA-G-expressing tumor cell lines and absence of killing against cancer cells lacking HLA-G membrane expression, highlighting the specificity against antigen-expressing tumor cells. JNJ-78306358 exhibited hallmarks of T cell engagement in vitro, including T cell proliferation and cytokine release. In addition, JNJ-78306358 showed HLA-G-expression-dependent anti-tumor activity in mice (humanized with human donor CD3+ pan-T cells or human umbilical cord-blood-derived CD34+ hematopoietic stem cells [HSCs]) bearing cell line- and patient-derived tumors. In these xenograft models, a dose-dependent increase in CD4+ and CD8+ T cell infiltration into tumors was observed with complete tumor regressions at low doses of JNJ-78306358 (0.03 mg/kg). JNJ-78306358’s safety, tolerability and preliminary anti-tumor activity are currently being evaluated in a first-in-human phase I study in advanced stage solid tumors with high prevalence of HLA-G protein expression (NCT04991740). This antigen-targeting approach may address an unmet medical need in patients with tumors expressing HLA-G. Citation Format: Nataša Obermajer, Adam Zwolak, Kelly Van De Ven, Shana Versmissen, Aleksandra Brajic, Ted Petley, Dan Weinstock, Jason Aligo, Fang Yi, Stephen Jarantow, Keith Schutsky, Ken Tian, Angelilo Lorraine, Diana Alvarez Arias, Kristel Buyens, Vince Torti, Krista Menard, Katharine Rogers, Brian Geist, Marjolein Van Heerden, Gerald Chu, Bie Verbist, Maté Ongenaert, Julien Hasler, Kathryn Packman, Jacintha Shenton, Dirk Brehmer, Josh Lauring, Regina J. Brown, James Greger, Daphne Salick Ryan, Sanjaya Singh, Matthew V. Lorenzi, Laurie Lenox, Sylvie Laquerre. JNJ-78306358: A first-in-class bispecific T cell redirecting HLA-G antibody [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr ND07.

Fibroblast growth factor (FGF) signaling plays critical roles in key biological processes ranging from embryogenesis to wound healing and has strong links to several hallmarks of cancer. Genetic alterations in FGF receptor (FGFR) family members are associated with increased tumor growth, metastasis, angiogenesis, and decreased survival. JNJ-42756493, erdafitinib, is an orally active small molecule with potent tyrosine kinase inhibitory activity against all four FGFR family members and selectivity versus other highly related kinases. JNJ-42756493 shows rapid uptake into the lysosomal compartment of cells in culture, which is associated with prolonged inhibition of FGFR signaling, possibly due to sustained release of the inhibitor. In xenografts from human tumor cell lines or patient-derived tumor tissue with activating FGFR alterations, JNJ-42756493 administration results in potent and dose-dependent antitumor activity accompanied by pharmacodynamic modulation of phospho-FGFR and phospho-ERK in tumors. The results of the current study provide a strong rationale for the clinical investigation of JNJ-42756493 in patients with tumors harboring FGFR pathway alterations. Mol Cancer Ther; 16(6); 1010–20. ©2017 AACR.

Small molecule inhibitors targeting mutant EGFR are standard of care in non–small cell lung cancer (NSCLC), but acquired resistance invariably develops through mutations in EGFR or through activation of compensatory pathways such as cMet. Amivantamab (JNJ-61186372) is an anti-EGFR and anti-cMet bispecific low fucose antibody with enhanced Fc function designed to treat tumors driven by activated EGFR and/or cMet signaling. Potent in vivo antitumor efficacy is observed upon amivantamab treatment of human tumor xenograft models driven by mutant activated EGFR, and this activity is associated with receptor downregulation. Despite these robust antitumor responses in vivo, limited antiproliferative effects and EGFR/cMet receptor downregulation by amivantamab were observed in vitro. Interestingly, in vitro addition of isolated human immune cells notably enhanced amivantamab-mediated EGFR and cMet downregulation, leading to antibody dose-dependent cancer cell killing. Through a comprehensive assessment of the Fc-mediated effector functions, we demonstrate that monocytes and/or macrophages, through trogocytosis, are necessary and sufficient for Fc interaction-mediated EGFR/cMet downmodulation and are required for in vivo antitumor efficacy. Collectively, our findings represent a novel Fc-dependent macrophage-mediated antitumor mechanism of amivantamab and highlight trogocytosis as an important mechanism of action to exploit in designing new antibody-based cancer therapies.

Objectives Treatment of non-small cell lung cancer (NSCLC) with immune checkpoint blockade (ICB) has resulted in striking clinical responses, but only in a subset of patients. The goal of this study was to evaluate transcriptional signatures previously reported in the literature in an independent cohort of NSCLC patients receiving ICB. Materials and methods This retrospective study analyzed transcriptional profiles from pre-treatment tumor samples of 52 chemotherapy-refractory advanced NSCLC patients treated with anti-PD1/PD-L1 therapy. Gene signatures based on published reports were created and examined for their association with response to therapy and progression-free and overall survival (PFS, OS). Results Two signatures predicting response and outcomes were identified. One reflected the degree of immune infiltration and upregulation of interferon-gamma-induced genes. A second reflected the EMT status. Compared to those not responding to therapy, patients whose tumors responded to ICB had higher scores in an inflammatory gene signature (6.0 ± 2.9 vs -5.5 ± 3.4, p = 0.014) or a more epithelial phenotype (-1.7 ± 1.0 vs 2.1 ± 1.2, p = 0.016). Both signatures demonstrated a satisfactory predictive accuracy for response: AUC of 0.69 (95% CI: 0.54, 0.84) for the inflammatory and 0.70 (95% CI: 0.55, 0.85) for EMT signatures, respectively. A weighted score combining EMT and inflammatory signatures showed increased predictive value with AUC of 0.92 (95% CI: 0.85, 0.99). Kaplan-Meier curves for patients above and below the median combined score showed a significant separation for PFS and OS (all p Conclusions The EMT/Inflammation signature score may be useful in directing checkpoint inhibitor therapy in lung cancer and suggests that reversal of EMT might augment efficacy of ICB.

Deep understanding of the genomic and immunological differences between Chinese and Western lung cancer patients is of great importance for target therapy selection and development for Chinese patients. Here we report an extensive molecular and immune profiling study of 245 Chinese patients with non-small cell lung cancer. Tumor-infiltrating lymphocyte estimated using immune cell signatures is found to be significantly higher in adenocarcinoma (ADC, 72.5%) compared with squamous cell carcinoma (SQCC, 54.4%). The correlation of genomic alterations with immune signatures reveals that low immune infiltration was associated with EGFR mutations in ADC samples, PI3K and/or WNT pathway activation in SQCC. While KRAS mutations are found to be significantly associated with T cell infiltration in ADC samples. The SQCC patients with high antigen presentation machinery and cytotoxic T cell signature scores are found to have a prolonged overall survival time., The relationship between genomic alteration and immune context in non-small cell lung cancer (NSCLC) is complex. Here, the authors analyse the molecular and immunological landscape of 245 Chinese patients with NSCLC and find low immune infiltration correlates with genomic alterations.

Small molecule EGFR inhibitors have proven effective in treatment of Non-Small Cell Lung Cancer (NSCLC) with activating EGFR mutations, however there is minimal to no efficacy in wild-type EGFR NSCLC. Amivantamab, an EGFR/MET bispecific antibody, has demonstrated clinical activity across a diverse range of EGFR activating mutations in an ongoing Phase I trial and differentiates from anti-EGFR antibodies tested previously in NSCLC clinical trials due to dual targeting and enhanced interaction with Fc receptors on immune cells. As candidate biomarkers to predict response in wt EGFR disease, EGFR and MET expression (immunohistochemistry (IHC)) and signaling (proximity ligation assays (PLA)), as well as tumor associated macrophage (TAM) content, were assessed in 39 NSCLC wt EGFR patient-derived xenograft (PDX) models. A strong correlation (EGFR r=0.63; p Citation Format: Benjamin Henley, Kristen Chevalier, Matthew Smith, Smruthi Vijayaraghaven, Barbara Bushey, Gerald Chu, Joshua Curtin, Nataša Obermajer, Kathryn Packman, Sylvie Laquerre, Matthew Lorenzi, Eric Haura, Sheri Moores. Efficacy of amivantamab, a bispecific EGFR/MET antibody, correlates with EGFR expression and signaling in NSCLC models with wild-type EGFR [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 953.

PRMT5 is a type II methyltransferase that specifically adds methyl groups to arginine as a long-lasting post-translational modification. The PRMT5/MEP50 complex regulates a plethora of cellular processes, such as epigenetics and splicing, which are notable events involved in tumorigenesis. Although not frequently mutated or amplified in tumors, elevated PRMT5 protein levels in lung and hematologic cancers are correlated with poorer survival. The PRMT5 inhibitor JNJ-64619178 has been selected as a clinical candidate based on its high selectivity and potency (subnanomolar range) under different in vitro and cellular conditions, paired with favorable pharmacokinetics and safety properties. JNJ-64619178 binds into the SAM binding pocket and reaches the substrate binding pocket to inhibit PRMT5/MEP50 function in a time-dependent manner. Broad cell line panel profiling of JNJ-64619178 revealed a wide range of sensitivity, which is indicative of a genomic dependency instead of a general cytotoxic on-target consequence of PRMT5 inhibition. Further investigations indicate a synthetic lethal correlation between PRMT5 inhibition and key cancer driver pathways. JNJ-64619178, dosed orally (10 mg/kg, every day), showed selective and efficient blockage of the methylation of SMD1/3 proteins, which are crucial components of the spliceosome and substrates of PRMT5/MEP50. JNJ-64619178 also demonstrated tumor regression in a biomarker-driven human small cell lung cancer xenograft model (NCI-H1048) and prolonged tumor growth inhibition after dosing cessation. In rodent and nonrodent toxicology studies, a tolerated dose of JNJ-64619178 has been identified, with the observed toxicity consistent with on-target activity. In summary, JNJ-64619178 has a favorable preclinical package that supports clinical testing in patients diagnosed with lung cancer and hematologic malignancies. Citation Format: Dirk Brehmer, Tongfei Wu, Geert Mannens, Lijs Beke, Petra Vinken, Dana Gaffney, Weimei Sun, Vineet Pande, Jan-Willem Thuring, Hillary Millar, Italo Poggesi, Ivan Somers, An Boeckx, Marc Parade, Erika van Heerde, Thomas Nys, Carol Yanovich, Barbara Herkert, Tinne Verhulst, Marc Du Jardin, Lieven Meerpoel, Christopher Moy, Gaston Diels, Marcel Viellevoye, Wim Schepens, Alain Poncelet, Joannes T. Linders, Edward C. Lawson, James P. Edwards, Dushen Chetty, Sylvie Laquerre, Matthew V. Lorenzi. A novel PRMT5 inhibitor with potent in vitro and in vivo activity in preclinical lung cancer models [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 DDT02-04. doi:10.1158/1538-7445.AM2017-DDT02-04

PURPOSE: Although EGFR exon20 insertion (ex20ins) mutations account for 4~12 % of EGFR mutant NSCLC patients, there is no effective and selectable anticancer drugs targeting ex20ins mutations so far due to various variant mutations in ex20ins mutations. Moreover, most EGFR ex20ins mutants show primary resistance to EGFR TKIs. JNJ-61186372 (JNJ-372), a bispecific antibody that targets the EGFR and cMet receptors, is currently being explored in a first-in-human study in patients with NSCLC. To better understand the mechanism of JNJ-372 activity in this patient population, we conducted preclinical studies exploring the activity of JNJ-372 in different EGFR ex20ins models. METHODS: To elucidate whether JNJ-372 has antitumor effect in EGFR ex20ins mutants via EGFR and c-MET inhibition, cell viability, western blot, cell cycle, colony formation assay, FACS analysis were performed in JNJ-372 treated BaF3 cells, PDCs, PDOs, and PDX expressing EGFR ex20ins mutation. For mouse tumor models, JNJ-372 was administered i.p. twice a week at 10 mg/kg or 30 mg/kg. Antibody dependent cellular cytotoxicity (ADCC) assay was assessed to figure out whether JNJ-372 had ADCC effects. Referenced patients with ex20ins disease were administered 1050 mg JNJ-372 i.v. weekly for the first 4-week cycle, then biweekly for each subsequent cycle RESULTS: JNJ-372 inhibited the growth of BaF3 cells, PDCs, and a PDO harboring a range of ex20ins, which were resistant to osimertinib and gefitinib. Mechanistic assays revealed the reduction of EGFR and cMet receptor levels and decreases in phospho-EGFR and c-Met, as well as inhibition of their downstream signaling pathways. Cleaved caspase-3 and BIMEL were upregulated at anti-proliferative doses, suggesting caspase-mediated cell death. JNJ-372 demonstrated corresponding antitumor activity in PDC and PDX models harboring different ex20ins; inhibition of signaling and engagement of the apoptotic pathway was confirmed in tumors of JNJ-372-treated mice. In PDCs with EGFR ex20ins mutation, we verified that JNJ-372 had a significant ADCC effect compared to EGFR antibody drug cetuximab. In the first-in-human trial, CT scans from two patients treated with JNJ-372 revealed reductions in tumor burden. A 58-year patient harboring H773delinsNPY showed -63% tumor reduction with progression-free survival of > 20 months and a 48-year patient harboring S768_D770dup showed -38.9% tumor reduction. CONCLUSION: JNJ-372 drives antitumor activity in preclinical models of EGFR ex20ins, which have no therapeutic options in clinic, by decreasing EGFR and cMet receptor levels, inhibiting downstream signaling cascades, activating apoptotic signaling as well as ADCC. These results provide a promising therapeutic option to patients with EGFR ex20ins mutations and an understanding of the activity of JNJ-372 being observed in the first-in-human study. Citation Format: Jiyeon Yun, Han Na Kang, Soo-Hwan Lee, Seo-Yoon Jeong, Chae Won Park, Jae-Hwan Kim, Kyoung-Ho Pyo, Ji Min Lee, Seok-Young Kim, Min Hee Hong, Hye Ryun Kim, Meena Thayu, Joshua Curtin, Roland Knoblauch, Matthew Lorenzi, Byoung Chul Cho. JNJ-61186372, an EGFR-cMet bispecific antibody, in EGFR Exon 20 insertion-driven advanced non-small cell lung cancer (NSCLC) [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5199.

Small molecule inhibitors targeting EGFR are now standard of care in NSCLC patients harboring EGFR mutations, but acquired resistance invariably develops through secondary mutations within EGFR and/or through activation of compensatory pathways such as cMet. JNJ-61186372 (JNJ-372) is an anti-EGFR and cMet bispecific antibody with enhanced binding to immune cell Fcγ receptors, designed to target tumors with activated EGFR and cMet signaling through a distinct mechanism of action. Ongoing first-in-human study in patients with advanced, treatment refractory EGFR mutant NSCLC revealed JNJ-372 to have clinical activity in patients with diverse EGFR-mutated NSCLC, including Exon 20 mutations, TKI resistance mutations (T790M, C797S), and resistance due to MET amplification. However preclinically, despite potent anti-tumor activity in NSCLC xenograft models, only modest anti-proliferative effects were observed with JNJ-372 in cell lines in vitro. Interestingly, the addition of isolated human immune cells (PBMCs) to the in vitro assays enhanced JNJ-372-mediated EGFR and cMet downregulation, and dose-dependent tumor cell killing. Through depletion or enrichment of individual immune cell types, we demonstrated that monocytes and/or macrophages are necessary for JNJ-372 Fc interaction-mediated EGFR/cMet downmodulation. Depletion of macrophages in mice showed that they are required for JNJ-372 anti-tumor efficacy. Finally, we showed that the down-modulation of EGFR and cMet receptors occurs through monocyte or macrophage-mediated trogocytosis. Collectively, these results demonstrate a novel Fc-dependent mechanism of action for JNJ-372 and support its continued clinical development in patients with aberrant EGFR and cMet signaling. Citation Format: Smruthi Vijayaraghavan, Lorraine Lipfert, Barbara Bushey, Kristen Chevalier, Benjamin Henley, Ryan Lenhart, Marilda Beqiri, Hillary J. Millar, Kathryn Packman, Matthew V. Lorenzi, Sylvie Laquerre, Sheri Moores. JNJ-61186372, an Fc enhanced EGFR/cMet bispecific antibody, mediates EGFR and cMet downmodulation and therapeutic efficacy preclinically through monocyte / macrophage mediated trogocytosis [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5651.

Cancer stem cells (CSCs) represent a unique subset of cells within a tumor that possess self-renewal capacity and pluripotency, and can drive tumor initiation and maintenance. First identified in hematological malignancies, CSCs are now thought to play an important role in a wide variety of solid tumors such as NSCLC, breast and colorectal cancer. The role of CSCs in driving tumor formation illustrates the dysregulation of differentiation in tumorigenesis. The Wnt, Notch and Hedgehog (HH) pathways are developmental pathways that are commonly activated in many types of cancer. While substantial progress has been made in developing therapeutics targeting Notch and HH, the Wnt pathway has remained an elusive therapeutic target. This review will focus on the clinical relevance of the Wnt pathway in CSCs and tumor cell biology, as well as points of therapeutic intervention and recent advances in targeting Wnt/β-catenin signaling.

Imatinib Mesylate (IM) and other tyrosine kinase inhibitor (TKI) therapies have had a major impact on the treatment of chronic myeloid leukemia (CML). However, TKI monotherapy is not curative, with relapse and persistence of leukemic stem cells (LSCs) remaining a challenge. We have recently identified an AHI-1-BCR-ABL-JAK2 protein complex that contributes to the transforming activity of BCR-ABL and IM-resistance in CML stem/progenitor cells. JAK2 thus emerges as an attractive target for improved therapies, but off-target effects of newly developed JAK2 inhibitors on normal hematopoietic cells remain a concern. We have examined the biological effects of a highly selective, orally bioavailable JAK2 inhibitor, BMS-911543, in combination with TKIs on CD34+ treatment-naive IM-nonresponder cells. Combination therapy reduces JAK2/STAT5 and CRKL activities, induces apoptosis, inhibits proliferation and colony growth, and eliminates CML LSCs in vitro. Importantly, BMS-911543 selectively targets CML stem/progenitor cells while sparing healthy stem/progenitor cells. Oral BMS-911543 combined with the potent TKI dasatinib more effectively eliminates infiltrated leukemic cells in hematopoietic tissues than TKI monotherapy and enhances survival of leukemic mice. Dual targeting BCR-ABL and JAK2 activities in CML stem/progenitor cells may consequently lead to more effective disease eradication, especially in patients at high risk of TKI resistance and disease progression.

JNJ-61186372 (JNJ-372) is an anti-EGFR and cMet bispecific antibody with an active Fc backbone (IgG1) designed to treat non-small cell lung cancer (NSCLC) disease. A first-in-human study is currently being conducted to assess the safety and preliminary efficacy of JNJ-372 in patients with advanced NSCLC. Early data suggests that JNJ-372 can induce partial responses in subjects with diverse populations of EGFR-mutated NSCLC, including Exon 20ins as well as TKI resistance mutations. Our previous pre-clinical in vivo studies showed that the Fc inactive version (IgG2sigma) of the EGFR/cMet antibody was significantly impaired in its ability to inhibit tumor growth compared to the Fc active JNJ-372. The IgG2sigma variant also reduced the ability of the bispecific antibody to mediate downregulation of EGFR, cMet and downstream signaling components. This suggested that the interaction of the Fc arm with the Fc receptors on the innate immune cells play a crucial role in the mechanism of action of JNJ-372. While JNJ-372 has demonstrated antibody dependent cellular cytotoxicity (ADCC) and antibody dependent cellular phagocytosis (ADCP) in vitro, the potential role of Fc interactions in downregulation of EGFR and cMet was not well understood. We explored the different Fc-mediated immune effector functions of JNJ-372 and interrogated how they contribute to EGFR and cMet downregulation and overall efficacy. In NSCLC cell lines, JNJ-372 induced Fc-mediated dose-dependent ADCC and ADCP but not complement-dependent cytotoxicity. Further, the presence of isolated human immune cells (PBMC) significantly enhanced JNJ-372 mediated EGFR and cMet downregulation and dose-dependent tumor cell killing. Studies are in progress to better understand which immune cells and which Fc receptor interactions are essential for the drug efficacy through immune cell depletion and FcR blocking studies; such studies may help guide clinical biomarker development. This work elucidates a novel Fc-dependent mechanism of action for the EGFR-cMet bispecific antibody. Citation Format: Smruthi Vijayaraghavan, Barbara Bushey, Lorriane Lipfert, Rupesh Nanjunda, Eilyn R. Lacy, Peter Buckley, Sylvie Laquerre, Matthew V. Lorenzi, Sheri Moores. Fc-mediated mechanism of action for the novel EGFR-cMET bispecific antibody (JNJ-61186372) in non-small cell lung cancer [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 4818.

9009 Background: JNJ-372 binds EGFR and cMet to block ligand binding, promote receptor degradation, and trigger antibody-dependent cellular cytotoxicity in models of EGFR-mutated (EGFRm) NSCLC. Here we describe the ongoing phase 1 safety, pharmacokinetics (PK), and activity of JNJ-372 in patients (pts) with NSCLC, including 3rd generation tyrosine kinase inhibitor (3GTKI)-relapsed EGFRm NSCLC and EGFR Exon20ins disease. Methods: Pts received JNJ-372 (140–1400 mg) IV weekly for the first 28-day cycle and biweekly thereafter. 1050–1400 mg doses are being explored in dose expansion. Blood samples were collected for PK analyses. Efficacy by investigator per RECIST v1.1 in pts with EGFRm NSCLC treated at ≥700 mg is presented. Tumors were characterized by next-generation sequencing of circulating tumor (ct)DNA and/or tumor tissue. Results: As of 17 Jan 2019, 116 enrolled pts with NSCLC were treated. Median age was 63 years, 38% were male, 77% were Asian, and 97% had EGFR mutations. Mean duration of treatment was 3.8 months, longest exposure was 20 cycles. The PK data set included pts from Korea (77%) and the US (23%). At the 1050 mg dose, 72% of pts achieved average concentrations above the EC90 based on preclinical models. Adverse events (AEs; ≥20%) were rash (59%), infusion related reaction (58%), paronychia (28%), and constipation (22%). Additional EGFR/cMet-related AEs include stomatitis (17%), pruritis (15%), peripheral edema (11%), and diarrhea (7%). Grade ≥3 AEs were reported in 34% (8% treatment-related) with dyspnea (6%) and pneumonia (3%) most frequently observed. Among response-evaluable pts, 25/88 (28%) achieved best timepoint response of partial response (PR). 10/47 pts with prior 3GTKI therapy had best timepoint response of PR (6 confirmed), including 4 with C797S, 1 with cMet amplification, and 5 without identifiable EGFR/cMet-dependent resistance. 6/20 pts with Exon20ins had best timepoint response of PR (3 confirmed). Conclusions: JNJ-372 has a manageable safety profile consistent with EGFR and cMet inhibition. Preliminary responses were achieved in 3GTKI-relapsed disease, including C797S and cMet amplification, and Exon20ins disease; enrollment in dose expansion is ongoing. Clinical trial information: NCT02609776.

9093 Background: The immunogenicity, safety, and efficacy of JNJ-757, a live attenuated, double-deleted Listeria monocytogenes-based immunotherapy expressing human mesothelin (MSLN), were evaluated in patients (pts) with advanced NSCLC (adenocarcinoma) as monotherapy (phase 1) and in combination with nivolumab (phase 1b/2). Methods: Adult pts with Stage IIIB/IV NSCLC who had received prior systemic therapy (including 1 platinum-based chemotherapy, prior PD-1/PD-L1 therapy allowed) were included. Dose-limiting toxicities, adverse events (AEs), tumor response, T cell response, and JNJ-757 bacterial shedding profile were evaluated in pts treated with JNJ-757 (108 or 109 colony forming units [CFU]) alone or JNJ-757 (109 CFU)+nivolumab 240 mg combination therapy until progression. Results: In the monotherapy trial, 18 pts (median age 63.5 years; women 61%) were treated with JNJ-757 108 or 109 CFU with a median duration of 1.4 months (range 0-29). Most common AEs were pyrexia (72%) and chills (61%), which were usually mild and resolved within 48 hours, suggesting transient activation of an innate immune response to JNJ-757. Treatment-related grade ≥3 AEs were infrequent (4 [22%]). Induction of peripheral proinflammatory cytokines and lymphocyte activation was observed post-treatment with transient MSLN-specific T cell responses in 10/13 evaluable pts, consistent with the mechanism of action of JNJ-757. With monotherapy, 4/18 response-evaluable pts had stable disease (SD) ≥16 weeks, including 1 pt with a 53% reduction in target lesions. In the combination therapy study, 12 pts were enrolled (median age 63.5 years; women 33%). The most common AEs were pyrexia (67%) and chills (58%); 6 pts had grade ≥3 AEs including 2 cases of treatment-related fatal pneumonitis. Best overall response for the combination was SD in 4/9 evaluable pts. JNJ-757 in combination with nivolumab suggested increased risk of pneumonitis. Conclusions: As monotherapy, JNJ-757 was tolerable with mild infusion-related fever and chills supporting the initiation of the combination therapy study. However, the risk-benefit profile of JNJ-757+nivolumab, did not support proceeding to phase 2. Clinical trial information: NCT02592967, NCT03371381.

420 Background: Plasma samples from a Phase 2 study of the pan-FGFR inhibitor erdafitinib in advanced UC patients (pts) with FGFR mutations (mut) or fusions, were tested using next generation sequencing (NGS) for circulating tumor DNA (ctDNA), and results compared to central FGFR status determination from tissue. Methods: FGFR alterations were measured in archival tissue using an RNA-based RT-PCR test and compared with FGFR alterations identified in pre-treatment plasma specimens using the Guardant360 ctDNA test. Sensitivity of the ctDNA test to detect the FGFR alterations identified by RT-PCR from tissue, the proportion of pts with a study-eligible FGFR alteration in ctDNA, and the ability to detect a tissue FGFR alteration in ctDNA in relation to clinical response were assessed. Results: Samples from 155 pts with detectable baseline ctDNA were included. Overall, concordance between ctDNA and tissue-based FGFR results was 56% (87/155): 63% (77/122) for tissue mut-positive pts vs 24% (7/29) in fusion-positive pts. 61% of pts (94/155) were positive for a study-eligible FGFR alteration from blood-based testing in the tissue-positive population. The response rate was 47% (36/77) for patients for which FGFR mutations could be detected in blood (ctDNA- FGFR-positive) compared with 32% (14/44) in patients for which mutations were not detected in blood (ctDNA- FGFR-negative), with an estimated odds ratio of 1.882 (95% CI: 0.866; 4.090) Conclusions: The 63% concordance rate for detecting FGFR mut in temporally unmatched blood and tissue supports potential for patient selection with blood-based testing, while ctDNA FGFR fusion detection may require further optimization. Although differences in clinical response rate were observed between ctDNA- FGFR-positive and negative patients, the results were not statistically significant. Importantly, a proportion of ctDNA-negative pts responded to erdafitinib, indicating that tissue-based testing may be warranted for pts negative for FGFR alterations via blood-based testing. (BLC2001, NCT02365597). Clinical trial information: NCT02365597.

Fibroblast growth factor receptor (FGFR) genetic alterations are frequently observed in cancer, suggesting that FGFR inhibition may be a promising therapy in patients harboring these lesions. Identification of predictive and pharmacodynamic biomarkers to select and monitor patients most likely to respond to FGFR inhibition will be the key to clinical development of this class of agents. Sensitivity to FGFR inhibition and correlation with FGFR pathway activation status were determined in molecularly annotated panels of cancer cell lines and xenograft models. Pathway inhibition in response to FGFR inhibitor treatment was assessed in cell lines (both in vitro and in vivo) and in samples from patients treated with the FGFR inhibitor JNJ-42756493 (erdafitinib). Frequency of FGFR aberrations was assessed in a panel of NSCLC, breast, prostate, ovarian, colorectal, and melanoma human tumor tissue samples. FGFR translocations and gene amplifications present in clinical specimens were shown to display potent transforming activity associated with constitutive pathway activation. Tumor cells expressing these FGFR activating mutants displayed sensitivity to the selective FGFR inhibitor erdafitinib and resulted in suppression of FGFR phosphorylation and downstream signal transduction. Clinically, patients receiving erdafitinib showed decreased Erk phosphorylation in tumor biopsies and elevation of serum phosphate. In a phase I study, a heavily pretreated bladder cancer patient with an FGFR3–TACC3 translocation experienced a partial response when treated with erdafitinib. This preclinical study confirmed pharmacodynamics and identified new predictive biomarkers to FGFR inhibition with erdafitinib and supports further clinical evaluation of this compound in patients with FGFR genetic alterations. Mol Cancer Ther; 16(8); 1717–26. ©2017 AACR.

We report the characterization of BMS-911543, a potent and selective small-molecule inhibitor of the Janus kinase (JAK) family member, JAK2. Functionally, BMS-911543 displayed potent anti-proliferative and pharmacodynamic (PD) effects in cell lines dependent upon JAK2 signaling, and had little activity in cell types dependent upon other pathways, such as JAK1 and JAK3. BMS-911543 also displayed anti-proliferative responses in colony growth assays using primary progenitor cells isolated from patients with JAK2(V617F)-positive myeloproliferative neoplasms (MPNs). Similar to these in vitro observations, BMS-911543 was also highly active in in vivo models of JAK2 signaling, with sustained pathway suppression being observed after a single oral dose. At low dose levels active in JAK2-dependent PD models, no effects were observed in an in vivo model of immunosuppression monitoring antigen-induced IgG and IgM production. Expression profiling of JAK2(V617F)-expressing cells treated with diverse JAK2 inhibitors revealed a shared set of transcriptional changes underlying pharmacological effects of JAK2 inhibition, including many STAT1-regulated genes and STAT1 itself. Collectively, our results highlight BMS-911543 as a functionally selective JAK2 inhibitor and support the therapeutic rationale for its further characterization in patients with MPN or in other disorders characterized by constitutively active JAK2 signaling.

NSCLC with activating mutations in the EGFR gene are associated with high response rates to EGFR tyrosine kinase inhibitors (TKIs), such as erlotinib and gefitinib, but invariably acquired resistance emerges over time. A primary cause of resistance is the emergence of secondary mutations in EGFR which neutralize the effectiveness of TKIs. In addition, the cMet pathway is often activated, either through MET gene amplification, overexpression of cMet protein, or an increase in the ligand HGF, to provide a compensatory survival pathway conferring resistance to EGFR TKIs. We have designed a bispecific EGFR-cMet antibody (JNJ-61186372) with a unique set of mechanisms of action resulting in anti-tumor activity in the EGFR mutant setting, with or without cMet pathway activation. We have demonstrated three mechanisms of action that contribute to the activity of JNJ-61186372: 1) inhibition of ligand-induced phosphorylation of both EGFR and cMet, 2) receptor degradation in vivo, and 3) enhanced ADCC activity. JNJ-61186372 inhibited EGF-induced phosphorylation of EGFR in cell lines with either wild-type (WT) EGFR or activating mutations in EGFR. In the same cell lines, JNJ-61186372 inhibited HGF-induced phosphorylation of cMet. JNJ-61186372 also blocked pERK and pAkt with similar IC50 values in EGFR-WT and EGFR mutant cell lines, indicating that downstream signaling pathways were inhibited. Total protein levels of both EGFR and cMet were decreased in xenograft tumor models following treatment with JNJ-61186372 compared to tumors from mice treated with PBS control suggesting that one mechanism by which JNJ-61186372 suppresses EGFR and cMet activity in vivo is through degradation of both receptors. The third mechanism of action is directing immune cells to kill tumor cells. JNJ-61186372 is produced with low levels of fucosylation, which translates to an enhanced antibody-dependent cellular cytotoxicity (ADCC). These three mechanisms of action of JNJ-61186372 provide a distinct preclinical profile for targeting both EGFR and cMET in a single bispecific antibody. JNJ-61186372 demonstrated efficacy in multiple in vivo tumor models with EGFR mutations, including both cell line and patient-derived xenografts. Importantly, JNJ-61186372 effectively inhibited tumor growth in models with mutant EGFR and cMet activation, whereas single agent EGFR inhibitors were less effective. The preclinical data support the clinical development of JNJ-61186372 in patients with lung cancer and other malignancies associated with aberrant EGFR and cMET signaling. Citation Format: Sheri L. Moores, Mark Chiu, Barbara Bushey, Kristen Chevalier, Peter Haytko, Joost Neijssen, Paul Parren, Janine Schuurman, Mark Anderson, Ricardo Attar, Robert Kramer, Matthew V. Lorenzi. Discovery and preclinical pharmacology of JNJ-61186372: A novel bispecific antibody targeting EGFR and cMET. [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 DDT01-03. doi:10.1158/1538-7445.AM2014-DDT01-03

PRMT5 is a type II methyltransferase that symmetrically di-methylates arginine residues on proteins involved in signal transduction and cellular transcription. For example, PRMT5 acts as the enzymatic machinery of the methylosome complex, crucial for spliceosome assembly and activity. Although not frequently mutated or amplified in tumors, an elevated PRMT5 protein level that leads to higher methylosome activity and promotes epithelial–mesenchymal transition, has recently been correlated with a poor survival of cancer patients. The PRMT5 inhibitor JNJ-64619178 has been selected as a clinical candidate based on its high selectivity and potency, paired with favorable oral pharmacokinetics and safety properties. JNJ-64619178 binds simultaneously to the SAM- and protein substrate- binding pockets of the PRMT5/MEP50 complex with a pseudo-irreversible mode-of-action. Chemical proteomics, methylomics and RNA-sequencing analyses of PRMT5 inhibitor treated cell line samples support the current biological understanding of PRMT5 as a regulator of alternative splicing events. JNJ-64619178 showed potent and broad inhibition of cellular growth, observed in several cell line panels that represent diverse cancer histologies. Ongoing investigations will explore the potential synthetic lethal correlation between PRMT5 inhibition and cancer driver pathways, including those addicted to altered splicing. Oral administration of JNJ-64619178 resulted in efficient inhibition of di-methylation of SMD1/3 proteins, components of the splicing machinery and direct substrates of the methylosome, in several human NSCLC and SCLC cancer mouse xenograft models. JNJ-64619178 demonstrated dose-dependent tumor growth inhibition and regression in several human NSCLC and SCLC cancer mouse xenograft models with sustained blockage of tumor re-growth after dosing cessation. In summary, JNJ-64619178 has a favorable pre-clinical profile supporting clinical testing in patients with lung cancer and other malignancies. Citation Format: Tongfei Wu, Hillary Millar, Dana Gaffney, Lijs Beke, Geert Mannens, Petra Vinken, Ivan Sommers, Jan-Willem Thuring, Weimei Sun, Christopher Moy, Vineet Pande, Junguo Zhou, Nahor Haddish-Berhane, Mark Salvati, Sylvie Laquerre, Matthew V. Lorenzi, Dirk Brehmer. JNJ-64619178, a selective and pseudo-irreversible PRMT5 inhibitor with potent in vitro and in vivo activity, demonstrated in several lung cancer models [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 4859.

Activating mutations in KRAS have a high prevalence in human cancer. The codon 12 glycine to cysteine missense mutation (KRASG12C) is among the most common KRAS mutations, present in non-small cell lung adenocarcinoma (~15 %), colorectal adenocarcinoma (~3 %), and pancreatic adenocarcinoma (~1 %). KRASG12C was previously identified as potentially druggable by allele-specific covalent targeting of cysteine 12 near the allosteric switch II pocket (S-IIP). Building on this early work, we recently described the ARS-853 series of S-IIP KRASG12C inhibitors that covalently react with the GDP-bound state of KRASG12C, trapping KRASG12C in this inactive state. In cells, ARS-853 series compounds profoundly deplete the signaling competent GTP-bound state of KRASG12C, thereby inhibiting downstream RAS signaling. However, this series of covalent KRASG12C inhibitors exhibited modest cellular potency and/or poor pharmacokinetic properties, making them unsuitable for further evaluation of covalent KRASG12C inhibition in animal models. We now describe in further detail the discovery and characterization of a new series of structurally distinct quinazoline based S-IIP KRASG12C inhibitors with substantially improved potency and pharmacologic properties that overcome limitations of the ARS-853 series. Through structure-guided medicinal chemistry optimization we identified compound ARS-1620, a potent, orally bioavailable covalent inhibitor of KRASG12C. The co-crystal structure of ARS-1620 covalently bound to KRASG12C reveals a distinct binding mode and additional interactions, compared to ARS-853. ARS-1620 rapidly engages KRASG12C, depletes KRASG12C-GTP in tumor cell lines, and inhibits downstream RAS signaling in a dose-dependent manner. The compound potently inhibits the growth of cell lines harboring the KRASG12C mutation with little or no effect on control cell lines. ARS-1620 demonstrates robust dose-dependent efficacy with once daily oral administration across a panel of KRASG12C-positive mouse cell line (CDX) and patient-derived (PDX) tumor xenograft models, with no response observed at all doses tested in KRASG12C-negative tumor models. The anti-tumor activity of ARS-1620 correlates with target engagement in the tumors as well as with inhibition of downstream RAS signaling. The in vivo efficacy and mutant selectivity observed with ARS-1620 across a wide range of KRASG12C mouse tumor models provides the first in vivo evidence that the S-IIP targeted approach may be a promising therapeutic strategy for patients with KRASG12C mutant cancers. Citation Format: Liansheng Li, Matthew R. Janes, Jingchuan Zhang, Rasmus Hansen, Ulf Peters, Xin Guo, Yuching Chen, Anjali Babbar, Sarah J. Firdaus, Jun Feng, Jeffrey H. Chen, Shuangwei Li, Shisheng Li, Carol Thach, Yuan Liu, Ata Zarieh, Jeff M. Kucharski, Tao Wu, Ke Yu, Yi Wang, Yvonne Yao, Xiaohu Deng, Patrick P. Zarrinkar, Dashyant Dhanak, Matthew V. Lorenzi, Dana Hu-Lowe, Pingda Ren, Yi Liu. Discovery of novel covalent KRASG12C inhibitors that display high potency and selectivity in vitro and in vivo [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 929.

Non–small cell lung cancers (NSCLC) with activating EGFR mutations become resistant to tyrosine kinase inhibitors (TKI), often through second-site mutations in EGFR (T790M) and/or activation of the cMet pathway. We engineered a bispecific EGFR-cMet antibody (JNJ-61186372) with multiple mechanisms of action to inhibit primary/secondary EGFR mutations and the cMet pathway. JNJ-61186372 blocked ligand-induced phosphorylation of EGFR and cMet and inhibited phospho-ERK and phospho-AKT more potently than the combination of single receptor–binding antibodies. In NSCLC tumor models driven by EGFR and/or cMet, JNJ-61186372 treatment resulted in tumor regression through inhibition of signaling/receptor downmodulation and Fc-driven effector interactions. Complete and durable regression of human lung xenograft tumors was observed with the combination of JNJ-61186372 and a third-generation EGFR TKI. Interestingly, treatment of cynomolgus monkeys with JNJ-61186372 resulted in no major toxicities, including absence of skin rash observed with other EGFR-directed agents. These results highlight the differentiated potential of JNJ-61186372 to inhibit the spectrum of mutations driving EGFR TKI resistance in NSCLC. Cancer Res; 76(13); 3942–53. ©2016 AACR.

Receptor tyrosine kinases (RTK) remain an area of therapeutic interest because of their role in epithelial tumors, and experimental models specific to these targets are highly desirable. Chimeric receptors were prepared by in-frame fusion of the CD8 extracellular sequence with the cytoplasmic sequences of RTKs. A CD8HER2 fusion protein was shown to form disulfide-mediated homodimers and to transform fibroblasts and epithelial cells. CD8RTK fusion proteins transform rat kidney epithelial cells and impart phenotypes that may reflect signaling specificity inherent in the native receptors. Transgenic expression of CD8HER2 and CD8Met in mice resulted in the formation of salivary and mammary gland tumors. The transgenic tumors allow the derivation of allograft tumors and cell lines that are sensitive to inhibition by small molecule kinase inhibitors. This approach provides excellent cell and tumor models for the characterization of signaling properties of diverse RTKs and for the evaluation of rationally designed antagonists targeting these kinases. [Mol Cancer Ther 2006;5(6):1571–6]

KRASG12C was recently identified to be potentially druggable by allele-specific covalent targeting of Cys-12 in vicinity to an inducible allosteric switch II pocket (S-IIP). Success of this approach requires active cycling of KRASG12C between its active-GTP and inactive-GDP conformations as accessibility of the S-IIP is restricted only to the GDP-bound state. This strategy proved feasible for inhibiting mutant KRAS in vitro; however, it is uncertain whether this approach would translate to in vivo. Here, we describe structure-based design and identification of ARS-1620, a covalent compound with high potency and selectivity for KRASG12C. ARS-1620 achieves rapid and sustained in vivo target occupancy to induce tumor regression. We use ARS-1620 to dissect oncogenic KRAS dependency and demonstrate that monolayer culture formats significantly underestimate KRAS dependency in vivo. This study provides in vivo evidence that mutant KRAS can be selectively targeted and reveals ARS-1620 as representing a new generation of KRASG12C-specific inhibitors with promising therapeutic potential.

Targeted therapies against activated oncogenes, such as receptor tyrosine kinases, have significantly prolonged non-small cell lung cancer (NSCLC) patient survival, but the development of resistance limits the durability of clinical response. Genetic alterations which constitutively activate Fibroblast Growth Factor Receptors (FGFR) have been observed in patients with NSCLC. Erdafitinib (JNJ-42756493), an orally bioavailable pan-FGFR inhibitor discovered as part of a collaboration between Janssen and Astex Pharmaceuticals, has been shown to inhibit FGFR signaling pathways resulting in cell death and tumor growth inhibition in both in vitro and in vivo models of FGFR pathway aberration. Further, erdafitinib has been shown to have favorable pharmaceutical properties with manageable side effects in humans and several clinical trials are currently underway. One potential strategy to enhance the durability of response to targeted therapies, such as FGFR inhibitors, is to couple them with immunotherapy. In this setting, T cell responses primed and activated by increased antigen release resulting from the tumor cell targeted therapy could be enhanced and maintained by T-cell directed checkpoint blockade. To test this hypothesis, we evaluated erdafitinib in combination with an anti-programmed death-1 (PD-1) blocking antibody in an autochthonous FGFR2K660N/p53 genetically engineered mouse model (GEMM) of lung cancer, in which tumors develop within the context of an intact immune microenvironment. Cohorts of tumor bearing FGFR2K660N/p53 mutant mice treated with erdafitinib with or without anti-PD-1 showed significant tumor regressions compared to control and anti-PD-1 alone groups. Despite lack of differences in acute tumor responses between erdafitinib monotherapy and combination therapy, we observed significant survival benefit in the combination group erdafitinib alone (median survival 19.7 weeks vs 13.4 weeks, p Citation Format: Sangeetha Palakurthi, Mari Kuraguchi, Sima Zacharek, Jeff Liu, Dennis Bonal, Wei Huang, Kristin Depeaux, Abha Dhaneshwar, Sam Regan, Dyane Bailey, Martha Gowaski, Mei Zheng, Roderick Bronson, Catherine Ferrante, Enrique Zudaire, Sylvie Laquerre, Mark Bittinger, Kirschmeier Paul, Kathryn Packman, Raluca I. Verona, Kwok-Kin Wong, Matthew V. Lorenzi. Improved survival with erdafitinib (JNJ-42756493) and PD-1 blockade mediated by enhancement of anti-tumor immunity in an FGFR2-driven genetically engineered mouse model of lung cancer. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2016 Oct 20-23; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2017;5(3 Suppl):Abstract nr B27.

Myeloproliferative diseases (MPDs) are a subset of myeloid malignancies that are characterized by the expansion of a multipotent hematopoietic stem cell. Chronic MPDs can be classified into two categories, those harboring the BCR-ABL oncogene and those that are negative. This later category of neoplasms encompasses polycythemia vera (PV), essential thrombocythemia (ET), and primary myelofibrosis (PMF). Recent discovery of activating mutations in the tyrosine kinase gene, JAK2 and constitutive activation of JAK2-STAT pathway, in large number of MPD patients has ignited considerable interest in MPD and has highlighted JAK2 as a therapeutic intervention point for drug discovery efforts. However, high-sequence homology with other JAK family members has posed a major challenge to design selective JAK2 inhibitors. Given that other JAK family members are involved in the regulation of immune function, it is important to maintain selectivity for JAK2 over these family members in order to mitigate the risks associated with undesired immunosuppression. Several JAK2 inhibitors with varying selectivity profiles are currently being evaluated in preclinical testing as well as in clinical trials for the treatment of MPD. Additionally, emerging genetic and pharmacologic evidence suggest that inhibition of the JAK2-STAT pathway may be an important therapeutic intervention point in other hematological malignancies as well as in certain solid tumors. We report here the discovery and characterization of BMS-911543, a functionally selective small molecule inhibitor of the Janus kinase family (JAK) member, JAK2. BMS-911543 is a potent and reversible inhibitor of JAK2 with a biochemical Ki of 0.48 nM. It has over 65-, 74- and 350-fold selectivity against the other JAK family members, TYK2, JAK3 and JAK1, respectively. Importantly, examination of > 450 other kinases in competition binding assays and in selected biochemical kinase assays did not reveal significant inhibitory activity for this JAK2 inhibitor, highlighting its high degree of biochemical selectivity for JAK2. Functionally, BMS-911543 displayed potent antiproliferative and pharmacodynamic (PD) effects in mutated JAK2-expressing cell lines dependent upon JAK2-STAT signaling and had little activity in cell types dependent upon other pathways such as JAK1 and JAK3. Further, single agent antiproliferative activity was not observed for BMS-911543 in a variety of solid tumor cell lines dependent upon other signaling pathways. In contrast, BMS-911543 was evaluated in colony growth assays using primary progenitor cells isolated from patients with JAK2V617F-positive myeloproliferative disease (MPD) and resulted in an increased antiproliferative response in MPD cells as compared with those from healthy volunteers. Similar to these in vitro observations, BMS-911543 was also highly active in in vivo models of JAK2-pSTAT signaling in multiple species with durable and potent pathway suppression observed after a single oral dose. Additionally, BMS-911543 was evaluated for effects in a JAK2V617F-expressing SET-2 xenograft model system and displayed a minimally effective dose of To test the hypothesis that a JAK2 selective inhibitor would have less effect on immune system function, BMS-911543 was compared to pan-JAK inhibitors in a mouse model of immunosuppression. At low dose levels active in JAK2-dependent PD models, no effects were observed on antigen-induced IgG and IgM production for BMS-911543 whereas a pan-JAK family inhibitor showed pronounced effects at all dose levels tested. The mechanistic selectivity of BMS-911543 to pan-JAK family inhibitors was extended through comparative analysis of these inhibitors in whole genome gene expression profiling experiments performed in sensitive and resistant cell types. In this comparison, BMS-911543 modulated a distinct subset of transcriptional changes as compared to pan-JAK inhibitors in clinical testing, thereby defining a minimal set of transcriptional changes underlying the pharmacologic effects of JAK2 inhibition. Collectively these results define the mechanistic basis for a differential therapeutic index between selective JAK2 and pan-JAK family inhibition pre-clinically and suggest a therapeutic rationale for the further characterization of BMS-911543 in patients with MPD and in other malignancies reliant upon constitutively active JAK2 signaling. References: Levine, R.L., et al. Role of JAK2 in the pathogenesis and therapy of myeloproliferative disorders (2007). Nature Rev. Cancer, 7, 673-683. Atallah, E. and Verstovsek, S. Prospect of JAK2 inhibitor therapy in myeloproliferative neoplasms. (2009). Expert Rev. Anticancer Ther. 9, 663-670. Ghoreschi, K., et al. Janus kinases in immune cell signaling. (2009). Immunol. Rev.,228, 273-287. Mesa, R.A. and Tefferi, A. Emerging drugs for the therapy of primary and post essential thrombocythemia, post polycythemia vera myelofibrosis (2009). Expert Opin. Emerging Drugs, 14, 1-9. Roll, J.D. and Reuther, G.W. CRLF2 and JAK2 in B-progenitor acute lymphoblastic leukemia: a novel association in oncogenesis. (2010) Cancer Res, 70, 7347-7352. Rui et al., Cooperative epigenetic modulation by cancer amplicon genes (2010). Cancer Cell, 18, 590-605. 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 DDT01-03. doi:10.1158/1538-7445.AM2011-DDT01-03

The tumor necrosis factor (TNF) receptor family are ligand-regulated transmembrane proteins that mediate apoptosis as well as activation of the transcription factor NF-kappaB. Exogenous expression of DR6, a recently identified member of the TNF receptor family, induced apoptosis in untransformed or tumor-derived cells and the apoptotic function of DR6 was inhibited by co-expression of Bcl-2, Bcl-x(L) or the inhibitor-of-apoptosis (IAP) family member, survivin. Expression of a dominant negative mutant of FADD failed to protect from DR6-mediated apoptosis indicating that unlike TNFR1 and Fas, DR6 induced apoptosis via a FADD-independent mechanism. Despite the ability of exogenous DR6 expression to induce apoptosis, DR6 mRNA and protein were found to be elevated in prostate tumor cell lines and in advanced stages of prostate cancer. Analysis of several anti-apoptotic proteins revealed that Bcl-x(L) levels and serine 32 phosphorylation of IkappaB, the natural inhibitor of NF-kappaB, were similarly elevated in cells expressing high levels of DR6, suggesting that NF-kappaB-regulated survival proteins may protect from DR6-induced apoptosis and that DR6 is a target of NF-kappaB regulation. Treatment of LnCAP cells with TNF-alpha resulted in increases in both DR6 mRNA and protein levels, and this induction was suppressed by inhibitors of NF-kappaB. Similarly, treatment of cells expressing high levels of DR6 with indomethacin and ibuprofen, compounds also known to perturb NF-kappaB function, resulted in a dose-dependent decrease in DR6 protein and mRNA levels. These results demonstrate that TNF-alpha signaling induces the expression of a member of its own receptor family through activation of NF-kappaB.

To assess the effect(s) of the C-terminal domain on FGFR2 function, we engineered a series of mutant FGFR2 cDNAs encoding deletions in the C-terminus of the receptor and compared their growth properties in NIH3T3 fibroblasts. In contrast to FGFR2-WT, receptors with C-terminal truncations induced ligand-independent transformation of NIH3T3 cells and transfectants expressing these mutant receptors efficiently formed colonies in semisolid medium. Introduction of point mutations (Y to F) into the C-terminus of FGFR2 at positions 813, 784 or 780 revealed that these mutant receptors also displayed activities similar to that of C-terminally truncated receptors. C-terminally altered FGF receptors did not show an increase in the basal level of receptor phosphorylation compared to that of FGFR2-WT suggesting that elevated receptor phosphorylation does not underlie the transforming activity of these receptors. Interestingly, expression of transforming FGFR2 derivatives, unlike H-Ras transformed cells, did not result in the activation of the mitogen-activated protein kinases (MAPKs), p42/ERK2 and p44/ERK1, indicating that this pathway is not constitutively active in FGFR2-transformed cells. Finally, we report the overexpression of FGFR2 mRNA and protein in several human tumor cell lines suggesting activation of the receptor in these tumors.

Targeted biologic therapeutics have become an important class of oncology drugs, and could be the predominant class of approved anti-tumor treatments in the coming years. Within the class of biologic therapies, multiple monoclonal antibodies have achieved approval for a variety of cancer indications (Table 78.1). This chapter will provide an overview of biologics therapy from an oncology drug-discovery perspective, including a review of monoclonal antibodies, oncology targets for antibody therapy, the challenges for antibodies as drug-discovery targets, and the future of protein therapeutics in oncology drug discovery. Overview of antibodies as cancer therapeutics An overview of antibody structure and function is essential to understanding their role as drugs (1). Antibodies are produced by B cells, having evolved as a defense mechanism mediated by their specific, high-affinity binding and subsequent neutralization of target pathogens or particles. Antibodies are comprised of two heavy chains and two light chains (Figure 78.1a). The heavy and light chains are tethered by disulfide bonds, and the heavy chains are themselves bound by additional disulfide bonds creating a four protein-chain molecule. The heavy and light chains each contain variable and constant regions, the variable regions differ between antibodies and are responsible for binding to the target; the constant regions’ amino-acid sequences remain fixed amongst antibodies. Each heavy and light chain contributes to the antibody binding domain, forming the complementarity determining region (CDR) which interacts with the target antigen. The binding characteristics of CDRs are defined by their specific amino-acid sequence. B cells initially produce low-affinity, high-avidity (decavalent) IgM molecules (Figure 78.1b). Antigen binding is enhanced through T-cell-directed affinity maturation. Isotype switching results in an exchange of the non-binding portion of the antibody to a bivalent IgG, establishing distinct effector functions, while retaining the CDR and thus target specificity. All marketed cancer therapeutics are one of the IgG class of antibodies. The Fc domain, which is made up of the region of the antibody distal to the CDRs and is comprised of the two associated heavy chains, is also important for antibody therapeutics in mediating effector functions, including the activation of immune-dependent activities that require the constant region of the antibody. Such activities are complement fixation, activation of immune cell cytotoxicity, and triggering of phagocytic activity of certain cells. These effector functions are responsible for many of the therapeutic outcomes of antibody treatment.

Fibroblast growth factors (FGFs) are a family of homologous secreted glycoproteins involved in signaling pathways responsible for embryonic development, cell proliferation, survival, and migration. FGF activity is mediated by four transmembrane fibroblast growth factor receptors (FGFRs) which are receptor tyrosine kinases. Typically, FGF binding induces FGFR dimerization, leading to phosphorylation of the intracellular tyrosine kinase domain. This leads to downstream activation of multiple signaling pathways, including the mitogen-activated protein kinase (MAPK), PI3K/AKT, signal transducer and activator of transcription (STAT), and phospholipase-C-γ cascades. Deregulated FGFR activity, through mutations or translocations, is often associated with oncogenic events. Aberrations in FGFR genes have been observed in several tumor types including bladder, gastric, colorectal, ovarian, and hematologic cancers. To date, several FGFR3 mutations have been identified in bladder cancer. In this study, we developed a TaqMan qRT-PCR-based approach to detect four FGFR3 mutations in formalin-fixed paraffin embedded tissue (FFPET) samples. Cell lines overexpressing FGFR3 mutations were generated to determine impact on cell signaling, and sensitivity to the small-molecule pan-FGFR inhibitor JNJ-42756493. To determine the role and significance of these FGFR3 mutations in cancer, mutation expression constructs were designed and individually transfected into normal rat kidney epithelial cells. Cells harboring the FGFR3 mutations exhibited anchorage-independent growth, increased proliferation, and showed increased sensitivity to the FGFR inhibitor JNJ-42756493 in vitro compared to parental lines. These findings underline the oncogenic potential of the FGFR3 mutation genes and highlight their unique potential as predictive biomarkers in the selection of patients for FGFR-targeted therapy. Citation Format: Gabriela Martinez Cardona, Dana Gaffney, Katherine Bell, Joseph Portale, Matthew Dunworth, Matthew Lorenzi, Suso Platero, Jayaprakash Karkera. FGFR3 mutations as novel oncogenic targets. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3990.

Janus kinases (JAKs) are a family of receptor-associated tyrosine kinases which are crucial for the survival and proliferation of immune and hematopoietic-derived cells. There are four mammalian JAKs identified to date: JAK1, JAK2, JAK3 and TYK2 (Lopez et al., 2010). JAKs are responsible for mediating the intracellular signaling of numerous growth factors and cytokines (Murray et al., 2007 & O’Shea et al., 2002). Once activated JAKs directly phosphorylate and activate the transcription factors signal transducers and activators of transcriptions (STATs) which transduce JAK signaling by translocating to the nucleus to modulate a subset of genes that are critical for cell proliferation and survival (Aaronson et al., 2002 & Levy et al., 2002). Dysregulated JAK signaling has been implicated in the pathogenesis of several blood-borne cancers but most notably in myeloproliferative neoplasms (MPNs) (Nelson et al., 2006; Lucia et al., 2011 & Patnaik et al., 2009). MPNs are hematological malignancies defined by the excessive proliferation of one or more myeloidderived cells. MPNs are classified into two clinical categories either BCR-ABL-positive (BCR-ABL (+)) or BCR-ABL-negative (BCR-ABL (-)) based on the presence of the BCR-ABL fusion protein. The major BCR-ABL (-) MPNs include polycythemia vera (PV), essential thrombocythemia (ET) and myelofibrosis (MF). The specific pathogenic role that JAK2 has in driving some of these MPNs is highlighted by the recent seminal discovery of the JAK2V617F mutation (Baxter et al., 2005; James et al., 2005; Kralovics et al., 2005; & Levine et al., 2005). The JAK2V617F mutation is a somatic, gain-of function point mutation which leads to constitutive activation of JAK2 and subsequent downstream activation of STATs (Baxter et al., 2005 & James et al., 2005). Current data demonstrates that JAK2 is a disease associated gene involved in the pathogenesis of BCR-ABL (-) MPNs. However, it remains unclear if JAK2 pathway mutations are the primary drivers of sustained disease progression. New data is emerging that suggests that targeting the JAK-STAT pathway may have implications beyond the treatment of MPN and can be useful for the treatment of other hematological and non-hematological cancers. This review will summarize the pathogenic role of JAKSTAT signaling in BCR-ABL (-) MPNs and introduce the potential broader implications by

Publisher Summary JAK2 is a member of the Janus kinase (JAK) family of nonreceptor tyrosine kinases comprising of four family members—JAK1, JAK2, JAK3, and TYK2. Each JAK is composed of two kinase domains— JH1 and JH2—in its carboxy terminal half, with JH2 domain representing a pseudokinase domain that negatively regulates enzymatic activity. The discovery of JAK2V617F as well as JAK2—STAT pathway mutations and their relevance to MPNs have fuelled research collaborations between academia and the pharmaceutical industry. These collaborations have resulted in the advancement of several candidates into early and midstage clinical trials. In addition, early clinical data has shown promise of JAK2 inhibitors to relieve constitutional symptoms and improve hematological parameters and the overall quality of life for moderately aggressive MPNs, particularly myelofibrosis; however, only modest effects on the disease pathology or molecular responses are observed. Success with kinase-targeted therapies, such as imatinib for the BCR-ABL mutation-driven CML, has raised hopes for using this same approach for JAK2 inhibitors. The ultimate success of a JAK2 inhibitor for the treatment of MPN would depend upon optimal kinase selectivity, long-term safety and tolerability of the agent, and its effect on the pathophysiology of the disease.

Myeloproliferative disorders (MPDs) are clonal malignancies that arise from hematopoietic progenitors and characterized by overproduction of mature, functional blood cells. These disorders can be broadly characterized into Philadelphia chromosome-positive (Ph(+)) or negative (Ph(-)) genetic groupings. Chronic myeloid leukemia (CML) is a Ph(+) MPD that is defined on the basis of its molecular lesion, the BCR-ABL fusion gene. Inhibitors directed at the constitutive kinase activity of BCR-ABL have been shown to be disease modifying in CML and have dramatically altered the standard of care for this leukemia. The three main Ph(-) MPDs are polycythemia vera (PV), essential thrombocythemia (ET) and primary myelofibrosis (PMF). The key features of these Ph(-) MPDs are an increased red blood cell mass in PV, a high platelet count in ET and bone marrow fibrosis in PMF, respectively. These disorders also share many clinical features such as long clinical course, increased risk for thrombosis, hemorrhage and elevated risk of leukemic transformation. Interest in these disorders has been ignited by the recent discovery of activating mutations in the tyrosine kinase gene, JAK2, in the predominance of Ph(-) MPD patients and has highlighted JAK2 as a therapeutic intervention point for drug discovery efforts with selective kinase inhibitors. This review will focus on the comparison of Ph(+) and Ph(-) MPDs, drug discovery and development efforts targeting these disorders, and will assess the new opportunities for targeted therapies for these diseases.

Non-small cell lung cancers (NSCLCs) with activating mutations in the Epidermal Growth Factor Receptor (EGFR) gene are associated with high response rates (70-80%) to EGFR tyrosine kinase inhibitors (TKIs), such as erlotinib and gefitinib, but most acquire resistance over time through numerous mechanisms. In these patients, the cMet pathway is often activated to compensate and provide resistance to the EGFR targeted monotherapy; this activation can occur by MET gene amplification, overexpression of cMet protein, or an increase in the ligand HGF. We have designed a bispecific EGFR-cMet antibody (JNJ-61186372) with multiple mechanisms of action resulting in anti-tumor activity in the EGFR mutant setting, with or without cMet pathway activation. Controlled Fab-arm exchange was used to produce JNJ-61186372, a technique that allows for efficient large-scale preparation of bispecific antibodies with a regular IgG1 structure. JNJ-61186372 was shown to bind EGFR and cMet and efficiently inhibited ligand-induced phosphorylation of both receptors. In addition to this important mechanism of action, we have engineered the antibody to contain lower than normal fucose levels to increase Fc-dependent effector mechanisms. JNJ-61186372 exhibited antibody dependent cellular cytotoxicity (ADCC) activity in vitro in a range of NSCLC cell lines with EGFR mutations, KRas mutation, and/or amplified MET gene. Furthermore, the low fucose form of JNJ-61186372 demonstrated more effective ADCC activity compared to its normal fucose counterpart. The bispecific JNJ-61186372 antibody showed increased potency (2-3 fold) compared to the combination of monovalent EGFR and monovalent cMet antibodies, demonstrating the beneficial effects of dual targeting in a single molecule. Antibody dependent cell-mediated phagocytosis (ADCP) activity of JNJ-61186372 was also confirmed in vitro. We have also demonstrated that Fc-dependent effector functions contributed to in vivo anti-tumor growth activity of JNJ-61186372, in a xenograft model with EGFR mutations and cMet activation. Our data demonstrate that the bispecific antibody JNJ-61186372, generated using controlled Fab-arm exchange, has in vitro ADCC and ADCP activity in EGFR mutant settings, either with or without cMet pathway activation, and with KRas mutation. In addition, the Fc-dependent effector mechanisms contributed to in vivo anti-tumor efficacy. The dual signaling inhibition of EGFR and cMet pathways by JNJ-61186372, combined with enhanced Fc effector function, may provide multiple mechanisms to combat resistance in EGFR mutant NSCLC patients. Citation Format: Keri L. Soring, Katharine D. Grugan, Randall J. Breszki, Jose Pardinas, Leopoldo Luistro, Barbara Bushey, Joost Neijssen, Paul Parren, Janine Schuurman, Mark Anderson, Ricardo Attar, Matthew V. Lorenzi, Mark Chiu, Sheri Moores. Activity of a bispecific antibody targeting EGFR and cMet with enhanced Fc effector function in EGFR mutant setting with cMet pathway activation. [abstract]. In: Proceedings of the AACR Special Conference: Tumor Immunology and Immunotherapy: A New Chapter; December 1-4, 2014; Orlando, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2015;3(10 Suppl):Abstract nr A11.

Fusion genes are chromosomal aberrations that are found in many cancers and can be used as prognostic markers and drug targets in clinical practice. Gene fusions can lead to the production of oncogenic fusion proteins or to enhanced expression of oncogenes. Advances in next-generation sequencing technologies have made possible to identify more efficiently novel fusion proteins in cancer. Recently, several FGFR fusion genes with intact kinase domains have been identified in bladder, lung, breast, thyroid, oral, and prostate cancers, as well as other tumor types. To date, several FGFR3 partner genes have been identified. In this study, we focused on the FGFR3-BAIAP2L1 and FGFR3-TACC3 gene fusions and investigated their tumorigenic activity, mechanism of activation, and sensitivity to the FGFR inhibitor JNJ-42756493. To determine the role and significance of FGFR fusion genes in cancer, FGFR fusion expression constructs were designed and individually transfected into normal rat kidney epithelial cells. FGFR fusion overexpressing cells not only showed increased cell proliferation, but also exhibited anchorage-independent cell growth. Cells harboring the FGFR fusions showed increased sensitivity to the FGFR inhibitor JNJ-42756493 in vitro, whereas the wild-type FGFR3 did not in the absence of FGF ligands. In addition, Western blotting analyses indicated that the overexpression of the FGFR fusions resulted in highly activated proteins that induce signaling via the MAPK pathway. These findings underline the oncogenic potential of the FGFR fusion genes and highlight their unique potential as predictive biomarkers in the selection of patients for FGFR-targeted therapy. Citation Format: Gabriela Martinez Cardona, Katherine Bell, Dana Gaffney, Joseph Portale, Suso Platero, Matthew Lorenzi, Jayaprakash Karkera. The role of FGFR fusion genes as novel oncogenic targets. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4325. doi:10.1158/1538-7445.AM2015-4325

The human ECT2 protooncogene encodes a guanine nucleotide exchange factor for the Rho GTPases and regulates cytokinesis. Although the oncogenic form of ECT2 contains an N-terminal truncation, it is not clear how the structural abnormality of ECT2 causes malignant transformation. Here we show that both the removal of the negative regulatory domain and alteration of subcellular localization are required to induce the oncogenic activity of ECT2. The transforming activity of oncogenic ECT2 was strongly inhibited by dominant negative Rho GTPases, suggesting the involvement of Rho GTPases in ECT2 transformation. Although deletion of the N-terminal cell cycle regulator-related domain (N) of ECT2 did not activate its transforming activity, removal of the small central domain (S), which contains two nuclear localization signals (NLSs), significantly induced the activity. The ECT2 N domain interacted with the catalytic domain and significantly inhibited the focus formation by oncogenic ECT2. Interestingly, the introduction of the NLS mutations in the S domain of N-terminally truncated ECT2 dramatically induced the transforming activity of this otherwise non-oncogenic derivative. Among the known Rho GTPases expressed in NIH 3T3 cells, RhoA was predominantly activated by oncogenic ECT2 in vivo. Therefore, the mislocalization of structurally altered ECT2 might cause the untimely activation of cytoplasmic Rho GTPases leading to the malignant transformation.

Autocrine or paracrine constitutive Wnt pathway activation occurs at a high frequency in several tumor types. The R-spondin (RSPO) protein family is comprised of four secreted growth factors. The four paralogs share 40-60% pairwise amino acid sequence identity and are predicted to share substantial structural homology. RSPO proteins are involved in vertebrate development and their ligand-type activities overlap substantially with those of canonical Wnt ligands. A characteristic feature of all four RSPO members is their ability to activate β-catenin signaling and enhance WNT-mediated β-catenin activation. It has recently been described that recurrent gene fusions involving RSPO family members RSPO2 and RSPO3 occur in ∼10% of colon tumors. In this study we developed a TaqMan qRT-PCR-based approach to evaluate the expression of these three (3) RSPO fusion transcripts in formalin-fixed paraffin embedded tissue (FFPET) samples. We examined 324 lung cancer, 81 colorectal cancer, 71 head & neck, 11 esophageal, 92 ovarian cancer, and 103 breast cancer FFPET samples for the presence of EIF3E(e1)-RSPO2(e1), PTPRK(e1)-RSPO3(e2), and PTPRK(e7)-RSPO3(e2). EIF3E(e1)-RSPO2(e1), a fusion which is expected to produce a functional RSPO2 protein driven by the EIF3E promoter, was identified in ∼1-2% of most of cancer types with the exception of breast cancer. The PTPRK(e1)-RSPO3(e2) fusion was expressed by ∼1-11% of the samples in the different cancers, making it the most prevalent of the fusions. PTPRK(e1)-RSPO3(e2) fusion is an in-frame fusion that preserves the entire coding sequence of RSPO3 and replaces its secretion signal sequence with that of PTPRK. The PTPRK(e7)-RSPO3(e2) fusion is also an in-frame fusion in which the RSPO3 native signal peptide is replaced by the secretion signal of PTPRK. The PTPRK(e7)-RSPO3(e2) was the least prevalent of all the fusions, positive samples were found exclusively in the head and neck (∼2%) and breast cancer samples (∼2%). All of the fusions detected were mutually exclusive. The RSPO gene fusions identified may provide new potential opportunities for therapeutic intervention. Citation Format: Gabriela Martinez Cardona, Katherine Bell, Joseph Portale, Dana Gaffney, Christopher Moy, Suso Platero, Matthew V. Lorenzi, Jayaprakash Karkera. Identification of R-Spondin fusions in various types of human cancer. [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 2408. doi:10.1158/1538-7445.AM2014-2408

Hepatocyte growth-factor (HGF) is a potent, widely produced, pleiotropic mediator of mesenchymal-epithelial interaction. In a study of changes in gene expression initiated by HGF in Balb/MK keratinocytes, we observed the induction of Neu-differentiation factor (NDF) mRNA (also known as heregulin, or HRG). Further characterization of the regulation of NDF expression in Balb/MK keratinocytes revealed potent induction by keratinocyte growth factor (KGF) and epidermal growth factor (EGF), but not by HGF/NK2, an alternative HGF isoform with motogenic but not mitogenic or morphogenic activities. Sustained treatment (8 h) of Balb/MK cells with KGF stimulated secretion of mature NDF protein into the culture medium, and Balb/ MK cells treated with purified recombinant NDF protein showed increased DNA synthesis. We also found evidence of NDF induction in two models of tissue repair in mice: in full-thickness skin wounds, following locally increased KGF production, and in kidney after partial hepatectomy, following elevation of circulating HGF levels. These results reveal that mesenchymally-derived HGF and KGF can activate autocrine NDF signaling in their epithelial targets, and suggest that this mechanism contributes to the coordination of stages of wound repair, and possibly development, where these growth factors act in concert to direct epithelial proliferation, morphogenesis and differentiation.

Parathyroid adenomas are benign tumors in the parathyroid glands, whose pathogenesis is largely unknown. We utilized an expression cDNA cloning strategy to identify oncogenes activated in parathyroid adenomas. An expression cDNA library was prepared directly from a clinical sample of parathyroid adenoma tissue, transfected into NIH3T3 cells, and foci of morphologically transformed cells were isolated. Following plasmid rescue, we identified cDNAs for the keratinocyte growth factor receptor at a high frequency. Interestingly, approximately half of the clones encoded a variant receptor containing an altered C-terminus. Analysis of the transforming activity of the variant receptor revealed that the altered C-terminus up-regulated the transforming activity in a ligand-independent manner. The higher transforming activity was not accompanied by increase of dimerization or overall autophosphorylation of the receptor. However, tyrosine phosphorylation of downstream receptor substrates, including Shc isoforms and possibly FRS2, are increased in the transfectants expressing the parathyroid tumor-derived receptor. Genomic analysis showed that a previously unidentified exon was used to form the novel isoform. This alternative splicing appears to occur preferentially in parathyroid adenomas.

A search for transforming genes expressed in brain led to the identification of a novel isoform of Ost, an exchange factor for RhoA and Cdc42. In addition to the Dbl-homology (DH) and pleckstrin-homology (PH) domains identified in the original Ost, this isoform contained a SH3 domain and a novel HIV-Tat related (TR) domain. The presence or absence of these domains in Ost defined multiple isoforms of the protein. RT - PCR and in situ hybridization analysis revealed that these isoforms were generated by tissue-specific and developmentally restricted alternative splicing events. Whereas deletion of the N-terminus activated the transforming properties of Ost, the presence of the SH3 domain reduced the transforming activity of the protein. This inhibition was relieved by the presence of a TR domain, which contained a potential SH3 ligand sequence. The transforming activity of all Ost isoforms was inhibited by dominant negative forms of the Rho family proteins. Expression of Ost isoforms potently induced the formation of actin stress fibers and filopodia as well as JNK activity and AP1- and SRF-regulated transcriptional pathways. Ost transfectants also displayed elevated levels of cyclins A and D1, suggesting that the de-regulation of these cyclins is linked to Ost-mediated transformation.

Members of the Ras subfamily of GTP-binding proteins, including Ras (H-, K-, and N-), TC21, and R-ras have been shown to display transforming activity, and activating lesions have been detected in human tumors. We have identified an additional member of the Ras gene family which shows significant sequence similarity to the human TC21 gene. This novel human ras-related gene, R-ras3, encodes for a protein of 209 amino acids, and shows approximately 60-75% sequence identity in the N-terminal catalytic domain with members of the Ras subfamily of GTP-binding proteins. An activating mutation corresponding to the leucine 61 oncogenic lesion of the ras oncogenes when introduced into R-ras3, activates its transforming potential. R-ras3 weakly stimulates the mitogen-activated protein kinase (MAPK) activity, but this effect is greatly potentiated by the co-expression of c-raf-1. By the yeast two-hybrid system, R-ras3 interacts only weakly with known Ras effectors, such as Raf and RalGDS, but not with RglII. In addition, R-ras3 displays modest stimulatory effects on trans-activation from different nuclear response elements which bind transcription factors, such as SRF, ETS/TCF, Jun/Fos, and NF-kappaB/Rel. Interestingly, Northern blot analysis of total RNA isolated from various tissues revealed that the 3.8 kilobasepair (kb) transcript of R-ras3 is highly restricted to the brain and heart. The close evolutionary conservation between R-ras3 and Ras family members, in contrast to the significant differences in its biological activities and the pattern of tissue expression, raise the possibility that R-ras3 may control novel cellular functions previously not described for other GTP-binding proteins.

The heterotrimeric G-protein, G alpha12, together with the closely-related G alpha13, are members of the G12 class of alpha-subunits important in mediating the signaling from seven transmembrane domain-spanning receptors. Recent evidence implicating both G alpha12 and G alpha13 in the activation of signaling pathways involving members of the RHO gene family led us to examine the role of Rac1, RhoA and Cdc42Hs in the transforming properties of G alpha12. Asparagine 17 (Asn 17) dominant inhibitory mutants of Rac1, and to a lesser extent RhoA, block focus forming ability of the GTPase-deficient mutant of G alpha12 (G alpha12 Leu 229) in NIH3T3 cells. In turn, wild-type G alpha12 cooperates well with Rac1 Val 12 but not with RhoA Leu 63 mutant in transforming NIH3T3 cells. Interestingly, the morphology of foci induced by G alpha12 and RhoA mutants are strikingly similar and is distinct from those displayed by Rac1 Val 12 mutant. The fact that G alpha12's ability to induce mitogenesis in NIH3T3 cells is not significantly perturbed by C3 ribosyltransferase suggested that RhoA does not play a major role in G alpha12-induced mitogenic events. Activated mutant of Rac1 has previously been demonstrated to stimulate the activity of the stress-induced c-Jun N-terminal kinase/stress-activated protein kinases (JNK/SAPKs). Transient co-transfection of Rac1 Val 12 mutant with the wild-type G alpha12 in COS7 cells leads to the further activation of an exogenously expressed hemagglutinin(HA)-tagged JNK. Furthermore, the cooperation between G alpha12 and Rac1 in cellular transformation is correlated with their ability to stimulate transcription from c-fos serum response element (SRE).