Prolonged Animal Survival Research Articles

Biodistribution and Efficacy of L-Annamycin in a Novel Imageable AML Mouse Model

Annamycin (Ann) is an anti-tumoral anthracycline whose anti-leukemia activity is relatively unaffected by P-glycoprotein-related multidrug resistance. Unlike for the related doxorubicin (DOX), Ann accumulates in multidrug resistant cell lines, which is accompanied by DNA damage and apoptosis. In preclinical toxicology studies, in contrast to DOX, free Ann displayed a greatly reduced cardiotoxicity, while L-Ann appeared to be non-cardiotoxic. A liposomal formulation of Ann, termed L-Annamycin (L-Ann), is currently evaluated in patients with acute myeloid leukemia (AML). Anti-leukemia activity of Ann was demonstrated in several leukemia models as judged by circulating blast cytoreduction and extension of overall survival. However, the efficacy of L-Ann in the microenvironment of the bone marrow and other organ tissues remains unclear. In the current study, we assessed the anti-AML efficacy of Ann in a novel AML model that allows visualizing the dynamics of individual AML cells in vivo by two-photon microscopy. In this model, mouse AML cells bearing the MLL/ENL-FLT3/ITD[p53-/-] mutations co-express high levels of the cyan fluorescent protein mTurquoise2. Upon intravenous infusion of several tens of thousands cells into syngeneic immunocompetent C57BL6 mice, lethal AML disease reliably develops within 2 weeks. Using host mice expressing appropriate fluorescence reporter genes, the bright cyan fluorescence enables sensitive intravital imaging of individual AML cells in the context of organ architecture. Using this model in Thy1-RFP reporter mice expressing red fluorescence in all organ tissues with the blood flow marked by BSA-AF647 fluorescence, we evaluated AML cellularity reduction in the bone marrow and other organs after a single dose of L-Ann as well as in response to chronic treatment. In addition, we assessed the localization of the surviving AML cells at a high spatial resolution. We evaluated the in vivo organ biodistribution of intravenously infused L-Ann in C57BL6 mice by flow cytometry and two-photon microscopy based on the intrinsic fluorescence of the drug. In addition, we visualized the intracellular compartmentalization of L-Ann using confocal microscopy. Consistent with in vitro findings, we observed a rapid and deep reduction of AML blasts in the peripheral blood after a single dose of L-Ann in a dose-dependent manner (1-4 mg/kg). This reduction was strongly correlated with prolongation of animal survival from 14 days (vehicle) to 37 days (L-Ann 4 mg/kg once weekly started on day 10). In vitro and intravital microscopy revealed a distinct pattern of L-Ann distribution in organ tissues, which correlated in part with the local index of AML cellularity reduction and residual disease localization. Interestingly, in addition to the expected uptake of Ann in the cell's nucleus, Ann was also accumulated in the cytosol of the cells. This bi-compartmental intracellular distribution pattern contrasted with the nuclear-only localization of DOX. Administration of L-Ann early in the course of AML resulted in occasional complete responses some of which associated with resistance to AML re-challenge, suggesting capacity for anti-AML immune memory induction. This study confirms the efficacy of the drug in the model setting of syngeneic, immune-competent AML. Besides reinforcing the rationale for further development of Annamycin in AML, this study demonstrates a highly advantageous AML mouse model that is highly informative in studies of AML pharmacology, minimum residual disease (MRD), microenvironment and immunology. Disclosures Fokt: Moleculin Biotech, Inc.: Equity Ownership, Research Funding. Andreeff:Oncoceutics: Equity Ownership; Senti Bio: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Daiichi Sankyo, Inc.: Consultancy, Patents & Royalties: Patents licensed, royalty bearing, Research Funding; Jazz Pharmaceuticals: Consultancy; Celgene: Consultancy; Amgen: Consultancy; AstaZeneca: Consultancy; 6 Dimensions Capital: Consultancy; Reata: Equity Ownership; Aptose: Equity Ownership; Eutropics: Equity Ownership; Leukemia Lymphoma Society: Membership on an entity's Board of Directors or advisory committees; NCI-RDCRN (Rare Disease Cliln Network): Membership on an entity's Board of Directors or advisory committees; CLL Foundation: Membership on an entity's Board of Directors or advisory committees; BiolineRx: Membership on an entity's Board of Directors or advisory committees; German Research Council: Membership on an entity's Board of Directors or advisory committees; NCI-CTEP: Membership on an entity's Board of Directors or advisory committees; Cancer UK: Membership on an entity's Board of Directors or advisory committees; Oncolyze: Equity Ownership; Breast Cancer Research Foundation: Research Funding; CPRIT: Research Funding; NIH/NCI: Research Funding; Center for Drug Research & Development: Membership on an entity's Board of Directors or advisory committees. Priebe:Moleculin Biotech, Inc.: Consultancy, Equity Ownership, Research Funding. Zal:VueBio.com: Equity Ownership; BioLineRx: Research Funding; Daiichi-Sankyo: Research Funding; Moleculin Biotech, Inc.: Research Funding; NIH-CTEP: Research Funding; CPRIT: Research Funding; NIH/NCI: Research Funding.

LAM-003, a Novel Oral Heat Shock Protein 90 Inhibitor for Treatment of Acute Myeloid Leukemia, Including Wild-Type and FMS-like Tyrosine Kinase 3 (FLT3)-Mutant Disease

Acute myeloid leukemia (AML) remains a disease with high unmet medical need. While most patients respond to initial therapy, few are cured, relapse rates are high, and most patients eventually develop life-threatening complications. FLT3-mutant disease is a particularly aggressive subtype. Recent approval of drugs targeting FLT3-mutant disease have improved short-term outcomes but not all patients respond, and duration of response is limited by secondary mutations that impede FLT3 inhibitor (FLT3i) binding (intrinsic factors) and by secreted stromal factors that activate alternative pro-survival pathways (extrinsic factors). Heat-shock protein 90 (HSP90) is a chaperone protein involved in many cellular processes and inhibition of HSP90 can have pleiotropic effects in targeting cancer cells such as degradation of oncoproteins that drive survival and proteins that mediate protective signaling. Here, we describe the nonclinical activity of LAM-003, an orally bioavailable HSP90 inhibitor (HSP90i) under clinical development for AML. To assess the anti-leukemic activity of LAM-003, we tested a panel of AML cell lines and primary AML samples. LAM-003 inhibited proliferation of both FLT3-mutant and wild-type cell lines, with preferential activity against cells harboring FLT3-ITD (geometric mean FLT3-ITD EC50 = 670 nM [n=8] vs FLT3 WT EC50 = 1400 nM [n=16]). Additionally, we observed that LAM-003 was potent in a subset of the FLT3 WT cells. To explore whether LAM-003 was effective against tumors driven by oncoproteins that are client proteins of HSP90, we focused on AML cells harboring FLT3-ITD. We confirmed that LAM-003 reduced cell surface FLT3-ITD expression and downstream signaling in MV-4-11 and MOLM-13 cells, consistent with HSP90i-mediated degradation of FLT3-ITD. In BA/F3 cells expressing FLT3-ITD with various secondary resistance mutations, we observed that LAM-003 elicited a dose-dependent reduction of FLT3 mutant cell surface expression. Moreover, BA/F3 cells expressing FLT3-ITD and the F691L mutation exhibited the expected resistance to crenolanib, yet LAM-003 retained anti-proliferative activity. Additionally, MOLM-13 cells harboring a FLT3 D835Y mutation demonstrated expected resistance to the FLT3i sorafenib and tandutinib yet remained sensitive to LAM-003. Finally, primary AML blasts harboring a D835 mutation displayed sensitivity to LAM-003 when tested ex vivo. To evaluate the potential of LAM-003 to overcome bone-marrow-stroma-derived resistance, FLT3-ITD AML cell lines (MV-4-11, MOLM-13, MOLM-14) were assayed in unconditioned or stromal-cell-conditioned medium. Conditioned medium dramatically reduced the potency of FLT3i but LAM-003 demonstrated equal potency under both conditions. We also showed that stromal cell co-culture induced FLT3i resistance in MOLM-13 cells whereas LAM-003 retained potent activity. Recognizing that the inherent genetic heterogeneity of AML blasts limits the curative potential of a single drug, we performed in vitro studies to identify drugs that synergize with LAM-003 in 3 FLT3-ITD AML cell lines. Synergy was demonstrated with FLT3i, daunorubicin, azacitidine or cytarabine, with the most robust synergy being observed with venetoclax. Extending the evaluation to AML cells wild type for FLT3 and cell lines from other hematologic indications (multiple myeloma, diffuse large B-cell lymphoma and mantle cell lymphoma), we found that the synergy was not limited to cells harboring FLT3-ITD, but rather correlated with BCL-2 abundance, suggesting a fundamental mechanism of action that depends on BCL-2 family-mediated survival. Mechanistic studies demonstrated that the combination of LAM-003 and venetoclax inhibited AKT-mediated regulation of GSK3B, resulting in MCL-1 degradation. In vivo studies using a MOLM-13 systemic model of FLT3-ITD AML demonstrated that LAM-003 monotherapy significantly improved animal survival and that the combination of LAM-003 and venetoclax significantly prolonged animal survival compared with each single agent. These nonclinical studies demonstrate that LAM-003 exhibits antileukemic activity, overcomes mechanisms of FLT3i resistance and potently synergizes with existing AML drugs. As such, our data provide strong rationale for evaluation of LAM-003 in an ongoing clinical trial in patients with AML (NCT03426605). Disclosures Beeharry: AI Therapeutics: Employment, Equity Ownership. Landrette:AI Therapeutics: Employment. Grotzke:AI Therapeutics: Employment. Gayle:AI Therapeutics: Equity Ownership. Young:AI Therapeutics: Employment, Equity Ownership. Miller:Incuron, Inc.: Consultancy; Cleveland Biolabs, Inc: Employment, Equity Ownership; Calistoga Pharmaceuticals, Inc.: Equity Ownership; AI Therapeutics: Consultancy, Equity Ownership; VelosBio Inc.: Employment, Equity Ownership; Acerta Pharma, Inc.: Equity Ownership. Xu:AI Therapeutics: Equity Ownership, Membership on an entity's Board of Directors or advisory committees. Rothberg:AI Therapeutics: Equity Ownership, Membership on an entity's Board of Directors or advisory committees. Lichenstein:AI Therapeutics: Employment, Equity Ownership.

Targeting Aurora Kinase B with AZD2811 Enhances Venetoclax Activity in TP53-Mutant AML

Background: Recent data suggests that older patients with acute myeloid leukemia (AML) have a higher frequency of poor risk TP53 mutations (TP53mut) than previously suspected. Patients with TP53mut have a very poor prognosis, making this AML sub-group an area of high unmet therapeutic need. Although recent clinical trials suggest promising response rates for the BCL-2 inhibitor venetoclax (Ven) in combination with DNA methyltransferase inhibitors (DNMTi) or low-dose cytarabine, survival outcomes remain poor among patients with TP53mut (Strickland et al, EHA 2018). Methods and Results: To identify novel therapies effective against TP53mut AML, isogenic TP53 knockout cells (TP53 KO) were generated by CRISPR/Cas9 in OCI-AML3, MV4;11 and MOLM-13 human AML cell lines. In a drug screen of 50 compounds, we identified AZD2811, an aurora kinase B inhibitor, which potently killed AML cells independently of TP53 genotype (Figure 1). We hypothesised that AZD2811 would synergise with Ven to overcome venetoclax-resistance in TP53-mutant AML. Consistent with this, AZD2811 and venetoclax showed strong synergy (Loewe score <4) in 6 AML cell lines independent of TP53 genotype, and combined treatment displayed highly efficacious activity in both TP53 KO cells and wild type control cells in vitro (Figure 2). Immunoblot of treated cells revealed that AZD2811 depleted MCL-1 expression in the majority of cases, a known cause of venetoclax resistance. AZD2811 was also active in killing Bax/Bak knockout cells, suggesting that the mechanism of AZD2811 may not be limited to the intrinsic apoptosis pathway. Using a competitive growth assay incorporating a fluorescent reporter for TP53 KO cells, we showed the relative resistance of TP53 defective cells to Ven and decitabine (DEC) alone, as well as Ven + DEC, compared to wild type cells. In contrast, TP53 KO cells showed no competitive growth advantage when exposed to an initially sub-lethal dose of AZD2811 (10 nM), compared to TP53 WT cells. Interestingly, continuous exposure to AZD2811 resulted in a catastrophic collapse in cell viability on day 10, unlike comparator anti-leukemic agents (Figure 4). In vivo efficacy for nanoparticle formulated AZD2811 (NP) was observed in SCID mice xenografted with HL-60 cells (known to be TP53 mutant), where combined treatment of AZD2811NP and Ven significantly prolonged animal survival (Figure 3). Remarkably, combined AZD2811NP and Ven treatment was more effective than Ven + azacitidine treatment in vivo. Patient-derived xenograft (PDX) models to verify the activity of AZD2811NP + venetoclax against primary AML cases with TP53 mutations will be presented at the meeting. Conclusions: We report for the first time that AZD2811NP can overcome venetoclax resistance in TP53-mutant AML in vitro and in vivo. These findings therefore, support the clinical investigation of combined aurora kinase and BCL-2 targeting in the clinic for patients with TP53-mutant AML, who currently lack effective treatment options. Disclosures Urosevic: AstraZeneca: Employment. Polanska:AstraZeneca: Employment. Cosaert:AstraZeneca: Employment. Pease:AstraZeneca: Employment, Equity Ownership. Travers:AstraZeneca: Employment. Wei:Pfizer: Honoraria; Janssen: Honoraria; Walter and Eliza Hall Institute: Other: former employee, Patents & Royalties: receives a fraction of its royalty stream related to venetoclax; AbbVie: Honoraria, Research Funding; Novartis: Honoraria, Research Funding; Macrogenics: Honoraria; Astellas: former employee, Honoraria; Genentech: Honoraria; Servier: Honoraria, Research Funding; Celgene: Honoraria, Research Funding; Amgen: Honoraria, Research Funding; AstraZeneca: Honoraria, Research Funding.

CD9 blockade suppresses disease progression of high-risk pediatric B-cell precursor acute lymphoblastic leukemia and enhances chemosensitivity.

CD9 has been implicated in cancer progression but its prognostic relevance and therapeutic potential in B-cell precursor acute lymphoblastic leukemia (BCP-ALL) are largely unknown. In a cohort of pediatric BCP-ALL patients, we found that CD9+ cases had a significantly lower 5-year relapse-free survival rate than CD9- cases. Multivariate analysis demonstrated that CD9 positivity independently predicted inferior survival outcomes, and could be applied with established prognostic features, including prednisone response and cytogenetic status, to refine patient stratification. Administration of CD9 antibody substantially suppressed disease progression in NOD/SCID mice xenografted with CD9+ cell lines and primary leukemic blasts from patients with high-risk and refractory BCP-ALL, without compromising hematopoietic stem cell engraftment. Combination of anti-CD9 with conventional chemotherapy further reduced leukemic burden and prolonged animal survival. Mechanistically, CD9 blockade inhibited leukemic cell proliferation, induced G0/G1 cell cycle arrest, activated p38, and enhanced chemotherapeutic agent-induced apoptosis. Further, CD9 physically interacted with integrin very late antigen-4, regulated affinity to vascular cell adhesion molecule-1, and was involved in leukemia-stroma interaction. Collectively, our study established CD9 as a new prognostic marker, validated the preclinical efficacy of CD9 antibody, and laid the foundation for clinical development of CD9-targeted therapy for high-risk and refractory pediatric BCP-ALL.

Preclinical Evaluation of Antitumoral and Cytotoxic Properties of Viscum album Fraxini Extract on Pediatric Tumor Cells.

In Europe, especially in German-speaking countries, administration of mistletoe extracts is the most common and popular complementary and alternative therapy approach reported in oncology. Mistletoe therapy is applied to children with cancer for curative and palliative therapeutic regimes with increasing frequency, but at the same time, there are only a few studies on the effectiveness of this therapy. Therefore, we have investigated the response of various pediatric cell lines (acute myeloid leukemia, Ewing's sarcoma, hepatocellular carcinoma, medulloblastoma, neuroblastoma, and osteosarcoma) to mistletoe extract, abnobaVISCUM Fraxini. Effects on cell proliferation, cell cycle distribution as well as on mitochondrial integrity and caspase-mediated apoptosis were investigated in neuroblastoma cell lines, SH-SY5Y and Kelly. Additionally, in vitro tumor cell migration and invasion were studied. In vivo effects of the mistletoe extract were investigated in a syngeneic neuroblastoma mouse model. We could show that tumor cell lines were from 5- to 640-fold more sensitive to abnobaVISCUM Fraxini treatment than non-tumorigenic fibroblasts, whereby neuroblastoma cell lines were the most sensitive. For two neuroblastoma cell lines, SH-SY5Y and Kelly, induction of caspase-9-mediated apoptosis, a decrease of mitochondrial integrity as well as attenuation of migration and invasion were observed. In vivo experiments revealed a reduction of tumor growth and a prolonged survival of tumor-bearing animals. In summary, we can state that these results provide the first preclinical data for cytotoxic activities of abnobaVISCUM Fraxini for a broad panel of pediatric tumor cell lines, in particular, neuroblastoma cells. Thus, it might be a potential remedy for the supportive treatment of neuroblastoma.

1881PD - Oral intestinal alkaline phosphatase improves efficacy of 5-FU in a colorectal cancer mouse model

BackgroundIntestinal alkaline phosphatase (IAP) is an endogenous intestinal enzyme that promotes gastrointestinal (GI) homeostasis by detoxifying inflammatory mediators, tightening the gut barrier and promoting a healthy microbiome. In previous reports, oral delivery of IAP ameliorated colitis in animals and in a human pilot study. The commonly used chemotherapy, 5-Fluorouracil (5-FU), can cause severe GI damage potentially resulting in dose limiting toxicities. Given the recent implication of the gut microbiome in modulating the anti-cancer response and the role of IAP in mitigating GI toxicity, we sought to evaluate the role of IAP in ameliorating 5-FU related GI side effects and to assess the impact of IAP therapy on anti-cancer efficacy. MethodsBALB/c mice were inoculated with CT26 murine colorectal cancer cells into the right flank. 5-FU (30mg/kg) was given IP QD for 5 consecutive days. IAP (0.5mg/kg) or vehicle was administered PO BID throughout the study. Mice were evaluated for stool consistency, tumor growth and survival. A cohort of mice was sacrificed at day 5 for histopathology analysis. Results5-FU administration caused loose stools/diarrhea in all the treated animals, which peaked at day 6 and recovered by day 9. Interestingly, mice that received IAP showed a significantly faster recovery (p<0.03). As expected, 5-FU slowed tumor progression compared to controls (no 5-FU). Surprisingly, IAP addition significantly improved anti-cancer efficacy. Mice treated with IAP showed diminished tumor growth compared to vehicle (at day 19, 1066 vs 1968mm3, p<0.05). Furthermore, IAP administration significantly prolonged animal survival when compared to vehicle-treated animals (p<0.02). Mechanistically, 5-FU led to goblet cell loss whereas IAP treatment protected the goblet cells. ConclusionsOral administration of IAP in mice not only improved some of the side effects associated with 5-FU, such as diarrhea and potentially mucositis, but also enhanced the anti-tumor efficacy of 5-FU leading to significantly prolonged survival. Overall, IAP has the potential to become a new therapeutic agent intended to mitigate cancer-treatment side effects and to enhance the overall anti-tumor response. Legal entity responsible for the studyThe authors. FundingSynthetic Biologics. DisclosureC. Furlan Freguia: Full / Part-time employment: Synthetic Biologics. M. Kaleko: Full / Part-time employment: Synthetic Biologics.

Quantitative Proteomics of Th-MYCN Transgenic Mice Reveals Aurora Kinase Inhibitor Altered Metabolic Pathways and Enhanced ACADM To Suppress Neuroblastoma Progression.

Neuroblastoma is a neural crest-derived embryonal tumor and accounts for about 15% of all cancer deaths in children. MYCN amplification is associated with aggressive and advanced stage of high-risk neuroblastoma, which remains difficult to treat and exhibits poor survival under current multimodality treatment. Here, we analyzed the transcriptomic profiles of neuroblastoma patients and showed that aurora kinases lead to poor survival and had positive correlation with MYCN amplification and high-risk disease. Further, pan-aurora kinase inhibitor (tozasertib) treatment not only induces cell-cycle arrest and suppresses cell proliferation, migration, and invasion ability in MYCN-amplified (MNA) neuroblastoma cell lines, but also inhibits tumor growth and prolongs animal survival in Th-MYCN transgenic mice. Moreover, we performed quantitative proteomics and identified 150 differentially expressed proteins after tozasertib treatment in the Th-MYCN mouse model. The functional and network-based enrichment revealed that tozasertib alters metabolic processes and identified a mitochondrial flavoenzyme in fatty acid β-oxidation, ACADM, which is correlated with aurora kinases and neuroblastoma patient survival. Our findings indicate that the aurora kinase inhibitor could cause metabolic imbalance, possibly by disturbing carbohydrate and fatty acid metabolic pathways, and ACADM may be a potential target in MNA neuroblastoma.

Bortezomib administered prior to temozolomide depletes MGMT, chemosensitizes glioblastoma with unmethylated MGMT promoter and prolongs animal survival

BackgroundResistance to temozolomide (TMZ) is due in part to enhanced DNA repair mediated by high expression of O6-methyl guanine DNA methyltransferase (MGMT) that is often characterised by unmethylated promoter. Here, we investigated pre-treatment of glioblastoma (GBM) cells with the 26S-proteasome inhibitor bortezomib (BTZ) as a strategy to interfere with MGMT expression and thus sensitise them to TMZ.MethodsCell lines and patient GBM-derived cells were examined in vitro, and the latter also implanted orthotopically into NOD-SCID C.B.-Igh-1b/lcrTac-Prkdc mice to assess efficacy and tolerability of BTZ and TMZ combination therapy. MGMT promoter methylation was determined using pyrosequencing and PCR, protein signalling utilised western blotting while drug biodistribution was examined by LC-MS/MS. Statistical analysis utilised Analysis of variance and the Kaplan–Meier method.ResultsPre-treatment with BTZ prior to temozolomide killed chemoresistant GBM cells with unmethylated MGMT promoter through MGMT mRNA and protein depletion in vitro without affecting methylation. Chymotryptic activity was abolished, processing of NFkB/p65 to activated forms was reduced and corresponded with low MGMT levels. BTZ crossed the blood–brain barrier, diminished proteasome activity and significantly prolonged animal survival.ConclusionBTZ chemosensitized resistant GBM cells, and the schedule may be amenable for temozolomide refractory patients with unmethylated MGMT promoter.

Deletion of the Miz-1 POZ Domain Increases Efficacy of Cytarabine Treatment in T- and B-ALL/Lymphoma Mouse Models.

Acute lymphoblastic leukemia (ALL) is an aggressive blood cancer that mainly affects children. Relapse rates are high and toxic chemotherapies that block DNA replication and induce DNA damage lead to health problems later in life, underlining the need for improved therapies. MYC is a transcription factor that is hyperactive in a large proportion of cancers including leukemia but is difficult to target in therapy. We show that ablation of the function of the BTB/POZ domain factor Zbtb17 (Miz-1), an important cofactor of c-Myc, significantly delayed T- and B-ALL/lymphoma in mice and interfered with the oncogenic transcriptional activity of c-Myc. Leukemic cells that still emerged in this system activated DNA replication pathways that could be targeted by current chemotherapeutic drugs such as cytarabine. Acute ablation of the Miz-1 POZ domain enhanced the effect of cytarabine treatment. The combined treatment was effective in both Eμ-Myc and Notch ICN-driven leukemia models and prolonged survival of tumor-bearing animals by accelerating apoptosis of leukemic cells. These observations suggest that targeting MIZ-1 could render current ALL chemotherapies more effective, with a better outcome for patients. SIGNIFICANCE: Ablation of the POZ domain of Miz-1 perturbs its interaction with c-MYC and delays the generation of T- and B-cell leukemias and lymphomas.

Combination PD-1 and PD-L1 Blockade Promotes Durable Neoantigen-Specific T Cell-Mediated Immunity in Pancreatic Ductal Adenocarcinoma

SUMMARYPancreatic ductal adenocarcinoma (PDA) is a lethal cancer resistant to immunotherapy. We create a PDA mouse model and show that neoantigen expression is required for intratumoral T cell accumulation and response to immune checkpoint blockade. By generating a peptide:MHC tetramer, we identify that PDA induces rapid intratumoral, and progressive systemic, tumor-specific T cell exhaustion. Monotherapy PD-1 or PD-L1 blockade enhances systemic T cell expansion and induces objective responses that require systemic T cells. However, tumor escape variants defective in IFNγ-inducible Tap1 and MHC class I cell surface expression ultimately emerge. Combination PD-1 + PD-L1 blockade synergizes therapeutically by increasing intratumoral KLRG1+Lag3−TNFα+ tumor-specific T cells and generating memory T cells capable of expanding to spontaneous tumor recurrence, thereby prolonging animal survival. Our studies support that PD-1 and PD-L1 are relevant immune checkpoints in PDA and identify a combination for clinical testing in those patients with neoantigen-specific T cells.

Indocyanine Green-Coated Gold Nanoclusters for Photoacoustic Imaging and Photothermal Therapy.

Traditional oncology treatment modalities are often associated with a poor therapeutic index. This has driven the development of new targeted treatment modalities, including several based on the conversion of optical light into heat energy (photothermal therapy, PTT) and sound waves (photoacoustic imaging, PA) that can be applied locally. These approaches are especially effective when combined with photoactive nanoparticles that preferentially accumulate in tissues of interest and thereby further increase spatiotemporal resolution. In this study, two clinically-used materials that have proven effective in both PTT and PA - indocyanine green and gold nanoparticles - were combined into a single nanoformulation. These particles, "ICG-AuNP clusters", incorporated high concentrations of both moieties without the need for additional stabilizing or solubilizing reagents. The clusters demonstrated high theranostic efficacy both in vitro and in vivo, compared with ICG alone. Specifically, in an orthotopic mouse model of triple-negative breast cancer, ICG-AuNP clusters could be injected intravenously, imaged in the tumor by PA, and then combined with near-infrared laser irradiation to successfully thermally ablate tumors and prolong animal survival. Altogether, this novel nanomaterial demonstrates excellent therapeutic potential for integrated treatment and imaging.

Pretreatment of Glioblastoma with Bortezomib Potentiates Natural Killer Cell Cytotoxicity through TRAIL/DR5 Mediated Apoptosis and Prolongs Animal Survival.

Background: Natural killer (NK) cells are potential effectors in anti-cancer immunotherapy; however only a subset potently kills cancer cells. Here, we examined whether pretreatment of glioblastoma (GBM) with the proteasome inhibitor, bortezomib (BTZ), might sensitize tumour cells to NK cell lysis by inducing stress antigens recognized by NK-activating receptors. Methods: Combination immunotherapy of NK cells with BTZ was studied in vitro against GBM cells and in a GBM-bearing mouse model. Tumour cells were derived from primary GBMs and NK cells from donors or patients. Flow cytometry was used for viability/cytotoxicity evaluation as well as in vitro and ex vivo phenotyping. We performed a Seahorse assay to assess oxygen consumption rates and mitochondrial function, Luminex ELISA to determine NK cell secretion, protein chemistry and LC–MS/MS to detect BTZ in brain tissue. MRI was used to monitor therapeutic efficacy in mice orthotopically implanted with GBM spheroids. Results: NK cells released IFNγ, perforin and granzyme A cytolytic granules upon recognition of stress-ligand expressing GBM cells, disrupted mitochondrial function and killed 24–46% of cells by apoptosis. Pretreatment with BTZ further increased stress-ligands, induced TRAIL-R2 expression and enhanced GBM lysis to 33–76% through augmented IFNγ release (p < 0.05). Blocking NKG2D, TRAIL and TRAIL-R2 rescued GBM cells treated with BTZ from NK cells, p = 0.01. Adoptively transferred autologous NK-cells persisted in vivo (p < 0.05), diminished tumour proliferation and prolonged survival alone (Log Rank10.19, p = 0.0014, 95%CI 0.252–0.523) or when combined with BTZ (Log Rank5.25, p = 0.0219, 95%CI 0.295–0.408), or either compared to vehicle controls (median 98 vs. 68 days and 80 vs. 68 days, respectively). BTZ crossed the blood–brain barrier, attenuated proteasomal activity in vivo (p < 0.0001; p < 0.01 compared to vehicle control or NK cells only, respectively) and diminished tumour angiogenesis to promote survival compared to vehicle-treated controls (Log Rank6.57, p = 0.0104, 95%CI 0.284–0.424, median 83 vs. 68 days). However, NK ablation with anti-asialo-GM1 abrogated the therapeutic efficacy. Conclusions: NK cells alone or in combination with BTZ inhibit tumour growth, but the scheduling of BTZ in vivo requires further investigation to maximize its contribution to the efficacy of the combination regimen.

PROTECTIVE EFFECT INDUCED BY THE NEW SUBUNIT TUBERCULOSIS VACCINE WHEN USED AS A BCG BOOST IS ASSOCIATED WITH INHIBITION OF MYCOBACTERIAL DISSEMINATION

Since 1924, BCG vaccine is used to protect children from the most severe forms of tuberculosis. At the same time, the protective effect of BCG in adults is variable. The potential for revaccination with live vaccine is further limited by the rapid spread of HIV infection. The early-secreted Mycobacterium tuberculosis proteins have been used extensively in TB vaccine development, due to their high immunogenicity and have shown protective effect in animal models. The aim of our study was to evaluate the opportunity to increase the anti-TB resistance in experimental animals by re-vaccination with a new subunit vaccine preparation following primary immunization with BCG. To perform such boost vaccination, we used a combination of the Ag85B-TB10.4-FliC chimeric protein, and the plasmid DNA encoding Ag85A antigen. Efficiency of the boost vaccination was evaluated in a model of M. tuberculosis H37Rv aerosol infection of C57BL / 6 laboratory mice, either in the intact animals, or those vaccinated with BCG only, or BCG followed by revaccination with the test vaccine. The data concerning mycobacteria outgrowth from the organs, and life-span of animals after infection were subject to comparative analysis. We have demonstrated that additional boost vaccination with the vaccine under study, as compared with conventional BCG vaccination, leads to further inhibition of mycobacteria dissemination from the site of infection, and significantly prolonged survival of infected animals.

Abstract 348: ABI-009 (nab-Sirolimus) improves tumor accumulation and antitumor activity over oral mTOR inhibitors

Abstract Background: mTOR pathway has been implicated in cell survival and proliferation and is an attractive target for cancer therapy. ABI-009 (nab-sirolimus) is an injectable nanoparticle form of human albumin-bound sirolimus with a mean particle size of approximately 100 nm developed with a proprietary nanoparticle albumin-bound (nab®) technology. ABI-009 is currently in phase 1 and 2 clinical studies for the treatment of multiple cancer types, including bladder cancer, soft-tissue sarcomas, neuroendocrine tumors, colorectal cancer, glioblastoma, and various childhood cancers, and a registrational phase 2 study for malignant perivascular epithelioid cell carcinoma (PEComa). In nonclinical and clinical studies, ABI-009 demonstrated a PK profile distinct from oral mTOR inhibitors. This study compared the antitumor activity, tissue penetration, and pharmacodynamics of ABI-009 with equal doses of oral mTOR inhibitors at clinically relevant doses in a mouse tumor xenograft model. Methods: Athymic mice bearing subcutaneous UMUC3 bladder cancer xenografts were treated with saline, ABI-009 (IV, 7.5 mg/kg, 2x/wk), sirolimus and everolimus (PO, 3 mg/kg/day, 5 days/wk). Body weights, tumor measurements, and clinical signs were recorded 3x/wk for up to 5 wks. Tumor and blood sirolimus concentrations were also obtained (tumor: D1 1h and 24h, D4 1h, D7; blood: D4 1h, D7). Markers for mTOR pathway activation (pS6, p4EBP1) will also be analyzed. Results: Overall, all treatments were well tolerated with no significant body weight loss in any group. ABI-009 IV resulted in significantly higher sirolimus concentrations in the tumor compared with oral mTOR inhibitors at all time points tested. Tumor AUC with ABI-009 IV over 1 week were significantly higher vs equal weekly dose of oral sirolimus (43 fold) and everolimus (12 fold) (p &amp;lt; 0.0001). The enhanced tumor drug delivery by ABI-009 corresponded with significantly greater tumor growth inhibition (TGI: ABI-009 69.6% vs oral sirolimus 24.3%, p &amp;lt; 0.00001; vs oral everolimus 36.2%, p = 0.0023) and longer animal survival (median OS: ABI-009 not reached vs oral sirolimus 21 days, p &amp;lt; 0.05; vs oral everolimus 19 days, p &amp;lt; 0.05). mTOR pathway inhibition in tissues is being assessed. Conclusions: Compared with equal weekly dose of oral mTOR inhibitors, ABI-009 at clinically relevant dose of administered IV demonstrated significantly greater tumor drug delivery, stronger antitumor activity, and prolonged animal survival. These advantages observed in this nonclinical study support the clinical investigation of ABI-009 in numerous oncology indications. Citation Format: Shihe Hou, Anita Schmid, Neil Desai. ABI-009 (nab-Sirolimus) improves tumor accumulation and antitumor activity over oral mTOR inhibitors [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 348.

Abstract 2103: NETs promote tumor resistance to anthracyclines

Abstract Multiple Myeloma (MM) is an incurable blood cancer characterized by the clonal proliferation of plasma cells that accumulate in the bone marrow (BM). Resistance to chemotherapy represents one of the main challenges in the treatment of this disease. We have previously shown that neutrophils, one of the major cellular populations in the BM, protect MM cells from chemotherapies including anthracyclines; however, the mechanisms of this effect remain unknown. Thus, we set out to investigate the mechanisms by which neutrophils reduce sensitivity of tumor cells to anthracyclines. Neutrophil extracellular traps (NETs) are structures composed of chromatin and protein formed by neutrophils that are a part of an innate immune mechanism of binding and killing pathogens. Recently, NETs have been shown to form in cancer patients as well as tumor-bearing animals and have been implicated in solid tumor progression; however, their role in hematological malignancies remain poorly understood. Utilizing an in vitro culture system, we demonstrated that MM cells were able to induce NET formation by BM neutrophils. Interestingly, neutrophils isolated from the BM of patients with MM have a significantly higher ability to produce NETs in response to tumor cells as compared to BM neutrophils from donors. NET formation was accompanied by an increase in the level of extracellular DNA. Using imaging flow cytometry and confocal microscopy we demonstrated that extracellular DNA is internalized by tumor cells and localizes to the cytoplasm. This internalized DNA was able to bind to doxorubicin and other anthracyclines thereby reducing their cytotoxic effects. Significantly increased cell-free DNA levels were detected in BM aspirates from MM patients as well as in tumor-bearing mice. Targeting the extracellular DNA in vivo using the clinically available DNase, Pulmozyme, restored the sensitivity of tumor cells to doxorubicin resulting in prolonged survival of tumor-bearing animals. Here, we have identified a novel mechanism of resistance to anthracyclines that is mediated by neutrophils in the tumor microenvironment and provided evidence to support the premise that targeting this mechanism would be of significant benefit in the treatment of MM. Citation Format: Cindy Lin, Sarah E. Herlihy, Marina Li, Hui Deng, Luca Bernabei, Dmitry I. Gabrilovich, Dan T. Vogl, Yulia Nefedova. NETs promote tumor resistance to anthracyclines [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 2103.

A Novel mTORC1/2 Inhibitor (MTI-31) Inhibits Tumor Growth, Epithelial–Mesenchymal Transition, Metastases, and Improves Antitumor Immunity in Preclinical Models of Lung Cancer

We aimed to investigate efficacy and mechanism of MTI-31 (LXI-15029), a novel mTORC1/mTORC2 inhibitor currently in human trial (NCT03125746), in non-small cell lung cancer (NSCLC) models of multiple driver mutations and tyrosine kinase inhibitor (TKI)-resistance. Gene depletion, inhibitor treatment, immunological, flow cytometry, cellular, and animal studies were performed to determine in vitro and in vivo efficacy in NSCLC models of driver mutations and elucidate roles by mTOR complexes in regulating migration, epithelial-mesenchymal transition (EMT), metastasis, intracranial tumor growth, and immune-escape. MTI-31 potently inhibited cell proliferation (IC50 <1 μmol/L) and in vivo tumor growth in multiple NSCLC models of EGFR/T790M, EML4-ALK, c-Met, or KRAS (MED <10 mg/kg). In EGFR-mutant and/or EML4-ALK-driven NSCLC, MTI-31 or disruption of mTORC2 reduced cell migration, hematogenous metastasis to the lung, and abrogated morphological and functional traits of EMT. Disruption of mTORC2 inhibited EGFR/T790M-positive tumor growth in mouse brain and prolonged animal survival correlating a diminished tumor angiogenesis and recruitment of IBA1+ microglia/macrophages in tumor microenvironment. MTI-31 also suppressed programmed death ligand 1 (PD-L1) in EGFR- and ALK-driven NSCLC, mediated in part by mTORC2/AKT/GSK3β-dependent proteasomal degradation. Depletion of mTOR protein or disruption of mTOR complexes profoundly downregulated PD-L1 and alleviated apoptosis in Jurkat T and primary human T cells in a tumor-T cell coculture system. Our results highlight mTOR as a multifaceted regulator of tumor growth, metastasis, and immune-escape in EGFR/ALK-mutant and TKI-resistant NSCLC cells. The newly characterized mechanisms mediated by the rapamycin-resistant mTORC2 warrant clinical investigation of mTORC1/mTORC2 inhibitors in patients with lung cancer.

PF164 CD9 IS AN ADVERSE PROGNOSTIC MARKER AND PROMISING THERAPEUTIC TARGET IN HIGH‐RISK PEDIATRIC B‐PRECURSOR ACUTE LYMPHOBLASTIC LEUKEMIA

Background:B‐precursor acute lymphoblastic leukemia (B‐ALL) is the commonest childhood malignancy. Despite advances in chemotherapy‐based treatment protocols, specific subtypes of high‐risk and relapsed/refractory B‐ALL have been associated with dismal outcomes, underscoring the need for development of novel targeted agents. CD9, a tetraspanin family protein, has been implicated in tumor progression but its prognostic relevance and therapeutic value in pediatric B‐ALL remain largely unknown.Aims:(1) To characterize the expression of CD9 in a pediatric B‐ALL patient cohort and its association with long‐term survival outcomes; (2) to assess the efficacy of targeting CD9 by a neutralizing antibody for treatment of pediatric B‐ALL in the NOD/SCID mouse xenograft model; and (3) to evaluate the potential toxicity of anti‐CD9 using a hematopoietic stem cell transplantation model.Methods:Primary B‐ALL cells were isolated from bone marrow (BM) aspirates of patients consecutively enrolled in the HKALL97, IC‐BFM‐ALL2002 and CCLG2008 studies. Immunophenotypes of leukemic blasts were characterized with CD9, CD19, CD34 and CD45 antibodies by flow cytometry. Patients were stratified into CD9+ and CD9‐ subgroups for comparison of overall survival (OS) and relapse‐free survival (RFS). NOD/SCID mice were infused with B‐ALL cell lines, patient‐derived leukemic blasts or cord blood CD34+ cells, and treated with IgG control or anti‐CD9 as a single‐agent or combined with standard chemotherapy. Leukemic load and stem cell engraftment in hematopoietic organs and CNS were measured by flow cytometry, bioluminescence imaging and immunohistochemistry.Results:Among 136 pediatric B‐ALL cases, blasts of 107 patients (78.7%) were CD9+. The 5‐year RFS rate of CD9+ patients was significantly lower than that of CD9‐ patients (65.4% vs.86.2%; P = 0.038). Subgroup analysis revealed significantly reduced RFS (33.3% vs.80.0%; P = 0.022) and a trend of lower OS (55.6% vs.90.0%; P = 0.055) in CD9+ patients of the high‐risk group. Multivariate analysis revealed that CD9 positivity independently predicted inferior survival (HR = 5.2; P = 0.004), and could be applied with established prognostic features, including prednisone response and cytogenetic status, for refinement of patient stratification. In NOD/SCID mice xenografted with CD9+ B‐ALL cell lines (n = 3) and primary leukemic blasts from patients with high‐risk and refractory B‐ALL (n = 5), administration of CD9 antibody substantially reduced leukemic burden in BM, spleen, blood, liver and CNS by 78.4–99.9% (P &lt; 0.01) and prolonged animal survival by 1.6–2.1‐fold (P &lt; 0.01). Anti‐CD9 did not compromise cord blood stem cell engraftment (n = 3), indicating leukemia‐selective activities. Combined treatment with anti‐CD9 and conventional chemotherapy (vincristine, dexamethasone and L‐asparaginase) further reduced leukemic load and extended survival, when compared with animals receiving anti‐CD9 (P = 0.043) or chemotherapy alone (P &lt; 0.001).Summary/Conclusion:Our study identified CD9 as a new prognostic marker for predicting survival outcomes in pediatric B‐ALL and validated the efficacy of anti‐CD9 for suppressing leukemia progression. We propose that inclusion of CD9 into the diagnostic marker panels would potentially identify patients who might benefit from targeted therapy. In addition, anti‐CD9 could be developed as an adjunct to standard chemotherapy for treatment of high‐risk or relapsed/refractory pediatric B‐ALL, and this strategy could be extended to adult B‐ALL and other malignancies that express the CD9 surface antigen.

The third-generation EGFR inhibitor AZD9291 overcomes primary resistance by continuously blocking ERK signaling in glioblastoma

BackgroundGlioblastoma (GBM) is a fatal brain tumor, lacking effective treatment. Epidermal growth factor receptor (EGFR) is recognized as an attractive target for GBM treatment. However, GBMs have very poor responses to the first- and second-generation EGFR inhibitors. The third-generation EGFR-targeted drug, AZD9291, is a novel and irreversible inhibitor. It is noteworthy that AZD9291 shows excellent blood–brain barrier penetration and has potential for the treatment of brain tumors.MethodsIn this study, we evaluated the anti-tumor activity and effectiveness of AZD9291 in a preclinical GBM model.ResultsAZD9291 showed dose-responsive growth inhibitory activity against six GBM cell lines. Importantly, AZD9291 inhibited GBM cell proliferation > 10 times more efficiently than the first-generation EGFR inhibitors. AZD9291 induced GBM cell cycle arrest and significantly inhibited colony formation, migration, and invasion of GBM cells. In an orthotopic GBM model, AZD9291 treatment significantly inhibited tumor survival and prolonged animal survival. The underlying anti-GBM mechanism of AZD9291 was shown to be different from that of the first-generation EGFR inhibitors. In contrast to erlotinib, AZD9291 continuously and efficiently inhibited the EGFR/ERK signaling in GBM cells.ConclusionAZD9291 demonstrated an efficient preclinical activity in GBM in vitro and in vivo models. AZD9291 has been approved for the treatment of lung cancer with good safety and tolerability. Our results support the possibility of conducting clinical trials of anti-GBM therapy using AZD9291.

Inflammatory activation of microglia by Staphylococcus aureus caused phenotypic alterations and affected glioblastoma growth.

As the most common type of tumour in brain, glioma has a high rate of morbidity and mortality and easily penetrates the surrounding normal brain parenchyma. The immunosuppressive microenvironment, which is similar to that in other neoplasms, is believed to participate in the tumorigenesis of glioma. Thus, many experts are seeking to exploit microenvironment as a therapeutic target. In the present study, we focused on microglia polarization to investigate the anti-glioma response of microglia inflammatory activation by Staphylococcus aureus in vitro and in vivo. First, we found that intratumor injection of S. aureus delayed glioma growth in C57/BL6 mice. Then, we showed that inflammatory microglia activated by S. aureus inhibited glioma cell proliferation, migration, and invasion. This inhibition was likely related to the phenotypic switch observed in microglia. In intracranial tumour models, the microglia activated by S. aureus exerted antitumour efficacy and prolonged animal survival. Taken together, our results suggest that microglia activated by S. aureus have antitumour efficacy, which may be a potential therapeutic agent for glioma. SIGNIFICANCE OF THE STUDY: In this study, we mainly demonstrated the antitumour efficacy of microglia after activated by S. aureus. Firstly, we found that intratumor injection of S. aureus inhibited the tumour growth in intracranial orthotopic glioma model. In addition, we found that the microglia around the glioma may exert antitumour efficacy, and its phenotype may be altered by stimulation of S. aureus. Our data manifested that S. aureus did not directly suppress cell proliferative, migration, and invasion capacity, but by activating microglia. And in mice GL261 GBMs, injection of microglia after cocultured with S. aureus inhibited tumour growth and prolonged animal survival.

Expression of a neoantigen renders pancreas cancer transiently susceptible to immunotherapy

Abstract Immune checkpoint blockade has been largely ineffective for treating pancreatic ductal adenocarcinoma (PDA). Here, we develop a novel model of pancreatic cancer to investigate reasons for response and failure to immune checkpoint blockade in this lethal malignancy. Primary tumor epithelial cell clones from the genetically engineered KPC mouse model transduced to express a specific luciferase antigen, commonly used for bioluminescent in mice, rapidly establish tumors similar to parental cells after orthotopic implantation into syngeneic mice. Unexpectedly, αPD-L1 or αPD-1, which fail to elicit responses to luciferase- KPC parental cells, induce objective responses to luciferase+ KPC clones and prolong animal survival. We identify the immunodominant luciferase H- 2Db-restricted epitope and generate a fluorescently labeled peptide:MHC tetramer that specifically binds PDA-specific CD8 T cells. Up to 40% of tumor-infiltrating T cells bind this tetramer, yet most of these cells co-express PD-1 and Lag-3 and are dysfunctional. αPD-L1 enhances the systemic expansion of tetramer+ T cells and induced the accumulation of PD-1+ Ki67+ and Ki67- tetramer+ T cells in PDA. αPD-L1 significantly reduced the frequency of intra-tumoral tetramer+ T cells that expressed Lag-3 resulting in enhanced ex vivo peptide-specific cytokine production. However, tumors rapidly returned despite T cell infiltration and prolonged immunotherapy. Current efforts are underway to study the evolution of PDA-specific T cells and tumor clones using single cell technologies to inform clinical immunotherapy design.