Treatment Of Tumor-bearing Animals Research Articles

Abstract 2659: Therapeutic administration of an engineered sphingosine-1-phosphate lyase improves T cell trafficking to tumors and enhances antitumor response

Abstract Introduction: Immunotherapy has revolutionized the landscape of cancer treatment; however, to date, only limited aspects of the tumor-immune interaction have been exploited successfully, e.g., relief of immune checkpoints, increase of tumor-associated antigen presentation. Although it is well-known that trafficking and entry of T cells from the circulation into tumors is often impaired, leading to an immune “cold”, checkpoint inhibitor-refractory state, there are no available therapies that address this important aspect of antitumor immunity. Sphingosine-1-phosphate (S1P) is a pleiotropic, bioactive lipid that has been shown to regulate lymphocyte egress from lymph nodes through its interaction with one of its several receptors, S1P1. Clinically, tumor expression levels of the sphingosine-1-phosphate-producing enzyme sphingosine kinase 1 (SPHK1) strongly and negatively correlates with survival of melanoma patients after immune checkpoint inhibitor (ICI) therapy, and pre-clinically correlates with reduced tumor infiltrating lymphocytes (TILs). We therefore hypothesize that aberrant S1P signaling in tumors and in their draining lymph nodes (TDLN) causes immune exclusion by impeding lymphocyte egress from the TDLN, ultimately restricting immune cell infiltration of tumors. Methods: We tested the therapeutic efficacy of an engineered S1P-degrading enzyme, S1P lyase (S1PL), on established (~100mm^3) syngeneic B16, CT26, and MC38 murine tumors. Using flow cytometry, we performed immune profiling of systemic, TDLN, and intratumoral T cell populations, assessing both activation status and expression of the S1P1 receptor. Results: Treatment of tumor-bearing animals with S1PL depleted systemic S1P, and dramatically increased both trafficking of lymphocytes out of TDLNs and T cell infiltration of tumors relative to control-treated animals. Mice treated with S1PL evinced a higher proportion of S1P1+ circulating and intratumoral T cells relative to control-treated mice, suggesting that the increase in liberation of T cells from the TDLN was effected through the S1P/S1P1 axis. This increased lymphocyte trafficking correlated with strong inhibition of tumor growth when S1PL was administered as a single agent. Conclusions: Therapeutic administration of S1PL may represent an attractive strategy for modulating lymphocyte trafficking to improve the efficacy of therapies that rely on the adaptive immune system to exert antitumor activity. This approach may be particularly potent in settings wherein tumor-mediated immune exclusion is a dominant method of immune escape. Citation Format: Alessandra Araujo, Max Rodnick-Smith, Ranya Al-Khaledy, George Georgiou, Mark Badeaux, Everett Stone. Therapeutic administration of an engineered sphingosine-1-phosphate lyase improves T cell trafficking to tumors and enhances antitumor response [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2659.

Biophysical phenotype mixtures reveal advantages for tumor muscle invasion in vivo

Bladder, colon, gastric, prostate, and uterine cancers originate in organs surrounded by laminin-coated smooth muscle. In human prostate cancer, tumors that are organ confined, without extracapsular extension through muscle, have an overall cancer survival rate of up to 97% compared with 32% for metastatic disease. Our previous work modeling extracapsular extension reported the blocking of tumor invasion by mutation of a laminin-binding integrin called α6β1. Expression of the α6AA mutant resulted in a biophysical switch from cell-ECM (extracellular matrix) to cell-cell adhesion with drug sensitivity properties and an inability to invade muscle. Here we used different admixtures of α6AA and α6WT cells to test the cell heterogeneity requirements for muscle invasion. Time-lapse video microscopy revealed that tumor mixtures self-assembled into invasive networks in vitro, whereas α6AA cells assembled only as cohesive clusters. Invasion of α6AA cells into and through live muscle occurred using a 1:1 mixture of α6AA and α6WT cells. Electric cell-substrate impedance sensing measurements revealed that compared with α6AA cells, invasion-competent α6WT cells were 2.5-fold faster at closing a cell-ECM or cell-cell wound, respectively. Cell-ECM rebuilding kinetics show that an increased response occurred in mixtures since the response was eightfold greater compared with populations containing only one cell type. A synthetic cell adhesion cyclic peptide called MTI-101 completely blocked electric cell-substrate impedance sensing cell-ECM wound recovery that persisted in vitro up to 20 h after the wound. Treatment of tumor-bearing animals with 10 mg/kg MTI-101 weekly resulted in a fourfold decrease of muscle invasion by tumor and a decrease of the depth of invasion into muscle comparable to the α6AA cells. Taken together, these data suggest that mixed biophysical phenotypes of tumor cells within a population can provide functional advantages for tumor invasion into and through muscle that can be potentially inhibited by a synthetic cell adhesion molecule.

Effective intravenous delivery of adenovirus armed with TNFα and IL-2 improves anti-PD-1 checkpoint blockade in non-small cell lung cancer

ABSTRACT Lung cancer remains among the most difficult-to-treat malignancies and is the leading cause of cancer-related deaths worldwide. The introduction of targeted therapies and checkpoint inhibitors has improved treatment outcomes; however, most patients with advanced-stage non-small cell lung cancer (NSCLC) eventually fail these therapies. Therefore, there is a major unmet clinical need for checkpoint refractory/resistant NSCLC. Here, we tested the combination of aPD-1 and adenovirus armed with TNFα and IL-2 (Ad5-CMV-mTNFα/mIL-2) in an immunocompetent murine NSCLC model. Moreover, although local delivery has been standard for virotherapy, treatment was administered intravenously to facilitate clinical translation and putative routine use. We showed that treatment of tumor-bearing animals with aPD-1 in combination with intravenously injected armed adenovirus significantly decreased cancer growth, even in the presence of neutralizing antibodies. We observed an increased frequency of cytotoxic tumor-infiltrating lymphocytes, including tumor-specific cells. Combination treatment led to a decreased percentage of immunosuppressive tumor-associated macrophages and an improvement in dendritic cell maturation. Moreover, we observed expansion of the tumor-specific memory T cell compartment in secondary lymphoid organs in the group that received aPD-1 with the virus. However, although the non-replicative Ad5-CMV-mTNFα/mIL-2 virus allows high transgene expression in the murine model, it does not fully reflect the clinical outcome in humans. Thus, we complemented our findings using NSCLC ex vivo models fully permissive for the TNFα and IL-2- armed oncolytic adenovirus TILT-123. Overall, our data demonstrate the ability of systemically administered adenovirus armed with TNFα and IL-2 to potentiate the anti-tumor efficacy of aPD-1 and warrant further investigation in clinical trials.

Oral IRAK-4 Inhibitor CA-4948 Is Blood-Brain Barrier Penetrant and Has Single-Agent Activity against CNS Lymphoma and Melanoma Brain Metastases.

An ongoing challenge in cancer is the management of primary and metastatic brain malignancies. This is partly due to restrictions of the blood-brain barrier and their unique microenvironment. These challenges are most evident in cancers such as lymphoma and melanoma, which are typically responsive to treatment in systemic locations but resistant when established in the brain. We propose interleukin-1 receptor-associated kinase-4 (IRAK-4) as a potential target across these diseases and describe the activity and mechanism of oral IRAK-4 inhibitor CA-4948. Human primary central nervous system lymphoma (PCNSL) and melanoma brain metastases (MBM) samples were analyzed for expression of IRAK-4 and downstream transcription pathways. We next determined the central nervous system (CNS) applicability of CA-4948 in naïve and tumor-bearing mice using models of PCNSL and MBM. The mechanistic effect on tumors and the tumor microenvironment was then analyzed. Human PCNSL and MBM have high expression of IRAK-4, IRAK-1, and nuclear factor kappa B (NF-κB). This increase in inflammation results in reflexive inhibitory signaling. Similar profiles are observed in immunocompetent murine models. Treatment of tumor-bearing animals with CA-4948 results in the downregulation of mitogen-activated protein kinase (MAPK) signaling in addition to decreased NF-κB. These intracellular changes are associated with a survival advantage. IRAK-4 is an attractive target in PCNSL and MBM. The inhibition of IRAK-4 with CA-4948 downregulates the expression of important transcription factors involved in tumor growth and proliferation. CA-4948 is currently being investigated in clinical trials for relapsed and refractory lymphoma and warrants further translation into PCNSL and MBM.

Local delivery of interleukin 7 with an oncolytic adenovirus activates tumor-infiltrating lymphocytes and causes tumor regression

ABSTRACT Cytokines have proven to be effective for cancer therapy, however whilst low-dose monotherapy with cytokines provides limited therapeutic benefit, high-dose treatment can lead to a number of adverse events. Interleukin 7 has shown promising results in clinical trials, but anti-cancer effect was limited, in part due to a low concentration of the cytokine within the tumor. We hypothesized that arming an oncolytic adenovirus with Interleukin 7, enabling high expression localized to the tumor microenvironment, would overcome systemic delivery issues and improve therapeutic efficacy. We evaluated the effects of Ad5/3-E2F-d24-hIL7 (TILT-517) on tumor growth, immune cell activation and cytokine profiles in the tumor microenvironment using three clinically relevant animal models and ex vivo tumor cultures. Our data showed that local treatment of tumor bearing animals with Ad5/3- E2F-d24-hIL7 significantly decreased cancer growth and increased frequency of tumor-infiltrating cells. Ad5/3-E2F-d24-hIL7 promoted notable upregulation of pro-inflammatory cytokines, and concomitant activation and migration of CD4+ and CD8 + T cells. Interleukin 7 expression within the tumor was positively correlated with increased number of cytotoxic CD4+ cells and IFNg-producing CD4+ and CD8+ cells. These findings offer an approach to overcome the current limitations of conventional IL7 therapy and could therefore be translated to the clinic.

Abstract 1235: Selective androgen receptor degraders for the treatment of androgen receptor-positive, triple-negative breast cancer

Abstract Introduction: Triple-negative breast cancer (TNBC) is an aggressive breast cancer with shorter overall survival compared to other breast cancer types. One of the six molecularly-classified TNBC subtypes is the luminal androgen receptor subtype (LAR), which overexpresses androgen receptor (AR) and is dependent on AR for its growth. About 10-20% of TNBCs belong to the LAR subtype. Competitive AR antagonists, enzalutamide and bicalutamide, were effective in preclinical models of LAR TNBC and in clinical trials. This led us to hypothesize that potent selective AR degraders (SARDs), due to their ability to inhibit and degrade the AR, could provide a novel therapeutic approach for the treatment of LAR subtype of TNBC. Description: Western blots, cell line proliferation assays, and gene expression analyses were performed to evaluate novel small molecule SARDs. LAR TNBC cell lines and patient-derived xenografts (PDX) were utilized for in vivo evaluation of the SARDs. Once tumors grew to ~100-300 mm3, mice were randomized and treated orally for four weeks with vehicle, SARD UT-34, SARD UT-105, enzalutamide, or bicalutamide. Tumor volumes were measured twice weekly and tumors were collected at sacrifice for further analyses. Summary: SARDs bind to the N-terminus of the AR and have been characterized in preclinical advanced prostate cancer models. In this study, the SARDs were evaluated in preclinical models of LAR TNBC. Western blot for AR in LAR MDA-MB-453 cells demonstrated degradation of the AR protein by SARDs at low micromolar concentrations. Gene expression studies showed a complete inhibition of androgen-induced AR target gene transcription by the SARDs. Androgen-induced proliferation of MDA-MB-453 cells was inhibited by SARDs. MDA-MB-453 cells implanted subcutaneously in NOD SCID Gamma female mice grew robustly to 100-300 mm3 in 15-20 days. Treatment of tumor-bearing animals with the SARDs completely inhibited or regressed the tumors. Conclusion: These results support the findings that AR is the driver of MDA-MB-453 cell and tumor growth. SARDs with their unique mechanism of action may provide a new therapeutic option to women affected by the LAR subtype of TNBC. Disclosure: This work was partially supported by Oncternal Therapeutics and by an NCI supplement award to R01 (CA229164S1 to author RN). The SARD program has been licensed to Oncternal Therapeutics, Inc. by the University of Tennessee Research Foundation. Author RN is a consultant to Oncternal Therapeutics. Citation Format: Sarah Asemota, Kirsten Young Young, Suriyan Ponnusamy, Thirumagal Thiyagarajan, Dong-Jin Hwang, Yali He, James B. Breitmeyer, Gunnar F. Kaufmann, Duane D. Miller, Ramesh Narayanan. Selective androgen receptor degraders for the treatment of androgen receptor-positive, triple-negative breast cancer [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 1235.

MyD88/CD40 Enhanced CD19-Specific CAR-T Cells Maintain Therapeutic Efficacy Following Resolution of Cytokine-Related Toxicity Using Inducible Caspase-9

Introduction: The efficacy of chimeric antigen receptor-modified T cells (CAR-T) targeting CD19+ leukemias and lymphomas is dependent on their in vivo expansion following adoptive transfer. Additional genetic augmentations to improve CAR-T expansion may improve therapeutic efficacy but risks increasing CAR-T toxicity. Here, we demonstrate that a highly active CD19-specific CAR-T cell constitutively expressing a MyD88/CD40 (MC) costimulatory fusion protein and the inducible caspase-9 (iC9) safety switch, is effective at eliminating tumors but induces acute cytokine-related toxicity in animal models. This toxicity, however, can be resolved by administration of rimiducid to induce partial CAR-T apoptosis preserving long-term anti-tumor effects.Methods: T cells were activated with anti-CD3/CD28 antibodies and transduced with a retrovirus encoding iC9, a first-generation CAR (with CD3ζ) targeting CD19, and a constitutively active MC costimulatory protein (SFG-iC9-CD19.ζ-MC). In vitro coculture assays were used to measure specific killing of CD19+ tumor cells (Daudi and Raji) and cytokine production. To assess anti-tumor efficacy, immunodeficient NSG mice were injected i.v. with 5x105 Raji-EGFPluciferase (EGFPluc) or 1x106 Daudi-EGFPluc tumor cells followed by i.v. administration of variable doses (1x106 to 1x107) non-transduced (NT) or iC9-CD19.ζ-MC-modified T cells on days 4 to 10 after tumor engraftment. Tumor growth was measured by in vivo imaging. CAR-T cell toxicity was assessed by weight loss and physical appearance. In initial experiments, CAR-T toxicity was resolved by i.p. administration of 0.5 mg/kg rimiducid after 15% weight loss. Serum cytokines were measured before and after rimiducid treatment. In addition, mice were treated with neutralizing antibodies to hIL-6, hIFN-γ and hTNF-α (100 ug i.p. twice weekly). Further, iC9-CD19.ζ-MC-modified T cells were sorted into CD4+ and CD8+ T cell fractions to assess the impact of cytokine production from CAR-T cells on animal health. Mice treated with iC9-CD19.ζ-MC-modified T cells that also received rimiducid to resolve toxicity were subsequently rechallenged with tumors to measure persistence of residual CAR-T cells, and at the end of the experiment were sacrificed and the spleens analyzed for the presence of human T cells and functional CAR-T activity using in vitro assays.Results: T cells were efficiently transduced (40-85%) with the iC9-CD19.ζ-MC retrovirus. In vitro assays showed that these CAR-T cells eliminated CD19+ Raji and Daudi tumor cells at low (1:20) effector-to-target (E:T) ratios in 7 day coculture assays, and produced high levels of IL-2 (669±324 pg/ml), IFN-γ (6715±2541 pg/ml), IL-6 (753±398 pg/ml) and TNF-α (2668±1867 pg/ml). NSG mice engrafted with CD19+ EGFPluc-expressing Raji or Daudi tumor cells were treated with variable doses of iC9-CD19.ζ-MC-modified T cells which were capable of eliminating tumor by day 21. However, acute toxicity (>15% body weight reduction) was observed in a CAR-T cell dose dependent manner occuring day 7 to 14 post-CAR-T injection. Systemic administration of 0.5 mg/kg rimiducid rapidly resolved toxicity and mice regained weight within 3-5 days. Serum cytokine concentrations of hIL-6 and hIFN-γ decreased to background levels within 24 hours post-rimiducid treatment. All mice treated with iC9-CD19.ζ-MC-modified T cells demonstrated long-term (>100 days) control of tumor growth and rejected CD19+ when rechallenged at day 150. Use of neutralizing antibodies against hIL-6, hIFN-γ and hTNF-α showed that blockade of hTNF-α resulted a significant reduction in weight loss. Further, iC9-CD19.ζ-MC-modified T cells were fractionated into CD4+ and CD8+ and measured for cytokine production, showing that CD4+ CAR-T cells produced higher levels of IL-2 (60-fold), IFN-γ (4-fold), IL-6 (37-fold) and TNF-α (10-fold) compared to CD8+ CAR-T cells. Furthermore, treatment of tumor-bearing animals with CD8+ selected iC9-CD19.ζ-MC-modified T cells reduced toxicity and the requirement of iC9 activation, while maintaining anti-tumor efficacy.Conclusions: Enhanced CAR-T cell activity can raise the risk of T cell toxicity. Constitutively active MC costimulation provides robust CAR-T cell proliferation and anti-tumor activity, but also increase cytokine-related toxicity. iC9 can be used to resolve toxicity without affecting long-term efficacy of CAR-T treatment in animals. DisclosuresFoster:Bellicum Pharmaceuticals: Employment, Other: stockholders . Lin:Bellicum Pharmaceuticals: Employment. Lu:Bellicum Pharmaceuticals: Employment. Chang:Bellicum Pharmaceuticals: Employment. Shinners:Bellicum Pharmaceuticals: Employment. Khalil:Bellicum Pharmaceuticals: Employment. Crisostomo:Bellicum Pharmaceuticals: Employment. Mahendravada:Bellicum Pharmaceuticals: Employment. Slawin:Bellicum Pharmaceuticals: Consultancy, Equity Ownership. Spencer:Bellicum Pharmaceuticals: Employment, Equity Ownership, Other: stockholders .

Exploiting KRAS-Driven Ferroaddiction in Cancer Through Ferrous Iron-Activatable Drug Conjugates (FeADC)

KRAS mutations cause a quarter of cancer mortality and currently are not sensitive to any targeted, FDA-approved agents. Several inhibitors of the MAPK pathway are FDA approved but exhibit low clinical tolerability at doses needed to adequately extinguish KRAS signaling. We discovered an avidity for ferrous iron (Fe2+) induced by and dependent on oncogenic KRAS signaling. We leveraged anFDA-approved MEK inhibitor to produce a prototypical Ferrous Iron–Activatable Drug Conjugate (FeADC) and with this novel agent achieved MAPK blockade in tumor cells while sparing normal tissues. These improvements allowed sustainable, effective treatment of tumor bearing animals with superior tolerability. Iron-activated therapeutics are unknown outside of antiparasitic therapy, but may hold significant promise for the treatment of KRAS-driven solid tumors.

Serum level of HMGB1 indicates outcome of the treatment of tumor bearing animals with antineoplastic drug NSC631570

BackgroundHMGB1 plays an important role in DNA architecture and transcriptional regulation in all nucleated cells. This protein is released by tumor cells during cellular necrosis and late apoptosis after the treatment with chemotherapeutic agents and acts as a danger associated molecular pattern (DAMP, endogenous alarmin) stimulating immune responses to pathogenic cell death products. Antineoplastic drug NSC631570 induces immunogenic tumor cell death in vitro that is accompanied by the release of HMGB1. This study was aimed at assessing the association of serum level of HMGB1 in tumor-bearing mice with the outcome of the treatment with NSC631570. MethodsEhrlich's carcinoma calls as well as two B16 melanoma sublines with different metastatic potentials were used. For in vivo experiments tumor cells were inoculated into Balb/c and C57BL/6 mice correspondingly. The anticancer effect of NSC631570 was characterized by the growth inhibition index. Cell cycle, apoptosis and necrosis were assessed by flow cytometry. The levels of HMGB1 in cell culture medium and in animal serum were determined by ELISA. ResultsTreatment of all mentioned tumor cells with NSC631570 at apoptogenic concentrations induced cell death coupled with dose-dependent HMGB1 release in vitro. The treatment of tumor-bearing mice with NSC631570 was also accompanied by the strong release of HMGB1 in the serum. Therapeutic efficacy of the drug was more pronounced in the case of B16, and was associated with the highest level of this alarmin in animal serum. ConclusionSerum level of HMGB1 can be considered as a biomarker of the outcome of the treatment with NSC631570.

A nanoparticle-based approach to improve the outcome of cancer active immunotherapy with lipopolysaccharides

This study sought to develop a simple nanoparticle-based approach to enhance the efficiency and tolerability of lipopolysaccharide (LPS), a potent ligand of Toll-like Receptor 4 (TLR4), for immunotherapy in cancer. Despite holding promise within this context, the strong pro-inflammatory properties of LPS also account for its low tolerability given localized and systemic side effects, which restrict the administrable dosage. Herein, we investigated the effect of LPS decoration as a surface-active molecule on a polymeric matrix upon its efficiency and tolerability. The LPS-decorated nanoparticles (LPS-NP) were about 150 nm in size, with slightly negative zeta potential (about −15 mV) and acceptable LPS incorporation (about 70%). In vitro, the particles accounted for a higher induction of apoptosis in tumor cells cultured with murine splenocytes compared to LPS solution. When used for the treatment of a murine syngeneic colorectal tumor model, higher intratumoral deposition of the particle-bound LPS was observed. Furthermore, unlike LPS solution, which accounted for localized necrosis at high concentrations, treatment of tumor-bearing animals with equivalent doses of LPS-NP was well tolerated. We propose that the observed localized necrosis can be Shwartzman phenomenon, which, due to modulated 24-h post-injection systemic TNF-α and LPS concentrations, have been avoided in case of LPS-NP. This has in turn enhanced the therapeutic efficiency and enabled complete tumor regression at concentrations at which LPS solution was intolerable. The findings indicate that nanoparticles can serve as beyond carriers for the delivery of superficially decorated LPS molecules, but impact their overall efficiency and tolerability in cancer therapy.

Abstract 3818: Therapeutic potential of blocking CCR5 by maraviroc in breast cancer bone metastasis

Abstract Introduction: Bone metastasis is a highly unfavorable condition observed in up to 70% of the patients with breast cancer. Treatment options for this condition are not curative, but palliative, only, which highlights the need for exploring new therapeutic options. In this study, we propose that targeting a chemokine receptor (CCR5), often upregulated in primary and secondary breast cancers, can contribute to a more successful treatment of breast cancer bone metastasis. Methodology: Following the blockage of CCR5 by maraviroc, cytotoxic effects were measured by MTT assay, while the migratory effects were determined by migration and scratch healing assays. Apoptosis related activities were investigated by nuclear staining and western blot analysis. Maraviroc mediated cytostatic changes were analyzed by a ready-made Human Cell Cycle Regulation Panel (Roche, Germany). In vivo experiments were performed by implanting MDA-MB-231 cells via the saphenous artery to the left hind limb of male nude rats (RNU strain) for inducing bone metastasis. Treatment with maraviroc was started from 2nd and 7th day of transplantation in two groups of rats (n = 6/group) designated as A and B, respectively, and was compared with an untreated control group (n = 8 rats). Results: CCR5 blockage by maraviroc (concentration > 100μM) induced concentration dependent cytotoxicity in MDA-MB-231 and MCF-7 cells. Maraviroc exposure also showed significant inhibition of migration of the cells, while nuclear staining indicated the condensation/fragmentation of nuclear content. Maraviroc exposure induced significant expression of cleaved caspase 7 and PARP, while real time RT-PCR showed pronounced inhibition of multiple genes (≥2fold) including cyclins, CDKs and their down-stream targets. Treatment of tumor bearing animals, with intra-peritoneal injections of maraviroc (25mg/kg, 3-4 weeks daily), reduced the tumor burden significantly (p < 0.05) in group A (50-75%), while the effects were minimal in group B (<25%). Concomitantly, no signs of toxicity were observed in rats of the two groups. Conclusion: CCR5 blockage by maraviroc induces significant anti-neoplastic effects in breast cancer cells. At mechanistic level, these effects included induction of apoptosis and alterations in cell cycle. Considerable inhibition in group A, but minimal inhibition of tumor burden in group B indicates that targeting CCR5 by maraviroc during early stages of breast cancer bone metastasis is a promising treatment option which should be further explored. Citation Format: Asim Pervaiz, Michael Zepp, Doaa M. Ali, Martin R. Berger, Hassan Adwan. Therapeutic potential of blocking CCR5 by maraviroc in breast cancer bone metastasis. [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 3818.

Novel Approach to the Potential Treatment of Patients with B-Cell Lymphomas Harboring the MYD88 L265P Mutation: Combination Treatment with TLR Antagonist and Rituximab

Introduction. Patients with B-cell lymphomas harboring the oncogenic MYD88 L265P mutation have poor response to standard of care regimens such as R-CHOP (Fernandez-Rodriguez et al, Leukemia, 2014, Jun 6). These patients also have not responded well to new therapies under development, including BTK inhibitors (Wilson et al, Blood 2012; 120). The MYD88 L265P oncogenic mutation has been shown to over-activate TLR7- and TLR9-mediated signaling pathways, including NF-κB, IRAK1/4 and STAT3, which in turn promote cell survival and proliferation (Lim et al., AACR 2013, Abstract #2332). Our approach to a potential treatment of B-cell lymphomas harboring the MYD88 L265P mutation is to block TLR7- and TLR9-mediated signaling through use of a TLR antagonist. IMO-8400 is an antagonist of TLR7, TLR8, and TLR9 that inhibits cell signaling and induces apoptosis specifically in B-cell lymphoma cell lines harboring the MYD88 L265P mutation (Bhagat et al, AACR 2014, Abstract #2570). IMO-8400 is currently in clinical development for the treatment of relapsed/refractory patients with Waldenström’s macroglobulinemia (WM) and diffuse large B-cell lymphoma (DLBCL).Methods. The current preclinical studies were designed to evaluate the combination of IMO-8400 with rituximab, which is a key component of R-CHOP. The studies were conducted in xenograft models of the activated B-cell like (ABC) DLBCL cell line OCI-Ly10 and the WM cell line MWCL-1, both positive for the MYD88 L265P mutation. For the ABC-DLBCL model, OCI-Ly10 cells were implanted s.c in NOD-SCID mice on day 0 and treatment was initiated on day 9 when mean tumor volume reached ~200 mm3. For the WM model, MWCL-1 cells were implanted s.c. in NOD-SCID mice on day 0 and treatment was initiated on day 6 when mean tumor volume reached ~250 mm3. In both models, the tumor-bearing mice were treated with PBS (control group), IMO-8400 (25 mg/kg, i.p.), rituximab (10 mg/kg, i.p.) or a combination of the two agents. IMO-8400 was administered twice per week for 3 weeks and continued further as maintenance treatment once per week. Rituximab was administered twice per week for three weeks in the ABC-DLBCL model whereas it was given only on days 3, 6, and 9 in the WM model.Results. Treatment of tumor-bearing animals with IMO-8400 or rituximab alone and in combination was well tolerated in both the models. In the ABC-DLBCL model, tumor growth inhibition compared to PBS control was 91.3% in the combination therapy group vs. 40.8% with IMO-8400 alone (p<0.001) and 51.6% with rituximab alone (p=0.003). Inhibition of tumor growth was associated with decreases in levels of several tumor-secreted circulating human cytokines including IL-10 (p<0.05 vs control) in all the three treatment regimens. In the WM model, tumor growth inhibition was 87% in combination group vs. 47% with IMO-8400 (p<0.001) and 72% with rituximab (p<0.001). Further, tumor growth inhibition in the WM model was associated with reduction in serum IgM levels (p<0.03 vs control) in the single-agent as well as the combination treatment groups. Importantly, combination therapy resulted in complete tumor regression in more than 80% of mice in both ABC-DLBCL and WM models. Histological analysis of the residual tumors showed disappearance of malignant lymphocytes with replacement by fibrotic stroma.Conclusions. Our data show that IMO-8400 in combination with rituximab was well tolerated in mouse xenograft models and exerted potent anti-tumor activity against B-cell lymphomas with the MYD88 L265P oncogenic mutation. These observations provide a strong rationale to evaluate IMO-8400 in combination with first-line rituximab-based therapies for the treatment of patients with B-cell lymphomas harboring the MYD88 L265P mutation. DisclosuresWang:Idera Pharmaceuticals: Employment. Jiang:Idera Pharmaceuticals: Employment. Sullivan:Idera Pharmaceuticals: Employment. Bhagat:Idera Pharmaceuticals: Employment.

Abstract P5-04-13: Antibody-based therapy targeting integrin a5 is an effective strategy to treat experimental breast cancer bone metastasis

Abstract Background: Integrin a5β1 is a specific fibronectin receptor that is often upregulated in breast cancer cells undergoing epithelial-to-mesenchymal transition. Preclinical evidence showed that α5β1-fibronectin interaction promotes in vitro the survival of growth-arrested breast cancer cells in the bone marrow. This is in line with the observation that disseminated tumor cells (DTCs) in the bone marrow, that are the earliest sign of development of metastatic disease in patients, express α5β1 integrin. However, the role of α5β1 in bone marrow micrometastasis formation remains unknown. Here, we investigated its role in experimental breast cancer bone metastasis, using an antibody-based strategy targeting the α5 subunit. Methods: Integrin α5 mRNA expression levels were quantified by qRT-PCR, using radically resected primary tumors of 427 breast cancer patients. Comparison between Kaplan-Meier curves was performed using the log-rank test. The Cox model was used for multivariate analysis. For experimental metastasis, female BALB/c immunodeficient mice were treated with a chimeric IgG4 monoclonal antibody that specifically binds to human integrin subunit α5 (M200;15mg/kg) or the vehicle. Treatment was performed 3 times per week starting the day before intra-arterial inoculation with luciferase-expressing human MDA-MB-231/B02 breast cancer cells, which selectively metastasize to bone. Vehicle and M200-treated mice were analyzed by radiography and bioluminescence. On day 28 after tumor cell inoculation, animals were sacrificed and histomorphometric analysis of metastatic legs was performed. Alternatively, animals were culled on day 7 after tumor cell inoculation, and the bone marrow was flushed for DTC colony assay. In-vitro cell-based assays were conducted to explore the functions of α5β1 on adhesion, invasion and survival. Statistical analysis was carried out by performing a Mann-Whitney U test. Results: Median durations of metastasis-free survival were 6.2 (high α5 expression) and 9.5 years (low α5 expression) (P = 0.0008). Additionally, compared with low expression, a high α5 expression was associated with shorter bone metastasis-free survival, both in univariate (P = 0.024) and multivariate analysis (HR = 1.65,95%CI = 1.02 to 2.67; P = 0.04).The treatment of tumor-bearing animals with M200 antibody statistically significantly delayed the onset of skeletal lesions (P = 0.02) and caused a 50% reduction in the extent of osteolytic lesions (P = 0.038), compared with vehicle. This difference was accompanied with a sharp reduction of tumor burden (P = 0.02), as determined by bioluminescence. Histomorphometric analysis of metastatic legs showed that M200 treatment decreased skeletal tumor burden (P = 0.027) and increased the bone volume (P = 0.02), compared with vehicle. Additionally, the number of DTC colonies in the bone marrow from M200-treated mice was dramatically decreased compared with vehicle (P&amp;lt;0.001). In vitro, M200 antibody did not affect tumor cell survival. By contrast, it specifically inhibited fibronectin-mediated tumor cell adhesion and invasion. Conclusion: Our results suggest that α5β1 integrin expression in breast cancer cells facilitates bone marrow micrometastasis formation and the subsequent development of osteolytic lesions. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P5-04-13.

Bioavailability and Efficacy of a Gap Junction Enhancer (PQ7) in a Mouse Mammary Tumor Model

The loss of gap junctional intercellular communication is characteristic of neoplastic cells, suggesting that the restoration with a gap junction enhancer may be a new therapeutic treatment option with less detrimental effects than traditional antineoplastic drugs. A gap junction enhancer, 6-methoxy-8-[(2-furanylmethyl) amino]-4-methyl-5-(3-trifluoromethylphenyloxy) quinoline (PQ7), on the normal tissue was evaluated in healthy C57BL/6J mice in a systemic drug distribution study. Immunoblot analysis of the vital organs indicates a reduction in Cx43 expression in PQ7-treated animals with no observable change in morphology. Next the transgenic strain FVB/N-Tg(MMTV-PyVT) 634Mul/J (also known as PyVT) was used as a spontaneous mammary tumor mouse model to determine the biological and histological effects of PQ7 on tumorigenesis and metastasis at three stages of development: Pre tumor, Early tumor, and Late tumor formation. PQ7 was assessed to have a low toxicity through intraperitoneal administration, with the majority of the compound being detected in the heart, liver, and lungs six hours post injection. The treatment of tumor bearing animals with PQ7 had a 98% reduction in tumor growth, while also decreasing the total tumor burden compared to control mice during the Pre stage of development. PQ7 treatment increased Cx43 expression in the neoplastic tissue during Pre-tumor formation; however, this effect was not observed in Late stage tumor formation. This study shows that the gap junction enhancer, PQ7, has low toxicity to normal tissue in healthy C57BL/6J mice, while having clinical efficacy in the treatment of spontaneous mammary tumors of PyVT mice. Additionally, gap junctional intercellular communication and neoplastic cellular growth are shown to be inversely related, while treatment with PQ7 inhibits tumor growth through targeting gap junction expression.

Targeted blockade of interleukin-8 abrogates its promotion of cervical cancer growth and metastasis

Surgery and radiotherapy have been used for decades to treat cervical cancer; however, high recurrence and lymph node metastasis rates are observed after these procedures. New therapeutic agents are needed to improve survival rates of patients by reducing tumor growth and metastasis. We previously demonstrated that interleukin-8 (IL-8) was associated with lymph node metastasis of early cervical squamous cell carcinoma (SCC). The current study assessed the role of IL-8 in growth and metastasis of cervical SCC and evaluated the effects of targeting IL-8 with small hairpin RNA (shRNA) and a human anti-IL-8 antibody. The human cervical SCC cell lines CaSki (high IL-8 producers), SiHa, HeLa, and SiHa transfected with the IL-8 gene were used for the studies. IL-8 stimulated proliferation, migration, and invasion but prevented apoptosis of SCC cells in vitro. Suppressing IL-8 expression with shRNA reduced cell growth and invasion of SCC cells in vitro. In a xenograft model, SCC cells were inoculated subcutaneously into athymic mice to evaluate the effect of IL-8 and its antibody on tumor growth and metastasis and animal survival. IL-8 enhanced tumor growth and metastasis in vivo concomitant with reduced animal survival. IL-8 antibody treatment of tumor-bearing animals resulted in smaller tumor volume, decreased lymph node metastasis, and longer animal survival. Blockade of IL-8 with an antibody demonstrated significant anti-tumor effects in a xenograft model and may thus provide a potential alternative approach for the treatment of cervical cancer.

Blocking IDO activity to enhance anti-tumor immunity

Tumors express potentially immunogenic antigens, yet the immune response to these antigens is typically profoundly suppressed. Patients with established tumors behave as if they were functionally tolerant to any antigens associated with the tumor. This tolerance reflects a process of active immune suppression elicited by the tumor, and represents a critical barrier to successful anti-tumor immunotherapy. Indoleamine 2,3-dioxygenase (IDO) is a natural immunoregulatory mechanism contributes to immune suppression and tolerance in a variety of settings. In tumor-bearing hosts, animal models suggest that tumor-induced IDO helps create a tolerogenic milieu within the tumor and the associated tumor-draining lymph nodes. IDO directly suppresses the proliferation and differentiation of effector T cells, and markedly enhances the suppressor activity of regulatory T cells (Tregs). Together, these effects contribute to the inability of the immune system to respond effectively to tumor antigens. Treatment of tumor-bearing animals with IDO-inhibitor drugs enhances anti-tumor immune responses, and IDO-inhibitors are synergistic with a variety of chemotherapeutic drugs, anti-tumor vaccines and other immunotherapy. Strategies to pharmacologically inhibit IDO may thus enhance immune-mediated responses following conventional chemotherapy, and may be synergistic with other forms of immunotherapy.

Blocking IDO activity to enhance anti-tumor immunity

Tumors express potentially immunogenic antigens, yet the immune response to these antigens is typically profoundly suppressed. Patients with established tumors behave as if they were functionally tolerant to any antigens associated with the tumor. This tolerance reflects a process of active immune suppression elicited by the tumor, and represents a critical barrier to successful anti-tumor immunotherapy. Indoleamine 2,3-dioxygenase (IDO) is a natural immunoregulatory mechanism contributes to immune suppression and tolerance in a variety of settings. In tumor-bearing hosts, animal models suggest that tumor-induced IDO helps create a tolerogenic milieu within the tumor and the associated tumor-draining lymph nodes. IDO directly suppresses the proliferation and differentiation of effector T cells, and markedly enhances the suppressor activity of regulatory T cells (Tregs). Together, these effects contribute to the inability of the immune system to respond effectively to tumor antigens. Treatment of tumor-bearing animals with IDO-inhibitor drugs enhances anti-tumor immune responses, and IDO-inhibitors are synergistic with a variety of chemotherapeutic drugs, anti-tumor vaccines and other immunotherapy. Strategies to pharmacologically inhibit IDO may thus enhance immune-mediated responses following conventional chemotherapy, and may be synergistic with other forms of immunotherapy.

Abstract A108: LY2606368, a second generation Chk1 inhibitor, inhibits growth of ovarian carcinoma xenografts either as monotherapy or in combination with standard-of-care agents.

Abstract Chk1 is a key component of the cellular response to DNA damage resulting from exposure to various genotoxic agents. When damaged DNA is present, Chk1 maintains genomic integrity by stalling cell cycle progression thereby providing time for the appropriate repair to occur. Inhibition of Chk1 kinase activity leads to a loss in the regulation of this damage response and results in abrogation of cell cycle arrest and ultimately to cell death as cells with damaged DNA proceed inappropriately into mitosis. Thus first generation small molecule inhibitors of Chk1 kinase were developed to act as companion compounds that would be used together with established standard -of-care (SoC) cancer treatments that target DNA to enhance the effectiveness of such treatments. As a second generation inhibitor, LY2606368 distinguishes itself from other molecules in this class in that it has been shown to inhibit tumor growth when used either as monotherapy or in combination with SoC agents. The single agent activity of LY2606368 is likely related to the ability of this molecule to potently inhibit Chk1 thereby effectively interfering with its function to support normal DNA replication via regulation of origin firing and/or aspects of chromosomal dynamics during mitosis which are thought to be dependent upon Chk1 kinase activity. The studies presented here have evaluated the potential of LY2606368 to inhibit ovarian tumor growth both as monotherapy and in combination with cisplatin or paclitaxel. Treatment of tumor-bearing animals on a BID X 3 schedule with LY2606368 alone resulted in profound growth inhibition such that greater than 80% inhibition was observed at doses of 12mg/kg in both subcutaneous and orthotopic models. Moreover, the studies in the orthotopic SKOV3 model demonstrated that treatment with LY2606368 not only robustly inhibited growth of the primary tumor but also significantly reduced the incidence of metastases and accumulation of ascites fluid. Subsequent studies in SKOV3 subcutaneous xenografts revealed that significant growth inhibition could be achieved with various combination strategies which include agents that are commonly used in the treatment of ovarian cancer. In particular, the extent of growth inhibition that could be achieved with cisplatin or paclitaxel alone was augmented by combining these agents with LY2606368. Overall these animal studies indicate the potential therapeutic benefit in ovarian cancer that could be achieved with LY2606368 either as monotherapy or in combination with SoC agents. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr A108.

Abstract B145: The novel Hedgehog pathway inhibitor IPI-926 delays metastatic spread and provides a survival benefit in an experimental model of pancreatic liver metastasis.

Abstract Metastasis, or the spread of disseminated tumor cells to and colonization of distal sites, is a major cause of cancer mortality. The Hedgehog (Hh) signaling pathway is normally involved in embryogenesis; however recent evidence suggests a role for this pathway in cancer and metastasis. Thus, inhibitors of Hh signaling may have potential utility as novel targeted anti-tumor therapies. IPI-926 is a novel, selective, small molecule that antagonizes the Hh pathway by binding to the Smoothened receptor, the major upstream facilitator of Hh signal transduction, and is currently in phase 2 clinical trials for pancreatic cancer, chondrosarcoma and primary myelofibrosis. Administration of IPI-926 in combination with gemcitabine inhibits incidence of metastases in a KRAS/p53 transgenic model of pancreatic cancer (Olive et al., 2009), supporting an integral role for the Hh pathway in this process. To further explore the activity of IPI-926 in the setting of metastasis, we developed an experimental model of pancreatic cancer liver metastasis where luciferase-tagged tumor cells were implanted in the liver via an intra-splenic injection, followed by splenectomy, resulting in liver metastases. Tumor burden, as assessed by bioluminescence and survival were employed to evaluate IPI-926 activity utilizing different dosing regimens. While treatment of tumor-bearing animals with established disease provided no survival benefit, IPI-926 administered on the day of cell implant provided modest but reproducible increase in survival. However, prophylactic administration of IPI-926 beginning 14 days (d) prior to cell implant resulted in a significant decrease in disease burden and enhanced survival. The activity was dependent on the schedule of IPI-926 administration. Treatment initiated 14d prior to cell implant was more effective than 7d before cell implant, and treatment started only 2d prior to cell implantation showed no difference compared to treatment initiated on the day of cell implant. Histological analysis of pre-treated vs control livers confirmed decreased metastatic nodule formation. In addition, analysis of serum harvested from IPI-926 14d pre-treated vs control animals showed down-regulation of components of the VEGF, PDGF and MMP pathways, suggesting that IPI-926 modulation of these pathways may be involved in the observed effects on metastatic spread. The activity of IPI-926 was also evaluated in the setting of sunitinib enhancement of metastases (Ebos et al., 2010) in the above model. As has been shown previously, short, high-dose sunitinib administration resulted in striking increases in disease burden and shortening of survival compared to control animals. However, when IPI-926 was administered 14d prior to cell implant and sunitinib treatment, disease burden and survival were returned to the levels observed in the control group. In summary, IPI-926 administration delays metastasis in an experimental model of pancreatic liver metastasis and in response to high-dose anti-angiogenic therapy. These results provide rationale for the evaluation of IPI-926 as adjuvant/neoadjuvant therapy to help control metastatic spread post surgical resection in pancreatic cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr B145.

Altered purinergic signaling in CD73‐deficient mice inhibits tumor progression

CD73/ecto-5'-nucleotidase dephosphorylates extracellular AMP into adenosine, and it is a key enzyme in the regulation of adenosinergic signaling. The contribution of host CD73 to tumor growth and anti-tumor immunity has not been studied. Here, we show that under physiological conditions CD73-deficient mice had significantly elevated ATPase and ADPase activities in LN T cells. In a melanoma model, the growth of primary tumors and formation of metastasis were significantly attenuated in mice lacking CD73. Among tumor-infiltrating leukocytes there were fewer Tregs and mannose receptor-positive macrophages, and increased IFN-γ and NOS2 mRNA production in CD73-deficient mice. Treatment of tumor-bearing animals with soluble apyrase, an enzyme hydrolyzing ATP and ADP, significantly inhibited tumor growth and accumulation of intratumoral Tregs and mannose receptor-positive macrophages in the WT C57BL/6 mice but not in the CD73-deficient mice. Pharmacological inhibition of CD73 with α,β-methylene-adenosine-5'-diphosphate in WT mice retarded tumor progression similarly to the genetic deletion of CD73. Together these data show that increased pericellular ATP degradation in the absence of CD73 activity in the host cells is a novel mechanism controlling anti-tumor immunity and tumor progression, and that the purinergic balance can be manipulated therapeutically to inhibit tumor growth.