Antitumor Activity In Preclinical Models Research Articles

Efficacy and Molecular Mechanisms of Differentiated Response to the Aurora and Angiogenic Kinase Inhibitor ENMD-2076 in Preclinical Models of p53-Mutated Triple-Negative Breast Cancer.

Triple-negative breast cancer (TNBC) is a subtype associated with poor prognosis and for which there are limited therapeutic options. The purpose of this study was to evaluate the efficacy of ENMD-2076 in p53-mutated TNBC patient-derived xenograft (PDX) models and describe patterns of terminal cell fate in models demonstrating sensitivity, intrinsic resistance, and acquired resistance to ENMD-2076. p53-mutated, TNBC PDX models were treated with ENMD-2076 and evaluated for mechanisms of sensitivity or resistance to treatment. Correlative tissue testing was performed on tumor tissue to assess for markers of proliferation, apoptosis, senescence, and pathways of resistance after treatment and at the time of acquired resistance. Sensitivity to ENMD-2076 200 mg/kg daily was associated with induction of apoptosis while models exhibiting intrinsic or acquired resistance to treatment presented with a senescent phenotype. Response to ENMD-2076 was accompanied by an increase in p53 and p73 levels, even within the background of mutant p53. Treatment with ENMD-2076 resulted in a decrease in pAurA and an increase in pHH3. We observed a TNBC subtype switch from the luminal androgen receptor to the basal-like subtype at acquired resistance. ENMD-2076 has antitumor activity in preclinical models of p53-mutated TNBC. Increased levels of p53 and p73 correlated with sensitivity whereas senescence was associated with resistance to ENMD-2076. The novel finding of a TNBC subtype switch at time of acquired resistance may provide mechanistic insights into the biologic effects of selective pressure of anticancer treatments on TNBC. ENMD-2076 is currently being evaluated in a Phase 2 clinical trial in patients with metastatic, previously treated TNBC where these biologic correlates can be further explored.

Abstract P3-04-22: RAD1901 demonstrates anti-tumor activity in multiple models of ER-positive breast cancer treatment resistance

Abstract Estrogen receptor positive (ER+) breast cancer makes up approximately 70% of all newly diagnosed breast cancers and ER remains the single most useful predictive and prognostic marker for these patients. In fact, dependence on ER signaling is retained in recurrent disease and even in disease treated with multiple lines of anti-endocrine agents. Indeed, inhibiting estrogen synthesis (e.g. with aromatase inhibitors) and modulating ER pathway activity (e.g. with tamoxifen) continue to be mainstays in the standard of care for ER+ breast cancer patients both in adjuvant and metastatic setting. While patients initially respond well to these agents, a majority of patients with metastatic disease will relapse. Therefore, it is critical to improve our understanding and model the specific mechanisms that can lead to treatment resistance. Mutations in the ligand binding domain of ESR1 have been described as a mechanism that contributes to clinical resistance to aromatase inhibitors. In addition, some ESR1 mutations can lead to a conformational change of the receptor, leading to decreased binding of tamoxifen, thereby reducing its activity. Recently, selective estrogen receptor degraders, or SERDs, have gained widespread attention as a novel treatment strategy for ER+ disease. Indeed, fulvestrant has been shown to degrade ER and cause tumor growth inhibition in many ER+ breast cancer models. However, in the clinic, fulvestrant appears to be limited by PK exposure properties and this, combined with its intramuscular route of administration underscore the need for an oral SERD with improved PK properties. Recently, we have described RAD1901, a novel, orally available, non-steroidal small molecule SERD that is able to degrade ER and effectively cause tumor growth inhibition in multiple patient-derived xenograft models (PDX) models, including two that harbor a Y537S mutation in ESR1. To further validate that RAD1901 has the potential to be used in the advanced disease setting, we tested its anti-tumor activity in preclinical models representing the most common ESR1 mutations (D538G, E380Q, S463P, Y537N/C). In addition, we have tested RAD1901 in multiple cell line models of anti-endocrine resistance. These include long-term estrogen deprived cells that model progression of disease on aromatase inhibitors, as well as tamoxifen-resistant models. Here, we present anti-tumor activity of RAD1901 both as a single agent and as part of rational clinical combinations (eg. CDK4/6, PI3K/Akt/mTOR inhibitors) in multiple models of anti-endocrine resistance. These results combined with previous studies demonstrating RAD1901 efficacy in treatment naïve models provide strong preclinical evidence for clinical testing of RAD1901 in both adjuvant and metastatic setting. RAD1901 is currently under clinical investigation for the treatment of ER+ breast cancers in post-menopausal women (NCT02650817, NCT02338349). Citation Format: Bihani T, Arlt H, Brechbuhl H, Patel H, Tao N, Purandare DM, Hattersley G, Kabos P, Garner F. RAD1901 demonstrates anti-tumor activity in multiple models of ER-positive breast cancer treatment resistance [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P3-04-22.

Denintuzumab Mafodotin Stimulates Immune Responses and Synergizes with CD20 Antibodies to Heighten Anti-Tumor Activity in Preclinical Models of Non-Hodgkin Lymphoma

Denintuzumab Mafodotin (SGN-CD19A) is an antibody-drug conjugate (ADC) composed of an anti-CD19 antibody attached to a synthetic cytotoxic agent, monomethyl auristatin F (MMAF). The ADC binds CD19, internalizes, and releases cys-mcMMAF, which ultimately results in G2-M growth arrest and induction of apoptosis in targeted cells (Law et. al. AACR 2011). SGN-CD19A has shown convincing antitumor activity in relapsed/refractory non-Hodgkin lymphoma patients, resulting in a significant number of objective responses, including complete responses with prolonged durability (Moskowitz et. al. Blood 2015). To further characterize the anti-tumor activity of SGN-CD19A, we evaluated whether immune-mediated cell killing and immunogenic cell death (ICD) act in addition to the previously described apoptotic cell death. We then examined whether these mechanisms of cell death are augmented by clinically approved CD20 antibodies.Our results show that SGN-CD19A supports immune-mediated killing of lymphoma cell lines in vitro through antibody-dependent cellular cytotoxicity (ADCC) and antibody-dependent cellular phagocytosis (ADCP). Assessment of disseminated and subcutaneous tumor xenografts showed that SGN-CD19A activity is reduced when the wild type antibody is replaced with IgG variants that lack effector function. We also found depletion of macrophage or NK cells eliminates the improved survival conferred by the parental antibody of SGN-CD19A. Together these data provide strong evidence that effector function contributes to activity of SGN-CD19A in vivo.Next, we implemented an in vitro system pairing lymphoblastoid cell lines (LCLs) with autologous PBMC to investigate the effect of SGN-CD19A-mediated cell death on immune activation. Treatment of LCL with SGN-CD19A drove hallmarks of ICD as indicated by activation of the ER stress response along with upregulation of surface calreticulin. In vitro co-culture of SGN-CD19A treated LCL with autologous PBMC resulted in heightened monocyte activation and expansion of LCL-reactive cytotoxic T cells supporting a role for SGN-CD19A in augmenting anti-tumor immune responses.Finally, we evaluated the anti-tumor activity of SGN-CD19A in the presence and absence of clinically relevant CD20 antibodies. Results showed that CD20-targeted antibodies impact internalization of CD19 antigen leading to improved drug delivery of SGN-CD19A. Blocking B cell signaling inhibits the effect of CD20 antibodies on CD19 and SGN-CD19A. The improved anti-tumor activity observed with SGN-CD19A plus CD20 therapeutics was confirmed in vivo using rituximab. In conclusion, our results demonstrate SGN-CD19A is a multifunctional ADC capable of eliciting cell killing through immune-mediated mechanisms as well as delivery of the cytotoxic payload, cys-mcMMAF. Importantly, the described mechanisms of SGN-CD19A activity are further augmented by CD20 antibodies. These studies support the ongoing clinical evaluation of SGN-CD19A, which is currently being tested in randomized Phase II clinical trials in combination with R-ICE (NCT02592876) as well as R-CHOP (NCT02855359). DisclosuresVan Epps:Seattle Genetics: Employment. Heiser:Seattle Genetics: Employment. Cao:Seattle Genetics: Employment. Klussman:Seattle Genetics: Employment. Yu:Seattle Genetics: Employment. Stone:Seattle Genetics: Employment. Neale:Seattle Genetics: Employment. Gardai:Seattle Genetics: Employment. Law:Seattle Genetics, Inc.: Employment, Equity Ownership. Ryan:Seattle Genetics: Employment.

258 - TPC-144, a novel reversible LSD1 inhibitor, exhibited strong antitumor activity in preclinical models of AML and SCLC

258 - TPC-144, a novel reversible LSD1 inhibitor, exhibited strong antitumor activity in preclinical models of AML and SCLC

Gene and microRNA modulation upon trabectedin treatment in a human intrahepatic cholangiocarcinoma paired patient derived xenograft and cell line.

Intrahepatic cholangiocarcinoma (ICC) is an aggressive and lethal malignancy with limited therapeutic options. Trabectedin has a high antitumor activity in preclinical models of biliary tract carcinoma (BTC), being a promising alternative treatment. Here, we studied the effect of trabectedin at transcriptomic level on an ICC patient derived xenograft (PDX) and on the derived cell line, MT-CHC01. Further, putative targets of trabectedin were explored in the in vitro model. In vitro, trabectedin inhibited genes involved in protein modification, neurogenesis, migration, and motility; it induced the expression of genes involved in keratinization, tissues development, and apoptotic processes. In the PDX model, trabectedin affected ECM-receptor interaction, focal adhesion, complement and coagulation cascades, Hedgehog, MAPK, EGFR signaling via PIP3 pathway, and apoptosis. Among down-regulated genes, we selected SYK and LGALS1; their silencing caused a significantly reduction of migration, but did not affect proliferation in in vitro models. In MT-CHC01 cells, 24 microRNAs were deregulated upon drug treatment, while only 5 microRNAs were perturbed by trabectedin in PDX. The target prediction analysis showed that SYK and LGALS1 are putative targets of up-regulated microRNAs. In conclusion, we described that trabectedin affected genes and microRNAs involved in tumor progression and metastatic processes, reflecting data previously obtained at macroscopically level; in particular, we identified SYK and LGALS1 as new putative targets of trabectedin.

Intratumoral CD4+ cell depletion sensitizes poorly immunogenic melanomas to immunotherapy with an OX40 agonist

OX40 which belongs to the TNF receptor superfamily and is expressed by activated T cells, have potent antitumor activities in preclinical models. However, previous studies have shown that the antitumor effects of OX40 agonists depend on the immunogenicity of the tumor and that poorly immunogenic tumors such as B16F10 melanomas do not respond to OX40 agonist treatment. In this study, we have shown that intratumoral administration of an anti-CD4 mAb (for CD4+ cell depletion) sensitized poorly immunogenic B16F10 melanomas to immunotherapy with an OX40 agonist. CD4+ cell depletion markedly enhanced the infiltrations of both CD8+ T cells and natural killer (NK) cells into tumor tissues. Cytokines with antitumor activities, such as IFN-γ, TNF-α, IL-2, and IL-12, were significantly upregulated by CD4+ cell depletion. However, unexpectedly, the number of CD11b+Gr-1+ myeloid-derived suppressor cells (MDSCs) within tumor tissues also significantly increased as a result of CD4+ cell depletion. As a countermeasure against CD8+ T cell accumulation, CCR2-positive CD11b+Gr-1int (monocytic) MDSCs predominantly increased. Treatment with an OX40 agonist under CD4+ cell depletion neither reduced MDSCs nor increased CD8+ T cells and NK cells, but further enhanced the expression of cytotoxic molecules from tumor-infiltrating effector cells. Our results suggest that combined immunotherapy using both an OX40 agonist and CD4+ cell depletion in situ could be a promising therapeutic strategy for poorly immunogenic tumors and might be more effective if further combined with a therapeutic strategy targeting MDSCs.

Abstract 4802: PB1, a DDR2 inhibitor with antitumor activity in preclinical models of squamous cell carcinoma and KRAS-mutated adenocarcinoma of the lung

Abstract Introduction: Discoidin domain receptor 2 (DDR2) is a receptor tyrosine kinase (RTK) activated by collagen I and identified as an oncogenic driver in squamous cell carcinoma of the lung (SCC) (1). DDR2 mutations have been reported in 4% of SCC patients and at lower frequencies in lung adenocarcinoma, gastric, breast and brain tumors (2). In addition, DDR2 is expressed during epithelial to mesenchymal transition (EMT) (3) and acts as an upstream activator of SHP2. SHP2 in turn is a key signaling effector which leads to activation of multiple signaling pathways (4). DDR2 has recently emerged as a potential therapeutic target in DDR2-mutated tumors and drugs such as dasatinib are being tested in this setting. Results and discussion: PB1 is a small organic molecule with a simple synthesis pathway that showed strong inhibitory activity against wild type (wt) and mutated purified DDR2 protein. The compound was subsequently tested in a panel of tumor cell lines from different origins and histologies. PB1 showed good antiproliferative activity: IC50<1 μM in (i) SCC cell lines with DDR mutations such as H2286 or H520, (ii) SCC lung tumor cell lines wt for DDR2, and (iii) some lung adenocarcinoma cell lines with KRAS mutations such as A549. Remarkably, the two cell lines tested carrying the DDR2 activating mutation I638F were extremely sensitive to PB1, with IC50 in the nanomolar range. The compound also showed good activity against cells harboring the DDR2 L239R mutation. We also generated two dasatinib-resistant cells lines from the H2286 and H520 cell lines. Cells had IC50s for dasatinib over 25 uM but remained sensitive to PB1, with IC50s of 0.6 and 5.92, respectively. Finally, in non tumor cell lines, no toxicity was observed with PB1 at concentrations tested (up to 50 μM), indicating a good therapeutic index. Western blotting was used to study the effects of PB1 on DDR2, ERK and AKT activation in H2286 and A549 cells. The compound strongly inhibited FBS-induced phosphorylation of the three proteins. Conclusion: PB1 shows significant antitumor activity in preclinical models of mutated and wt DDR2 in SCC of the lung and some KRAS mutated adenocarcinoma cell lines. PB1 is also active against tumor cells with secondary resistance to dasatinib.

Abstract LB-237: BMS-986012, a fully human anti-fucosyl-GM1 antibody has potent in vitro and in vivo antitumor activity in preclinical models of small cell lung cancer

Abstract Fucosyl-GM1, is a sphingolipid monosialoganglioside and tumor-associated antigen with a high prevalence in human small cell lung cancer (SCLC), while its expression is minimal in most normal tissue. Here, we report results of our preclinical studies with BMS-986012, a novel fully human, non-fucosylated, IgG1 monoclonal antibody that specifically binds to FucGM1. The binding affinity of BMS-986012 was evaluated in Biacore® and cell-based assays. BMS-986012 binds with a high affinity to FucGM1 and demonstrates no significant binding to other closely related gangliosides. The antibody was engineered in a non-fucosylated IgG1 format, resulting in a 40-fold increased affinity for FcγRIIIa (CD16). In in vitro assays, BMS-986012 binds to FucGM1 on cancer cells and activates antibody-dependent cellular cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC), and antibody-mediated phagocytosis (ADCP). The antitumor activity of BMS-986012 was profiled in a panel of SCLC lung cancer models in immunodeficient mice with varying FucGM1 target expression levels qualified using immunohistochemistry. Dose-dependent antitumor activity was observed in a panel of SCLC xenograft models with tumor regressions occurring at BMS-986012 doses greater than 0.3 mg/kg against DMS79 tumors. BMS-986012 efficacy generally correlated with FucGM1 target expression in tumor models. In mice, the antibody was well tolerated, with no adverse events observed in long term efficacy studies. Preclinical studies also explored combinations of BMS-986012 with standard-of-care (SOC) chemotherapeutics, including cisplatin, etoposide, topotecan and irinotecan. Combination therapy of BMS-986012 with SOC resulted in significantly enhanced antitumor activity compared to monotherapy. Finally, studies conducted with BMS-986012 in combination with anti-CD137 antibody resulted in significant improvement in efficacy. These data provide preclinical support for evaluation of BMS-986012 in SCLC. BMS-986012 is currently in a phase I trial. Citation Format: Bing Chen, Chin Pan, Paul Ponath, Miho Oyasu, Daniel Menezes, Pina Cardarelli. BMS-986012, a fully human anti-fucosyl-GM1 antibody has potent in vitro and in vivo antitumor activity in preclinical models of small cell lung cancer. [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 LB-237.

Abstract 3022: BAY 1143572, a first-in-class, highly selective, potent and orally available inhibitor of PTEFb/CDK9 currently in Phase I, shows convincing anti-tumor activity in preclinical models of acute myeloid leukemia (AML)

Abstract PTEFb/CDK9 mediated transcription of short-lived anti-apoptotic survival proteins like Mcl-1 and Myc, plays a critical role in cancer cell growth and survival in various tumor entities including AML. In addition, these survival proteins exhibit important functions in the development of resistance to chemotherapy. In contrast to pan-CDK inhibitors, to our knowledge PTEFb selective inhibitors have not been explored for clinical utility. We report the preclinical activity of BAY 1143572, a novel selective PTEFb/CDK9 inhibitor (AACR; Cancer Res 2015;75(15 Suppl):Abstract nr DDT02-02) currently being investigated in Phase I clinical trials in advanced cancer (NCT01938638) and acute leukemia (NCT02345382) in various in vitro, ex vivo and in vivo models of AML. BAY 1143572 inhibited the proliferation of 7 MLL-rearrangements positive and negative AML cell lines with a median IC50 of 385 nM (range 230-1100 nM) and induced apoptosis. Furthermore, BAY 1143572 showed potent in vitro activity in 8 out of 10 non-MLL-rearranged patient derived AML samples incl. NPM1 mutant and Flt3-ITD positive samples derived from intermediate and high risk patients. Moreover, we elucidated the dynamic changes of the cellular proteome/phosphoproteome upon pharmacological and genetic PTEFb inhibition and identified PTEFb interaction partners in various AML in vitro models. These analyses uncover the oncogenic PTEFb-dependent signaling networks and substantiate the molecular rationale for the use of PTEFb inhibitors in this indication. When applied in vivo, BAY 1143572 exhibited single agent efficacy at tolerated doses in 4 out of 5 AML xenograft tumor models in mice and in 2 out of 2 AML xenograft tumor models in rats upon once daily oral administration. Of note, partial or even complete remissions could be achieved in several models. Furthermore, intermittent dosing schedules with up to 4 days treatment pauses were feasible in terms of efficacy and tolerability. Using MOLM-13 xenografts in mice and rats to address the in vivo MoA of BAY 1143572, a transient inhibition of RNA polymerase II phosphorylation, MYC mRNA and protein levels, MCL-1 mRNA and protein levels, and an induction of apoptosis was documented. In conclusion, our data provide the rationale for the initiation of clinical development of BAY 1143572 as a potent and highly selective inhibitor of PTEFb/CDK9 with first-in-class potential for the treatment of AML patients. A phase I clinical trial to determine the safety, tolerability and recommended Phase 2 dose in this indication is ongoing (NCT02345382). Citation Format: Arne Scholz, Thomas Oellerich, Akhtar Hussain, Sarah Lindner, Ulrich Luecking, Annette O. Walter, Peter Ellinghaus, Ray Valencia, Franz von Nussbaum, Dominik Mumberg, Michael Brands, Stuart Ince, Hubert Serve, Karl Ziegelbauer. BAY 1143572, a first-in-class, highly selective, potent and orally available inhibitor of PTEFb/CDK9 currently in Phase I, shows convincing anti-tumor activity in preclinical models of acute myeloid leukemia (AML). [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 3022.

Abstract LB-053: Towards new cryptophycins as promising payloads for ADC

Abstract Cryptophycins are a class of macrocyclic depsipeptides produced as secondary metabolites by cyanobacteria of the genus Nostoc and were found to bind to microtubules at the vinca site. The natural product C-1 isolated in 1990 and the synthetic derivative C-52 displayed potent in vitro and in vivo antitumor activity in preclinical models. Cryptophycin C-52 (LY355703) produced marginal antitumor activity at MTD in two phase II lung cancer trials and was therefore discontinued. Considering its higher potency versus other tubulin binders such as maytansine and auristatin, this chemical series was selected for an ADC approach. Several conjugates were evaluated in vitro and in vivo based on the conjugation of C52 derivatized at the para-benzylic position of the macrocycle. As described in this study, this optimization led to the discovery of cryptophycin ADC which displayed potent antitumor activity in vivo. However, these conjugates were found unstable in mice plasma while being stable in the plasma of non-rodent species. This species-dependent instability was shown to be the result of a mice plasmatic metabolization of C52 macrocycle once conjugated to the antibody. We therefore designed and synthesized new cryptophycin ADC which exhibited improved plasmatic stability when tested in mice and enhanced therapeutic index in comparison with C52 ADC. In conclusion, these data demonstrated the potent in vitro and in vivo antitumor activity of these new cryptophycin ADC and warrant further development of this cytotoxic payload for an ADC approach. Citation Format: Marie-Priscille Brun, Hervé Bouchard, François Clerc, Jidong Zhang, Pierre-Yves Abecassis, Céline Amara, Eric Beys, Florence Efremenko, Céline Nicolazzi, Marie-Hélène Pascual, Olivier Pasquier, Alain Krick, Pierre-François Berne, Laurence Gauzy, Nathalie Karst, Sylvain Huille, Christophe Henry, Anne-Marie Lefebvre, Ingrid Sassoon. Towards new cryptophycins as promising payloads for ADC. [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 LB-053.

Abstract 2214: GITR ligand fusion protein (GITRL-Fc) induces T cell mediated anti-tumor immune response and can combine with anti-PDL1 to enhance anti-tumor immunity and long-term immune memory

Abstract GITRL (Glucocorticoid-Induced Tumor Necrosis Factor Receptor Ligand, TNFSF18) is a member of the TNF superfamily and naturally exists as a membrane-anchored type II protein that self assembles as a trimer. GITRL activates the co-stimulatory receptor GITR. GITR is found primarily on activated T effector (Teff) cells and regulatory T (Treg) cells. Co-stimulation of GITR by agonist agents is hypothesized to promote anti-tumor immunity by enhancing Teff cell activity and inhibiting Treg suppression. We generated a novel single-gene GITRL trimer fused to an immunoglobulin Fc domain (GITRL-Fc). GITRL-Fc activated GITR signaling more effectively than prototype GITR agonist antibody DTA-1. GITRL-Fc promoted a robust anti-tumor immune response in multiple syngeneic mouse tumor models. GITRL-Fc enhanced tumor specific T-cell responses, particularly of the Th1 type, and also led to reduction in Treg-mediated immunesuppressive activity. GITRL-Fc displayed single agent activity in inhibiting tumor growth and promoting complete tumor rejection in the murine CT26 colon carcinoma model and combination activity with anti-PDL1 as compared to anti-PDL1 and control IgG2a alone. Mice “cured” with GITRL or GITRL/anti-PDL1 combination treatments were protected from re-challenge with tumor cells, suggesting the existence of immunologic memory. More mice were protected from tumor re-challenge with the combination of GITRL-Fc and anti-PDL1, as compared to GITRL-Fc alone. Our results demonstrate that agonist GITRL-Fc induces potent T cell responses, overcomes Treg inhibition, and promotes anti-tumor activity in preclinical models as a single agent or in combination with anti PDL1. The mechanism of tumor eradication and induction of long-term immune memory response by the combination is under investigation and will be discussed at the presentation. Citation Format: Minu K. Srivastava, Rui Yun, Erin Mayes, Hyun_Bae Jie, Fumiko Axelrod, Jorge Monteon, Ming-Hong Xie, John Lewicki, Tim Hoey, Austin Gurney, Angie Inkyung Park. GITR ligand fusion protein (GITRL-Fc) induces T cell mediated anti-tumor immune response and can combine with anti-PDL1 to enhance anti-tumor immunity and long-term immune memory. [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 2214.

Abstract 2232: Immune-inflammatory markers predict the outcome of metastatic colorectal cancer patients treated with the thymidylate synthase poly-epitope peptide (TSPP) vaccine: results from a multi-arm TSPP/VAC phase Ib program

Abstract Thymidylate synthase (TS) is a tumor-associated enzyme critical for DNA replication. TSPP is a poly-epitope-peptide vaccine to TS, which elicits a multi-epitopic CTL response with antitumor activity in preclinical models. TSPP has been tested in 50 advanced cancer patients enrolled in a multi-arm dose-finding TSPP/VAC1 phase Ib trial program (Eudract:# 2009-016897-33)(Table1) between May 2011 and January 2013. The trial results revealed that TSPP is safe and immune-biologically active, and provided preliminary evidence of a antitumor activity in pretreated mCRC patients (Cusi MG et al 2015 and Correale P et al 2015). This population of 41 patients presented a progression-free-survival and an overall-survival (OS) of 6.9 and 11.3 months, respectively, with a one-year survival rate of 32% (13 patients). We focused on mCRC patients as they represented the most relevant and homogeneous population in the trial. We performed Kaplan-Meier curves, log-rank tests and regression curves to correlate immunobiological findings and patients’ outcome. We found that their OS correlated with lower (under the median) ECOG scores (p:0.039), CEA levels (p:0.021), and serum levels of inflammatory markers (NLR, CRP, ESR, and LDH/LDHNR and ENA; p inferior 0.04) at baseline. Patients’ OS also correlated with greater (over the median) baseline levels of IL4 (p:0.028) and 17 (P:0.049), and with a post-treatment increase in anti-neutrophil-cytoplasm-antibodies/anti-proteinase-3 (Fold change to baseline values grater than 1; p:0.039). Activating K-ras mutations did not correlate with survival, however, inflammatory markers, cytokines, and autoantibodies lost their correlation with the survival in patients bearing these mutations. These results suggest that TSPP-antitumor activity in mCRC patients is affected by their baseline inflammatory/immunological status at baseline. TSPPVAC1 Phase Ib program; patients’ distributionARM A1. ADJ + TSPP 100 μg (n = 3)2. ADJ + TSPP 200 μg (n = 3)3. ADJ + TSPP 300 μg (n = 6)ARM B1. ADJ + TSPP 100 μg + IG-1 (n = 3)2. ADJ + TSPP 200 μg + IG-1 (n = 3)3. ADJ + TSPP 300 μg + IG-1 (n = 3)ARM C (DL1-3)1. GOLFIG + ADJ+TSPP 100 μg + IG-1 (n = 3)2. GOLFIG + ADJ+TSPP 200 μg + IG-1 (n = 3)3. GOLFIG + ADJ+TSPP 300 μg + IG-1 (n = 11)ARM C(DL0) 1. GOLFIG (12 courses) -> ADJ + TSPP 300 μg + IG-1 (n = 10)Figure legend: ADJ = montanide; n = number of patients; IG1 = sc. GM-CSF and sc. Aldesleukine; GOLFIG = gemcitabine + FOLFOX4 polychemotherapy + IG1 regimen Citation Format: Pierpaolo Correale, Cirino Botta, Elodia Claudia Martino, Valerio Nardone, Cristina Ulivieri, Claudia Gandolfo, Tatiana Cosima Baldari, Stefano Lazzi, Alessandro Ginori, Antonella Fioravanti, Giacomo Maria Guidelli, Luigi Pirtoli, Antonio Giordano, Pierfrancesco Tassone, Pierosandro Tagliaferri, Maria Grazia Cusi. Immune-inflammatory markers predict the outcome of metastatic colorectal cancer patients treated with the thymidylate synthase poly-epitope peptide (TSPP) vaccine: results from a multi-arm TSPP/VAC phase Ib program. [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 2232.

Herbacetin Is a Novel Allosteric Inhibitor of Ornithine Decarboxylase with Antitumor Activity.

Ornithine decarboxylase (ODC) is a rate-limiting enzyme in the first step of polyamine biosynthesis that is associated with cell growth and tumor formation. Existing catalytic inhibitors of ODC have lacked efficacy in clinical testing or displayed unacceptable toxicity. In this study, we report the identification of an effective and nontoxic allosteric inhibitor of ODC. Using computer docking simulation and an in vitro ODC enzyme assay, we identified herbacetin, a natural compound found in flax and other plants, as a novel ODC inhibitor. Mechanistic investigations defined aspartate 44 in ODC as critical for binding. Herbacetin exhibited potent anticancer activity in colon cancer cell lines expressing high levels of ODC. Intraperitoneal or oral administration of herbacetin effectively suppressed HCT116 xenograft tumor growth and also reduced the number and size of polyps in a mouse model of APC-driven colon cancer (ApcMin/+). Unlike the well-established ODC inhibitor DFMO, herbacetin treatment was not associated with hearing loss. Taken together, our findings defined the natural product herbacetin as an allosteric inhibitor of ODC with chemopreventive and antitumor activity in preclinical models of colon cancer, prompting its further investigation in clinical trials.

Abstract P5-03-01: Therapeutic targeting of Rac GTPases in ER+ and HER2+ breast cancer

Abstract As crucial regulators of cell motility, survival, and proliferation, Rac GTPases (Rac1/2/3) have been implicated in cancer. We previously found that P-REX1, a guanine nucleotide exchange factor that activates Rac GTPases, forms a PI3K-driven positive feedback loop to promote activation of Rac enzymes, receptor tyrosine kinases, PI3K/AKT/mTOR, and MEK/ERK in ER+ breast cancer cells. Importantly, inhibition of Rac GTPases with a small molecule (EHT1864) simultaneously suppressed activation of both the PI3K/AKT/mTOR and MEK/ERK pathways. While co-targeting of the PI3K/AKT/mTOR and MEK/ERK pathways with drug combinations has anti-tumor activity in preclinical models and is being tested in ongoing clinical trials, targeting Rac as a common upstream signaling node may be a more efficient means of simultaneously targeting these two oncogenic pathways. In breast cancer cell lines, treatment with EHT1864 decreased activation of AKT, mTOR, p70S6K, and S6 in a dose-dependent manner. Pulldown of activated Rac from cell lysates revealed that GTP-bound Rac1 and/or Rac3 bind MEK1/2, ERK1/2, p70S6K, S6, Raptor, Rictor, and mTOR. Temporal analysis indicated that EHT1864 inhibits phosphorylation of p70S6K (an mTORC1 substrate) before AKT is inhibited, suggesting that Rac may directly activate p70S6K and/or mTORC1. Mining of sensitivity data from ∼700 cell lines to a panel of 138 drugs (COSMIC/GDSC database) revealed that the growth-suppressive effects of EHT1864 correlate most strongly with growth-suppressive patterns induced by a p70S6K inhibitor, supporting the notion that Rac directly activates p70S6K. Additionally, EHT1864 treatment dose-dependently decreased phosphorylation of ERK1/2 and MEK1/2, and induced apoptosis in breast cancer cell lines. In a panel of 16 breast cancer lines, cells with activating mutations in PIK3CA (encodes the p110-alpha subunit of PI3K) exhibit increased EHT1864 sensitivity. Pharmacokinetic analysis of EHT1864 (100 mg/kg, i.p.) in plasma in NSG mice revealed that drug was present at >50 uM for 1 h after administration. In mice bearing s.c. ER+/HER2+ BT-474 breast cancer xenografts, EHT1864 (100 mg/kg BID) significantly slowed tumor growth compared to vehicle control. Thus, therapeutically targeting Rac could be an effective means of reducing cancer cell proliferation and survival by simultaneously suppressing both the PI3K/AKT/mTOR and MEK/ERK signaling pathways in ER+ and HER2+ breast cancers. Citation Format: Hampsch RA, Shee K, Miller TW. Therapeutic targeting of Rac GTPases in ER+ and HER2+ breast cancer. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P5-03-01.

Preclinical evaluation of WYE-687, a mTOR kinase inhibitor, as a potential anti-acute myeloid leukemia agent

Mammalian target of rapamycin (mTOR) as a potential drug target for treatment of acute myeloid leukemia (AML). Here, we investigated the potential anti-leukemic activity by WYE-687, a potent mTOR kinase inhibitor. We demonstrated that WYE-687 potently inhibited survival and proliferation of established (HL-60, U937, AML-193 and THP-1 lines) and human AML progenitor cells. Yet, same WYE-687 treatment was non-cytotoxic to the primary peripheral blood mononuclear leukocytes (PBMCs) isolated from healthy donors. WYE-687 induced caspase-dependent apoptotic death in above AML cells/progenitor cells. On the other hand, the pan-caspase inhibitor (Z-VAD-FMK), the caspase-3 specific inhibitor (Z-DEVD-FMK) or the caspase-9 specific inhibitor (z-LEHD-fmk) attenuated WYE-687-induced cytotoxicity. At the molecular level, WYE-687 concurrently inhibited activation of mTORC1 (p70S6K1 and S6 phosphorylations) and mTORC2 (AKT Ser-473 and FoxO1/3a phosphorylations), whiling downregulating mTORC1/2-regulated genes (Bcl-xL and hypoxia-inducible factor 1/2α) in both HL-60/U937 cells and human AML progenitor cells. In vivo, oral administration of WYE-687 potently inhibited U937 leukemic xenograft tumor growth in severe combined immunodeficient (SCID) mice, without causing significant toxicities. In summary, our results demonstrate that targeting mTORC1/2 by WYE-687 leads to potent antitumor activity in preclinical models of AML.

CD19xCD3 DART protein mediates human B-cell depletion in vivo in humanized BLT mice

Novel therapeutic strategies are needed for the treatment of hematologic malignancies; and bispecific antibody-derived molecules, such as dual-affinity re-targeting (DART) proteins, are being developed to redirect T cells to kill target cells expressing tumor or viral antigens. Here we present our findings of specific and systemic human B-cell depletion by a CD19xCD3 DART protein in humanized BLT mice. Administration of the CD19xCD3 DART protein resulted in a dramatic sustained depletion of human CD19+ B cells from the peripheral blood, as well as a dramatic systemic reduction of human CD19+ B-cell levels in all tissues (bone marrow, spleen, liver, lung) analyzed. When human CD8+ T cells were depleted from the mice, no significant B-cell depletion was observed in response to CD19xCD3 DART protein treatment, confirming that human CD8+ T cells are the primary effector cells in this in vivo model. These studies validate the use of BLT humanized mice for the in vivo evaluation and preclinical development of bispecific molecules that redirect human T cells to selectively deplete target cells.

Antroquinonol A: Scalable Synthesis and Preclinical Biology of a Phase 2 Drug Candidate.

The fungal-derived Taiwanese natural product antroquinonol A has attracted both academic and commercial interest due to its reported exciting biological properties. This reduced quinone is currently in phase II trials (USA and Taiwan) for the treatment of non-small-cell lung carcinoma (NSCLC) and was recently granted orphan drug status by the FDA for the treatment of pancreatic cancer and acute myeloid leukemia. Pending successful completion of human clinical trials, antroquinonol is expected to be commercialized under the trade name Hocena. A synthesis-enabled biological re-examination of this promising natural product, however, reveals minimal in vitro and in vivo antitumor activity in preclinical models.

Preclinical Efficacy and Safety Assessment of an Antibody-Drug Conjugate Targeting the c-RET Proto-Oncogene for Breast Carcinoma.

The RET proto-oncogene has been implicated in breast cancer, and the studies herein describe the preclinical and safety assessment of an anti-RET antibody-drug conjugate (ADC) being developed for the treatment of breast cancer. RET protein expression was analyzed in breast tumor samples using tissue microarrays. The fully human anti-RET antibody (Y078) was conjugated to the DM1 and DM4 derivatives of the potent cytotoxic agent maytansine using thioether and disulfide linkers, respectively. The resulting compounds, designated Y078-DM1 and Y078-DM4, were evaluated for antitumor activity using human breast cancer cell lines and established tumor xenograft models. A single-dose, 28-day, safety study of Y078-DM1 was performed in cynomolgus monkeys. By immunohistochemistry, RET expression was detected in 57% of tumors (1,596 of 2,800 tumor sections) and was most common in HER2-positive and basal breast cancer subtypes. Potent in vitro cytotoxicity was achieved in human breast cancer cell lines that have expression levels comparable with those observed in breast cancer tissue samples. Dose-response studies in xenograft models demonstrated antitumor activity with both weekly and every-3-weeks dosing regimens. In cynomolgus monkeys, a single injection of Y078-DM1 demonstrated dose-dependent, reversible drug-mediated alterations in blood chemistry with evidence of on-target neuropathy. RET is broadly expressed in breast cancer specimens and thus represents a potential therapeutic target; Y078-DM1 and Y078-DM4 demonstrated antitumor activity in preclinical models. Optimization of the dosing schedule or an alternate cytotoxic agent with a different mechanism of action may reduce the potential risk of neuropathy. Clin Cancer Res; 21(24); 5552-62. ©2015 AACR.

Significant blockade of multiple receptor tyrosine kinases by MGCD516 (Sitravatinib), a novel small molecule inhibitor, shows potent anti-tumor activity in preclinical models of sarcoma

Sarcomas are rare but highly aggressive mesenchymal tumors with a median survival of 10–18 months for metastatic disease. Mutation and/or overexpression of many receptor tyrosine kinases (RTKs) including c-Met, PDGFR, c-Kit and IGF1-R drive defective signaling pathways in sarcomas. MGCD516 (Sitravatinib) is a novel small molecule inhibitor targeting multiple RTKs involved in driving sarcoma cell growth. In the present study, we evaluated the efficacy of MGCD516 both in vitro and in mouse xenograft models in vivo. MGCD516 treatment resulted in significant blockade of phosphorylation of potential driver RTKs and induced potent anti-proliferative effects in vitro. Furthermore, MGCD516 treatment of tumor xenografts in vivo resulted in significant suppression of tumor growth. Efficacy of MGCD516 was superior to imatinib and crizotinib, two other well-studied multi-kinase inhibitors with overlapping target specificities, both in vitro and in vivo. This is the first report describing MGCD516 as a potent multi-kinase inhibitor in different models of sarcoma, superior to imatinib and crizotinib. Results from this study showing blockade of multiple driver signaling pathways provides a rationale for further clinical development of MGCD516 for the treatment of patients with soft-tissue sarcoma.

Clinical Evaluation of Combined Epigenetic Therapies on the Induction of Fetal Hemoglobin in Patients with Hematologic Malignancies

Fetal hemoglobin induction with hydroxyurea (HU) is a mainstay of therapy for β-hemoglobinopathies, especially sickle cell disease (SCD). A high level of fetal hemoglobin (HbF) has a direct relationship with acute clinical status in SCD patients including pain crises, acute chest syndrome, and death. However, not all patients benefit from HU, and more effective HbF induction strategies are needed. DNA methyl transferase (DNMT) inhibitors and histone deacetylase (HDAC) inhibitors have been shown in vitro to induce HbF production through epigenetic modification of the β-globin gene cluster. Azacitidine (AZA) is a DNMT already used in some SCD patients resistant to HbF modulation with HU. Entinostat (MS-275) is an orally available histone deacetylase inhibitor with a long half-life and established antitumor activity in preclinical models. Recent studies suggest that drugs, which act with different molecular and epigenetic mechanisms, have synergistic effects on induction of fetal hemoglobin (Fard et al. IJHOSCR 2013). In this study, we evaluated the effects of a combination of AZA and MS-275 on HbF levels. This was preformed as a correlative study of a phase I clinical trial (J0443 trial) of these drugs in patients with myelodysplastic syndrome (MDS) or acute myeloid leukemia (AML). We sequentially measured the level of HbF the peripheral blood in 33 patients receiving different doses of AZA (range: 30mg/m2 to 50mg/m2 per day for 10 doses) and MS-275 (range: 2 to 8 mg/m2 orally on days 3 and 10). Patients completed a minimum of four 28-day cycles of combined therapy. HbF levels were measured in peripheral blood at baseline, at day 15 or 16 and day 29 or 30 of cycle 1, and after cycles 2, 4, and if applicable 6. Azacitidine dose positively correlated with HbF fold increase (mean of 1.1, 2.3, and 2.1 for doses of 30, 40, and 50 mg respectively, p=0.07) while MS-275 dose had a slightly negative correlation with HbF level (mean of 3.0, 1.8, and 1.3 for doses of 2, 4, and 6mg respectively, p=0.13). There was no correlation between baseline HbF and HbF fold increase after exposure to treatment (p=NS) and no correlation between baseline HbF levels and clinical disease response (p=0.19). Interestingly, we demonstrated a correlation between HbF fold increase and clinical disease response: median fold increase of 3.5 for patients achieving hematologic normalization (complete response, partial response, or trilineage hematological improvement) versus 1.4 in non-responders (p=0.006). The positive correlation between AZA dose and HbF increase is consistent with prior work showing that this drug induces HbF production. The correlation between clinical response and HbF induction could reflect a greater susceptibility to AZA potentially related to differing methylomes. Alternatively, it may also represent a known increase in HbF in the setting of stress erythropoiesis. The slight inverse correlation between MS-275 and HbF level was surprising, as other HDAC inhibitors are known to induce HbF in vitro. However, these results are in line with the methylation data found in the more recent randomized phase 2 trial of AZA +/- MS-275 (E1905 trial) that showed a potential pharmacodynamic antagonism of the combination (Prebet et al. J Clin Oncol. 2014). Overall, this work supports the use of AZA as a clinical inducer of HbF. It also shows the importance of trialing various combinations of HbF inducers, as not all drugs work synergistically and some may even be antagonistic in combination. Disclosures Off Label Use: Azacitidine (AZA) is a DNA methyl transferase (DNMT) inhibitor. Entinostat (MS-275) is an orally available histone deacetylase inhibitor. Both drugs were used in a phase I clinical trial (J0443 trial) of these drugs in patients with myelodysplastic syndrome (MDS) or acute myeloid leukemia (AML). . Keefer:MAST therapeutics: Employment. Gore:Celgene: Consultancy, Honoraria, Research Funding. Prebet:CELGENE: Research Funding.