Survival In Preclinical Models Research Articles

Valproic acid decreases resuscitation requirements after hemorrhage in a prolonged damage-control resuscitation model.

Hemorrhage is the leading cause of preventable death in trauma. Future military conflicts are likely to be in austere environments, where prolonged damage-control resuscitation (p-DCR) may be required for 72 hours before evacuation. There is a need to demonstrate that p-DCR is feasible and to optimize its logistics. Dried plasma (DP) is a practical alternative to conventional blood products in austere settings, and valproic acid (VPA) improves survival in preclinical models of trauma and hemorrhage. We performed the current experiment to study the synergistic effects of VPA and DP and hypothesized that VPA treatment would decrease the fluid resuscitation requirements in p-DCR. Female swine were subjected to 50% hemorrhage (associated with 20% survival using non-plasma-based p-DCR) and left unresuscitated for 1 hour to simulate medic response time. They were then randomized to receive VPA (150 mg/kg + DP 250 mL; DP-VPA group; n = 5) or DP alone (DP group; n = 6). All animals were resuscitated to a systolic blood pressure of 80 mm Hg with lactated Ringer according to the Tactical Combat Casualty Care Guidelines for 72 hours, after which packed red blood cells were transfused to simulate evacuation to higher levels of care. The DP-VPA group needed significantly (p = 0.002) less volume of lactated Ringer to reach and maintain the target systolic blood pressure. This would translate to a 4.3 L volume sparing effect for a 70-kg person. Addition of a single dose of VPA significantly decreases the volume of resuscitation required in a p-DCR model.

Abstract PR08: Inhibition of ATR as a therapeutic strategy to overcome cisplatin resistance in HNSCC

Abstract Background: Standard treatment for head and neck squamous cell carcinoma (HNSCC) includes platinum-based therapy given in combination with radiation. Although platinum has been a key drug of HNSCC treatment for decades, many patients develop resistance and the prognosis is unsatisfactory, and therefore development of novel treatment strategies is needed. Targeting ATR has been shown to be effective as a monotherapy in various type of cancers. Thus, inhibition of ATR could be a new approach when combined with DNA damaging agents to kill cancer cells. Here, we hypothesize that addition of ATR inhibitor AZD6738 can enhance the antitumor effects of cisplatin in HNSCC cells and result in better survival in preclinical models of HNSCC. Materials and Methods: Clonogenic survival assays, reverse phase protein array (RPPA), Western blotting, cell cycle analysis, and apoptosis assay were used to evaluate the in vitro sensitivity of HNSCC cells to AZD6738 and cisplatin and the molecular mechanisms associated with antitumor response. An orthotopic mouse model of oral cancer was used to explore in vivo efficacy of the combination treatment. Results: HNSCC cells treated with various concentrations of AZD6738 showed an IC50 value of 0.24-3.00 μmol/L and no differential sensitivity was observed among cells with different TP53 mutational status. Treatment with constant drug concentration ratios of AZD6738 and cisplatin (2:1) resulted in a combination index of <1, indicating synergistic effects. RPPA pointed out proteins involved in DNA repair, DNA replication, and cell cycle to be predominantly changed in the combination groups compared to single treatments. Among these, the replication factors Cdc6 and Cdt1 were both reduced with the combination treatment. Furthermore, Western blot results revealed that combination treatments lead to enhanced DNA damage indicated by increased phosphorylation of H2AX and induction of replication stress marker (decrease of phospho-CDK1 and phospho-CHK1). Moreover, levels of RRM2 and Rad51 were diminished, suggesting insufficient nucleotide supply and impaired homologous replication. Combining both drugs also resulted in a time-dependent increase of cleaved PARP. This apoptotic death was further confirmed by Annexin V/PI assay. Cell cycle analysis and live cell imaging revealed that an accumulation of cells in the S phase was more than doubled after 12 and 48 hours, indicating replicative stress. In a pilot in vivo study, cisplatin combined with AZD6738 reduced tumor growth in mice bearing oral tumors. Conclusions: Addition of AZD6738 synergizes with cisplatin in vitro and potentiates cisplatin response in vivo. Mechanistically, combination of cisplatin and AZD6738 induces DNA damage and replication stress, resulting in apoptotic cell death. Interestingly, proteins essential for the origin licensing as part of the DNA replication initiation machinery were decreased following combination treatment, offering a potential mechanistic insight and warranting further investigation. Citation Format: Antje Lindemann, Hideaki Takahashi, Ameeta A. Patel, Walter N. Hittelman, Abdullah A. Osman, Jeffrey N. Myers. Inhibition of ATR as a therapeutic strategy to overcome cisplatin resistance in HNSCC [abstract]. In: Proceedings of the AACR-AHNS Head and Neck Cancer Conference: Optimizing Survival and Quality of Life through Basic, Clinical, and Translational Research; 2019 Apr 29-30; Austin, TX. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(12_Suppl_2):Abstract nr PR08.

Actinomycin D downregulates Sox2 and improves survival in preclinical models of recurrent glioblastoma.

BackgroundGlioblastoma (GBM) has been extensively researched over the last few decades, yet despite aggressive multimodal treatment, recurrence is inevitable and second-line treatment options are limited. Here, we demonstrate how high-throughput screening (HTS) in multicellular spheroids can generate physiologically relevant patient chemosensitivity data using patient-derived cells in a rapid and cost-effective manner. Our HTS system identified actinomycin D (ACTD) to be highly cytotoxic over a panel of 12 patient-derived glioma stemlike cell (GSC) lines. ACTD is an antineoplastic antibiotic used in the treatment of childhood cancers. Here, we validate ACTD as a potential repurposed therapeutic for GBM in 3-dimensional GSC cultures and patient-derived xenograft models of recurrent glioblastoma.MethodsTwelve patient-derived GSC lines were screened at 10 µM, as multicellular spheroids, in a 384-well serum-free assay with 133 FDA-approved compounds. GSCs were then treated in vitro with ACTD at established half-maximal inhibitory concentrations (IC50). Downregulation of sex determining region Y–box 2 (Sox2), a stem cell transcription factor, was investigated via western blot and through immunohistological assessment of murine brain tissue.ResultsTreatment with ACTD was shown to significantly reduce tumor growth in 2 recurrent GBM patient-derived models and significantly increased survival. ACTD is also shown to specifically downregulate the expression of Sox2 both in vitro and in vivo.ConclusionThese findings indicate that, as predicted by our HTS, ACTD could deplete the cancer stem cell population within the tumor mass, ultimately leading to a delay in tumor progression.Key Points1. High-throughput chemosensitivity data demonstrated the broad efficacy of actinomycin D, which was validated in 3 preclinical models of glioblastoma.2. Actinomycin D downregulated Sox2 in vitro and in vivo, indicating that this agent could target the stem cell population of GBM tumors.

CD11b is a novel alternate receptor for CD154 during alloimmunity.

Antagonism of the CD154/CD40 pathway is a highly effective means of inducing long-term graft survival in preclinical models. Using a fully allogeneic murine transplant model, we found that CD154 blockade was more effective in prolonging graft survival than was CD40 blockade, raising the possibility that CD154 binds a second receptor. To test this, we queried the impact of CD154 antagonism in the absence of CD40. Data indicated that anti-CD154 functioned to reduce graft-infiltrating CD8+ T cells in both WT and CD40-/- hosts. Because it has recently been reported that CD154 can ligate CD11b, we addressed the impact of blocking CD154-CD11b interactions during transplantation. We utilized a specific peptide antagonist that prevents CD154 binding of CD11b but has no effect on CD154-CD40 interactions. CD154:CD11b antagonism significantly increased the efficacy of anti-CD40 in prolonging allograft survival as compared to anti-CD40 plus control peptide. Mechanistically, CD154:CD11b antagonism functioned to reduce the frequency of graft-infiltrating CD8+ T cells and innate immune cells. These data therefore demonstrate that blocking CD154 interactions with both CD40 and CD11b is required for optimal inhibition of alloimmunity and provide an explanation for why CD40 blockers may be less efficacious than anti-CD154 reagents for the inhibition of allograft rejection.

ΔNp73/ETS2 complex drives glioblastoma pathogenesis— targeting downstream mediators by rebastinib prolongs survival in preclinical models of glioblastoma

Glioblastoma (GBM) remains one of the least successfully treated cancers. It is essential to understand the basic biology of this lethal disease and investigate novel pharmacological targets to treat GBM. The aims of this study were to determine the biological consequences of elevated expression of ΔNp73, an N-terminal truncated isoform of TP73, and to evaluate targeting of its downstream mediators, the angiopoietin 1 (ANGPT1)/tunica interna endothelial cell kinase 2 (Tie2) axis, by using a highly potent, orally available small-molecule inhibitor (rebastinib) in GBM. ΔNp73 expression was assessed in glioma sphere cultures, xenograft glioblastoma tumors, and glioblastoma patients by western blot, quantitative reverse transcription PCR, and immunohistochemistry. Immunoprecipitation, chromatin immunoprecipitation (ChiP) and sequential ChIP were performed to determine the interaction between ΔNp73 and E26 transformation-specific (ETS) proto-oncogene 2 (ETS2) proteins. The oncogenic consequences of ΔNp73 expression in glioblastomas were examined by in vitro and in vivo experiments, including orthotopic zebrafish and mouse intracranial-injection models. Effects of rebastinib on growth of established tumors and survival were examined in an intracranial-injection mouse model. ΔNp73 upregulates both ANGPT1 and Tie2 transcriptionally through ETS conserved binding sites on the promoters by interacting with ETS2. Elevated expression of ΔNp73 promotes tumor progression by mediating angiogenesis and survival. Therapeutic targeting of downstream ΔNp73 signaling pathways by rebastinib inhibits growth of established tumors and extends survival in preclinical models of glioblastoma. Aberrant expression of ΔNp73 in GBM promotes tumor progression through autocrine and paracrine signaling dependent on Tie2 activation by ANGPT1. Disruption of this signaling by rebastinib improves tumor response to treatment in glioblastoma.

IMGN632, a CD123-Alkylating ADC Bearing a DNA-Alkylating IGN Payload, Combines Effectively with Azacitidine and Venetoclax In Vivo, Prolonging Survival in Preclinical Models of Human Acute Myeloid Leukemia (AML)

Introduction: IMGN632, a novel CD123-targeting ADC, demonstrates a favorable safety profile and complete remissions as a monotherapy in patients with relapsed/refractory AML and BPDCN (NCT03386513). We have previously reported the synergy of combining IMGN632 with venetoclax (BCL-2 inhibitor) (EHA, 2019, abstract #PF201). Given the recent approval of azacitidine (AZA, a hypomethylating agent) and venetoclax (VEN) in AML patients unfit for intensive chemotherapy, we investigated the combination of IMGN632 with AZA and the triple combination of IMGN632, AZA and VEN. Methods: The IMGN632 and AZA +/- VEN combinations were evaluated in the following AML models: EOL-1, subcutaneous (SC); Molm-13, disseminated; MV4-11, disseminated; and one patient derived AML xenograft (PDX) model. In SC models, tumor volume was measured serially, complete regressions (CRs) were noted, and endpoint was determined by either clinical observations or when tumor volume reached 1000 mm3. Tumor growth inhibition (T/C) was calculated as (median tumor volume of the treated / median tumor volume of the control) x 100%. In disseminated models, the percent Increased Life Span (%ILS) was calculated as [(T-C, tumor growth delay) / C] x 100%, where T is the median survival time (days) of a treated group and C is the median survival time (days) of the vehicle control group. In AML PDX models, tumor burden was monitored by hCD45/hCD123 flow cytometry of serial peripheral blood (PB) samples. Results: In EOL-1, treatment with AZA (3 mg/kg, daily x5) was inactive, producing only a 4% reduction in T/C and 0/6 CRs treatment. IMGN632, at a sub-optimal single dose of 0.08 mg/kg resulted in a 36% reduction in T/C and 2/6 long-term CRs at study end. The combination of IMGN632 and AZA generated a robust 100% reduction in T/C and 4/6 CRs, while addition of VEN (100 mg/kg, daily x28) to AZA was not as active with a 54% reduction in T/C and 1/6 CRs. The triple combination with the addition of IMGN632 to the VEN + AZA doublet was the most active, with a 100% reduction in T/C and 5/6 CRs. Similarly, in Molm-13, AZA (3.5 mg/kg, daily x5) was minimally active, with in a 6-day T-C and 30% ILS. IMGN632 (0.024 mg/kg, weekly x3) was active and generated a 9.5-day T-C and a 47.5% ILS. Strikingly, the combination of IMGN632+AZA was highly active, resulting in a 61-day T-C and a 303% ILS (Figure 1A), demonstrating a clear survival benefit over single agent treatments. In a separate Molm-13 study, the combination of AZA+ VEN produced a 14.5-day T-C and a 67.4% ILS (highly active), while the triple combination including IMGN632 was clearly the most active combination, with a >116-day T-C and a >537% ILS (highly active) 50% of mice surviving beyond 4.5 months (Figure 1B). Similarly, in the MV4-11 model, the two AZA (either 4.5 mg/kg or 3 mg/kg, daily x5) + VEN (100 mg/kg, daily x28) combinations resulted in a 28-day T-C (62.2% ILS) and a 20-day T-C (44.4% ILS), respectively. The addition of IMGN632 (0.08 mg/kg) to these resulted in a superior regimen with a 64-day T-C (142.2% ILS) and a 76-day T-C (168.9% ILS), respectively. The PDX AML model, 4023126, responded very well to treatment with the triple combination (AZA (2.5mg/kg, daily x 5), VEN (100 mg/kg, 5 days/week x3 weeks) and sub-optimal IMGN632 (0.12 mg/kg once a week x3 weeks), with circulating leukemia burden lower than 1% after two weeks of treatment, compared to greater than 50% in the vehicle-treated group (Figure 1C). In model 4023126, IMGN632 was active and resulted in a 33-day T-C and 57% ILS, and AZA+VEN resulted in a 52-day T-C and 90% ILS. The triple combination group survived the longest and was highly active resulting in a 74-day T-C and 128% ILS demonstrating highest survival benefit (Figure 1D). Conclusions: The addition of IMGN632 to AZA alone and to AZA+VEN in multiple AML xenograft and PDX models leads to improved survival. These data support the addition of a CD123-targeted ADC with a novel DNA damaging payload to standard of care AZA and AZA+VEN in AML patients Disclosures Kuruvilla: The University of Texas M.D.Anderson Cancer Center: Employment. McCarthy:ImmunoGen: Employment. Zhang:The University of Texas M.D.Anderson Cancer Center: Employment. Sloss:ImmunoGen: Employment. Zweidler-McKay:ImmunoGen: Employment. Romanelli:ImmunoGen: Employment. Konopleva:Calithera: Research Funding; Stemline Therapeutics: Consultancy, Honoraria, Research Funding; Forty-Seven: Consultancy, Honoraria; Eli Lilly: Research Funding; AbbVie: Consultancy, Honoraria, Research Funding; Cellectis: Research Funding; Amgen: Consultancy, Honoraria; F. Hoffman La-Roche: Consultancy, Honoraria, Research Funding; Genentech: Honoraria, Research Funding; Ascentage: Research Funding; Kisoji: Consultancy, Honoraria; Reata Pharmaceuticals: Equity Ownership, Patents & Royalties; Ablynx: Research Funding; Astra Zeneca: Research Funding; Agios: Research Funding.

A novel therapeutic approach for anaplastic thyroid cancer through inhibition of LAT1

A novel therapeutic approach is urgently needed for patients with anaplastic thyroid cancer (ATC) due to its fatal and rapid progress. We recently reported that ATC highly expressed MYC protein and blocking of MYC through its selective inhibitor, JQ1, decreased ATC growth and improved survival in preclinical models. One of the important roles of MYC is regulation of L-neutral amino acid transporter 1 (LAT1) protein and inhibition of LAT1 would provide similar anti-tumor effect. We first identified that while the human ATC expresses LAT1 protein, it is little or not detected in non-cancerous thyroidal tissue, further supporting LAT1 as a good target. Then we evaluated the efficacy of JPH203, a LAT1 inhibitor, against ATC by using the in vitro cell-based studies and in vivo xenograft model bearing human ATC cells. JPH203 markedly inhibited proliferation of three ATC cell lines through suppression of mTOR signals and blocked cell cycle progression from the G0/G1 phase to the S phase. The tumor growth inhibition and decrease in size by JPH203 via inhibition of mTOR signaling and G0/G1 cell cycle associated proteins were further confirmed in xenograft models. These preclinical findings suggest that LAT1 inhibitors are strong candidates to control ATC, for which current treatment options are highly limited.

P12.03 Bi-specific T cell engagers targeting IL13Rá2 activate tumor-infiltrating lymphocytes and improve survival in pre-clinical models of glioblastoma

Abstract BACKGROUND The outstanding efficacy of bi-specific T-cell engagers (BiTE against hematological malignancies offers hope that they can similarly target solid tumors like GBM. In this study, we have designed a BiTE protein with specificity to the tumor-associated antigen, IL13Rα2, and investigated how BiTE protein engages a host’s T cell immune response to promote anti-glioma activity in pre-clinical models of GBM. MATERIAL AND METHODS BiTE molecule consisting of two single chain variable regions (scFv) of antibodies against either murine or human CD3ε and scFv47 against human IL13Rα2 connected through a flexible linker (BiTEIL13Rα2) were sub-cloned in a lentiviral expression cassette. The BiTE molecule (BiTEIL13Rα2off) modified to abrogate the interaction of scFv47 with IL13Rα2 served as a negative control. The BiTE proteins were isolated from the supernatants of HEK293T cells using His-Tag affinity chromatography and validated in SDS-PAGE, Western Blot, and ELISA assays. The BiTEIL13Rα2-induced T cells activation was measured in (i) cytotoxicity assay against IL13Rα2+ glioma cells, (ii) flow cytometry measuring for CD69 and CD25 T cells’ activation markers, and (iii) the production of cytokines, IFNγ and TNFα. For in vivo analysis, VmDk and C57Bl/6 mice bearing established intracranial glioma were treated systemically with BiTE proteins. The survival of the mice was recorded and analyzed using the log-rank test. RESULTS Here we show that BiTEIL13Rα2 specifically binds to IL13Rα2 but not to IL13Rα1, whereas BiTEIL13Rα2off has no binding activity to both IL13 receptors. The co-culture of naïve murine or donor’s human CD3+ T cells with IL13Rα2+ glioma cells in the presence of BiTEIL13Rα2 but not with BiTEIL13Rα2off (i) activates CD3+CD8+ T cells as judged by upregulation of CD69, CD25, and production of IFNγ and TNFα and (ii) results in concentration- and antigen-dependent cytotoxicity in glioma cells. Furthermore, a direct comparison of CD3+ T cells obtained from the peripheral blood and tumor tissue of GBM patients revealed that BiTEIL13Rα2 induces a potent cytotoxic activity of CD3+ T cells against IL13Rα2+ glioma cells. Finally, treatment of immunocompetent mice bearing IL13Rα2+ murine glioma with BiTEIL13Rα2 resulted in a higher frequency of intratumoral CD8+ T cells, and significant (p<0.05) improvement of survival over a negative control, BiTEIL13Rα2off group of mice. CONCLUSION Our data demonstrate that BiTEIL13Rα2 protein activates CD3+ T cells in an antigen-specific fashion. Furthermore, systemic treatment with BiTEIL13Rα2 protein confers a significant survival benefit in pre-clinical syngeneic glioma models, warranting investigations in other IL13Rα2-expressing cancers and translation to clinical settings.

Mitochondrial fusion exploits a therapeutic vulnerability of pancreatic cancer.

Pancreatic ductal adenocarcinoma (PDAC) requires mitochondrial oxidative phosphorylation (OXPHOS) to fuel its growth, however, broadly inhibiting this pathway might also disrupt essential mitochondrial functions in normal tissues. PDAC cells exhibit abnormally fragmented mitochondria that are essential to its oncogenicity, but it was unclear if this mitochondrial feature was a valid therapeutic target. Here, we present evidence that normalizing the fragmented mitochondria of pancreatic cancer via the process of mitochondrial fusion reduces OXPHOS, which correlates with suppressed tumor growth and improved survival in preclinical models. Mitochondrial fusion was achieved by genetic or pharmacologic inhibition of dynamin related protein-1 (Drp1) or through overexpression of mitofusin-2 (Mfn2). Notably, we found that oral leflunomide, an FDA-approved arthritis drug, promoted a two-fold increase in Mfn2 expression in tumors and was repurposed as a chemotherapeutic agent, improving the median survival of mice with spontaneous tumors by 50% compared to vehicle. We found that the chief tumor suppressive mechanism of mitochondrial fusion was enhanced mitophagy, which proportionally reduced mitochondrial mass and ATP production. These data suggest that mitochondrial fusion is a specific and druggable regulator of pancreatic cancer growth that could be rapidly translated to the clinic.

Sequential Anti-PD1 Therapy Following Dendritic Cell Vaccination Improves Survival in a HER2 Mammary Carcinoma Model and Identifies a Critical Role for CD4 T Cells in Mediating the Response.

Patients with metastatic HER2 breast cancer (MBC) often become resistant to HER 2 targeted therapy and have recurrence of disease. The Panacea trial suggested that HER2 MBC patients were more likely to respond to checkpoint therapy if TIL were present or if tumor expressed PD-L1. We assessed whether type I polarized dendritic cells (DC1) could improve checkpoint therapy in a preclinical model of HER2+ breast cancer. TUBO bearing mice were vaccinated with either MHC class I or class II HER2 peptide pulsed DC1 (class I or class II HER2-DC1) concurrently or sequentially with administration of anti-PD-1 or anti-PDL1. Infiltration of tumors by immune cells, induction of anti-HER2 immunity and response to therapy was evaluated. Class I or class II HER2-DC1 vaccinated mice generated anti-HER2 CD8 or CD4+ T cell immune responses and demonstrated delayed tumor growth. Combining both MHC class I and II HER2-pulsed DC1 did not further result in inhibition of tumor growth or enhanced survival compared to individual administration. Interestingly class II HER2-DC1 led to both increased CD4 and CD8 T cells in the tumor microenvironment while class I peptides typically resulted in only increased CD8 T cells. Anti-PD-1 but not anti-PD-L1 administered sequentially with class I or class II HER2-DC1 vaccine could improve the efficacy of HER2-DC1 vaccine as measured by tumor growth, survival, infiltration of tumors by T cells and increase in systemic anti-HER2 immune responses. Depletion of CD4+ T cells abrogated the anti-tumor efficacy of combination therapy with class II HER2-DC1 and anti-PD-1, suggesting that tumor regression was CD4 dependent. Since class II HER2-DC1 was as effective as class I, we combined class II HER2-DC1 vaccine with anti-rat neu antibodies and anti-PD-1 therapy. Combination therapy demonstrated further delay in tumor growth, and enhanced survival compared to control mice. In summary, Class II HER2-DC1 drives both a CD4 and CD8 T cell tumor infiltration that leads to increased survival, and in combination with anti-HER2 therapy and checkpoint blockade can improve survival in preclinical models of HER2 positive breast cancer and warrants exploration in patients with HER2 MBC.

Abstract 2006: Targeting AXL kinase with TP-0903 successfully reverses the mesenchymal phenotype and extends survival in preclinical models of advanced ovarian cancer

Abstract Epithelial-to-mesenchymal transition (EMT) is a critical step in the progression of ovarian cancer (OVCA), which drives local invasion, metastasis and drug resistance leading to poor survival. AXL, a receptor tyrosine kinase, functions as a molecular driver of EMT in multiple cancers, including OVCA. The high mortality of this tumor is explained by the fact that the majority (75%) of patients present at an advanced stage, with widely metastatic disease within the peritoneal cavity often associated with ascites. The extent of ascites is a prognostic factor for survival, impacts quality of life, and correlates with the EMT phenotype. Approaches to block or reverse EMT are needed to improve the prognosis of OVCA patients, particularly those with refractory or early relapse (<6 months) disease following debulking surgery and conventional adjuvant treatments. TP-0903 is a potent, small molecule AXL inhibitor that has activity in reversing EMT in multiple models of disease and embryonic development. The objective of this study was to explore the anti-tumor activity in preclinical mesenchymal OVCA-derived models, which may provide rationale to evaluate TP-0903 in treatment-resistant or refractory OVCA patients. TP-0903 showed potent effects on cell viability in multiple OVCA-derived cell lines. The IC50 values in cell viability studies ranged from 33 nM (OVTOKO cells) to 840 nM (OVMANA cells). Kuramochi cells represent the most common ovarian cancer subtype, high-grade serous ovarian cancer and was particularly platinum-resistant (>5 μM), yet sensitive to TP-0903 (210 nM). Baseline phospho-AXL (Y702) was exceptionally high in Kuramochi cells and TP-0903 inhibited this phosphorylation at concentrations as low as 100 nM. TP-0903 reversed EMT in OVCA cells as evident by the down-regulation of mesenchymal markers including Snail and Slug. Consistent with the reversal of the EMT, TP-0903 inhibited the migration of ES-2 OVCA cells in a scratch assay. TP-0903 suppressed ascites development in preclinical models of OVCA and inhibited metastatic intraperitoneal tumor dissemination. Treatment suppressed ascites development (ascites volume, vehicle: 3.34 mL, TP-0903: 0.28 mL; abdominal circumference, vehicle: 7.96 cm (day 15), TP-0903: 6.82 cm (day 15), p<0.05) in an orthotopic ES-2 model and extended overall survival compared to vehicle or cisplatin alone. Phospho-AXL modulation and changes in other mechanism-validating biomarkers were observed in the ES-2 model. Similar preclinical activities, including decreased ascites volume, were observed in A2780cis and OVCAR-3 xenograft models. These results support further clinical evaluation of TP-0903 in either single-agent or combination regimens treating recurrent or refractory OVCA. TP-0903 is investigated in a Phase I trial that includes an expansion cohort of this patient population (NCT02729298). Citation Format: Nozomi Tomimatsu, Ken Fujimura, Yuta Matsumura, Hiroki Umehara, Lars Mouritsen, Steven L. Warner, David J. Bearss. Targeting AXL kinase with TP-0903 successfully reverses the mesenchymal phenotype and extends survival in preclinical models of advanced ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2006.

Abstract 3053: Development of novel cell-based bioassays for the development of biologics targeting LAG-3 and PD-1xLAG-3

Abstract Programmed cell death protein 1 (PD-1) and Lymphocyte-activation gene 3 (LAG-3) are immune checkpoint receptors that bind to PD-L1/PD-L2 or MHCII, respectively, on antigen presenting cells or tumor cells. PD-1 or LAG-3 activation results in the inhibition of T cell receptor (TCR) signaling and reduced T cell activation. However, co-blockade of PD-1 and LAG-3 leads to a synergistic enhancement in T cell responses, suggesting functionally divergent inhibitory signaling mechanisms between the two receptors. Preclinical studies suggest that blocking LAG-3 re-activates the immune system to kill cancer cells, and co-blockade of both PD-1 and LAG-3 lead to a synergistic increase in tumor control and survival in preclinical models. Current methods for measuring the potency of LAG-3 and PD-1xLAG-3 blocking antibodies measure cytokine production from human PBMCs upon Staphylococcal Enterotoxin B (SEB) superantigen stimulation. These protocols are tedious, highly variable, and require the use of highly toxic superantigen. Herein, we describe the development of reporter-based bioassays for the development of LAG-3 and PD-1xLAG-3 combination-targeted biologics. Engineered T effector cell lines expressing LAG-3 alone or in combination with PD-1 are activated in an antigen-dependent manner by APC cell lines. The assays are specific for LAG-3 or PD-1xLAG-3-targeted biologics and are robust as demonstrated by Design of Experiments analyses. In addition, the assays can be used to measure the relative potencies of test samples compared with reference sample in a detection range of 50% to 200% and show appropriate precision, accuracy and linearity. The assays can detect potency changes for heat-stressed samples, and are tolerant to human serum, demonstrating the potential to be further developed into neutralizing antibody (NAb) assays. Therefore, these bioassays can serve as valuable tools for the development of biologics targeting LAG-3 and PD-1xLAG-3. Citation Format: Jamison Grailer, Denise Garvin, Jim Hartnett, Frank Fan, Mei Cong, Zhi-jie Jey Cheng. Development of novel cell-based bioassays for the development of biologics targeting LAG-3 and PD-1xLAG-3 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3053.

Transcriptomic analysis reveals that BMP4 sensitizes glioblastoma tumor-initiating cells to mechanical cues

The poor prognosis of glioblastoma (GBM) is associated with a highly invasive stem-like subpopulation of tumor-initiating cells (TICs), which drive recurrence and contribute to intra-tumoral heterogeneity through differentiation. These TICs are better able to escape extracellular matrix-imposed mechanical restrictions on invasion than their more differentiated progeny, and sensitization of TICs to extracellular matrix mechanics extends survival in preclinical models of GBM. However, little is known about the molecular basis of the relationship between TIC differentiation and mechanotransduction. Here we explore this relationship through a combination of transcriptomic analysis and studies with defined-stiffness matrices. We show that TIC differentiation induced by bone morphogenetic protein 4 (BMP4) suppresses expression of proteins relevant to extracellular matrix signaling and sensitizes TIC spreading to matrix stiffness. Moreover, our findings point towards a previously unappreciated connection between BMP4-induced differentiation, mechanotransduction, and metabolism. Notably, stiffness and differentiation modulate oxygen consumption, and inhibition of oxidative phosphorylation influences cell spreading in a stiffness- and differentiation-dependent manner. Our work integrates bioinformatic analysis with targeted molecular measurements and perturbations to yield new insight into how morphogen-induced differentiation influences how GBM TICs process mechanical inputs.

Ex vivo Hsp70-Activated NK Cells in Combination With PD-1 Inhibition Significantly Increase Overall Survival in Preclinical Models of Glioblastoma and Lung Cancer.

Heat shock protein 70 (Hsp70) which is expressed on the plasma membrane of highly aggressive tumors including non-small cell lung carcinoma and glioblastoma multiforme serves as a target for Hsp70-targeting NK cells. Herein, we aimed to investigate the antitumor effects of a combined therapy consisting of ex vivo Hsp70-peptide TKD/IL-2-activated NK cells in combination with mouse/human anti-PD-1 antibody in a syngeneic glioblastoma and a xenograft lung cancer mouse model. Mice with membrane Hsp70 positive syngeneic GL261 glioblastoma or human xenograft A549 lung tumors were sham-treated with PBS or injected with ex vivo TKD/IL-2-activated mouse/human NK cells and mouse/human PD-1 antibody either as a single regimen or in combination. Tumor volume was assessed by MR scanning and tumor-infiltrating CD8+ T, NK, and PD-1+ cells were quantified by immunohistochemistry (IHC). We could show that the adoptive transfer of ex vivo TKD/IL-2-activated mouse NK cells or the inhibition of PD-1 resulted in tumor growth delay and an improved overall survival (OS) in a syngeneic glioblastoma mouse model. A combination of both therapies was well-tolerated and significantly more effective with respect to both outcome parameters than either of the single regimens. A combined treatment in a xenograft lung cancer model showed identical effects in immunodeficient mice bearing human lung cancer after adoptive transfer of TKD/IL-2-activated human effector cells and a human PD-1 antibody. Tumor control was associated with a massive infiltration with CD8+ T and NK cells in both tumor models and a decreased in PD-1 expression on immune effector cells. In summary, a combined approach consisting of activated NK cells and anti-PD-1 therapy is safe and results in a long-term tumor control which is accompanied by a massive tumor immune cell infiltration in 2 preclinical tumor models.

Glucocorticoids promote breast cancer metastasis.

Diversity within or between tumours and metastases (known as intra-patient tumour heterogeneity) that develops during disease progression is a serious hurdle for therapy1-3. Metastasis is the fatal hallmark of cancer and the mechanisms of colonization, the most complex step in the metastatic cascade4, remain poorly defined. A clearer understanding of the cellular and molecular processes that underlie both intra-patient tumour heterogeneity and metastasis is crucial for the success of personalized cancer therapy. Here, using transcriptional profiling of tumours and matched metastases in patient-derived xenograft models in mice, we show cancer-site-specific phenotypes and increased glucocorticoid receptor activity in distant metastases. The glucocorticoid receptor mediates the effects of stress hormones, and of synthetic derivatives of these hormones that are used widely in the clinic as anti-inflammatory and immunosuppressive agents. We show that the increase in stress hormones during breast cancer progression results in the activation of the glucocorticoid receptor at distant metastatic sites, increased colonization and reduced survival. Our transcriptomics, proteomics and phospho-proteomics studies implicate the glucocorticoid receptor in the activation of multiple processes in metastasis and in the increased expression of kinase ROR1, both of which correlate with reduced survival. The ablation of ROR1 reduced metastatic outgrowth and prolonged survival in preclinical models. Our results indicate that the activation of the glucocorticoid receptor increases heterogeneity and metastasis, which suggests that caution is needed when using glucocorticoids to treat patients with breast cancer who have developed cancer-related complications.

Targeting mitochondrial dysfunction in amyotrophic lateral sclerosis: a systematic review and meta-analysis.

Interventions targeting mitochondrial dysfunction have the potential to extend survival in preclinical models of amyotrophic lateral sclerosis. The aim of this systematic review was to assess the efficacy of targeting mitochondria as a potential therapeutic target in amyotrophic lateral sclerosis. Preclinical studies written in the English language were identified with no restrictions on publication date from PubMed, Medline and EMBASE databases. All studies adopting interventions targeting mitochondria to treat amyotrophic lateral sclerosis in genetic or drug-induced organism models were considered for inclusion. A total of 76 studies were included in the analysis. Survival data were extracted, and the meta-analysis was completed in RevMan 5 software. We show that targeting mitochondrial dysfunction in amyotrophic lateral sclerosis results in a statistically significant improvement in survival (Z = 5.31; P < 0.00001). The timing of administration of the intervention appears to affect the improvement in survival, with the greatest benefit occurring for interventions given prior to disease onset. Interventions at other time points were not significant, although this is likely to be secondary to a lack of publications examining these timepoints. The quality score had no impact on efficacy, and publication bias revealed an overestimation of the effect size, owing to one outlier study; excluding this led to the recalculated effect size changing from 5.31 to 3.31 (P < 0.00001). The extant preclinical literature indicates that targeting mitochondrial dysfunction may prolong survival in amyotrophic lateral sclerosis, particularly if the intervention is administered early. A limitation of current research is a significant bias towards models based on superoxide dismutase 1, with uncertainty about generalisability to amyotrophic lateral sclerosis with an underlying TAR DNA binding protein 43 proteinopathy. However, further mechanistic research is clearly warranted in this field.

Crosstalk Between Sensory Nerves and Cancer in Bone.

Sensory nerves (SNs) richly innervate bone and are a component of bone microenvironment. Cancer metastasis in bone, which is under the control of the crosstalk with bone microenvironment, induces bone pain via excitation of SNs innervating bone. However, little is known whether excited SNs in turn affect bone metastasis. Cancer cells colonizing bone promote neo-neurogenesis of SNs and excite SNs via activation of the acid-sensing nociceptors by creating pathological acidosis in bone, evoking bone pain. Denervation of SNs or inhibition of SN excitation decreases bone pain and cancer progression and increases survival in preclinical models. Importantly, patients with cancers with increased SN innervation complain of cancer pain and show poor outcome. SNs establish the crosstalk with cancer cells to contribute to bone pain and cancer progression in bone. Blockade of SN excitation may have not only analgesic effects on bone pain but also anti-cancer actions on bone metastases.

The 2017 IXA Presidential Lecture: Recent developments in xenotransplantation.

The last few years have seen significant progress in xenotransplantation. Porcine xenograft survival in preclinical models continues to improve, accompanied by the adjustment of immunosuppression to more clinically realistic levels. The rapid uptake of CRISPR/Cas9 technology has accelerated the generation of new knock-in and knockout pigs, including animals null for the endogenous retrovirus PERV. This brief review presents a personal view of recent developments in the field.

A phase IB/II randomized study of mFOLFIRINOX (mFFOX) + pegylated recombinant human hyaluronidase (PEGPH20) versus mFFOX alone in patients with good performance status metastatic pancreatic adenocarcinoma (mPC): SWOG S1313 (NCT #01959139).

208 Background: PEGPH20 degrades hyaluronan (HA), a major component of the stroma, increases delivery of gemcitabine and prolongs survival in preclinical models. We evaluated the activity of PEGPH20 in combination with mFFOX in mPC , unselected for tumor HA. Methods: Pertinent eligibility: untreated mPC, PS of 0-1 and adequate organ function. Standard FFOX was modified to add prophylactic growth factor support and omit bolus 5FU. Due to increased thromboembolic (TE) events with PEGPH20, an amendment instituted LMWH prophylaxis in the PEGPH20 arm only. Following a dose finding cohort of mFFOX + PEGPH20, the Phase II study randomized patients (1:1) to the combination arm or mFFOX alone (n = 138). The primary endpoint was overall survival (OS), with a null median OS of 10 mo and an alternative of 15 mo (1-sided type 1 error 0.1, 80% power). Results: PEGPH20 at 3 mcg/kg, q2 weeks was more tolerable than twice weekly dosing. The randomized phase II study began May 2015. The planned interim futility analysis when 35 deaths occurred gave a HR of 0.44 favoring the standard arm, thus the study was closed to accrual (March 2017). As of August 22, 2017 (n = 111), the estimated median OS in the mFFOX arm was 15.1 mo (95% CI 10.1-15.7) vs 7.6 mo (95% CI 4.6-9.2) in the PEGPH20 arm. Conclusions: OS in the mFFOX control arm (15.1 mo) is longest yet reported. Addition of PEGPH20 to mFFOX is not recommended for further study and appears to be detrimental (HR = 0.48). The impact of PEGPH20 on OS was unexpected and in contrast to favorable results reported for the combination of gemcitabine/nab-paclitaxel + PEGPH20 especially in the HA high cohort (Hingorani S et al. A 4008, ASCO 2017). PEGPH20 with mFFOX caused increased toxicity (mostly GI and TE events) and decreased treatment duration compared to mFFOX alone. Tumor HA content will be analyzed. Funding: NIH/NCI grants CA180888, CA180819; and in part by Halozyme Inc. Clinical trial information: 01959139. [Table: see text]

Liposomal Irinotecan Accumulates in Metastatic Lesions, Crosses the Blood-Tumor Barrier (BTB), and Prolongs Survival in an Experimental Model of Brain Metastases of Triple Negative Breast Cancer.

The blood-tumor barrier (BTB) limits irinotecan distribution in tumors of the central nervous system. However, given that the BTB has increased passive permeability we hypothesize that liposomal irinotecan would improve local exposure of irinotecan and its active metabolite SN-38 in brain metastases relative to conventional irinotecan due to enhanced-permeation and retention (EPR) effect. Female nude mice were intracardially or intracranially implanted with human brain seeking breast cancer cells (brain metastases of breast cancer model). Mice were administered vehicle, non-liposomal irinotecan (50 mg/kg), liposomal irinotecan (10 mg/kg and 50 mg/kg) intravenously starting on day 21. Drug accumulation, tumor burden, and survival were evaluated. Liposomal irinotecan showed prolonged plasma drug exposure with mean residence time (MRT) of 17.7 ± 3.8 h for SN-38, whereas MRT was 3.67 ± 1.2 for non-liposomal irinotecan. Further, liposomal irinotecan accumulated in metastatic lesions and demonstrated prolonged exposure of SN-38 compared to non-liposomal irinotecan. Liposomal irinotecan achieved AUC values of 6883 ± 4149 ng-h/g for SN-38, whereas non-liposomal irinotecan showed significantly lower AUC values of 982 ± 256 ng-h/g for SN-38. Median survival for liposomal irinotecan was 50 days, increased from 37 days (p<0.05) for vehicle. Liposomal irinotecan accumulates in brain metastases, acts as depot for sustained release of irinotecan and SN-38, which results in prolonged survival in preclinical model of breast cancer brain metastasis.