SCID Mouse Model Research Articles

Sp3 -Rich Glycyrrhetinic Acid Analogues Using Late-Stage Functionalization as Potential Breast Tumor Regressing Agents.

Late-stage functionalization (LSF) aids drug discovery efforts by introducing functional groups onto C-H bonds on pre-existing skeletons. We adopted the LSF strategy to synthesize analogues of the abundantly available triterpenoid, glycyrrhetinic acid (GA), by introducing aryl groups in the A-ring, expanding the A-ring and selectively activating one methyl group of the gem-dimethyl groups. Intriguingly, two compounds were found to preferentially accumulate in the mitochondrial compartment of MDA-MB-231 breast cancer cells, to cause depolarization of mitochondrial membrane potential and to induce antiproliferative and anti-invasive effects through enhanced mitochondrial superoxide production with parallel depletion of GSH levels. Furthermore, intraperitoneal administration of these two compounds, in comparison with GA, greatly regressed breast tumor growth and metastasis in a SCID mouse model bearing labeled MDA-MB-231 cells.

Abstract 2887: Bispecific antibody drug conjugates targeting CD7 and CD33 for the treatment of acute myeloid leukemia

Abstract Phenotyping of AML patient samples shows that CD33, a myeloid cell marker, and CD7, a lymphoid cell marker, are specifically co-expressed in 25% of AML cases. Development of a bispecific antibody drug conjugate (ADC) dependent on co-expression of CD7 and CD33 may lead to an ADC with better tumor-cell selectivity than the anti-CD33 ADC, gemtuzumab ozogamicin. Anti-CD7 and -CD33 Fabs were expressed in HEK293 cells, and affinities assessed using surface plasmon resonance. Chemically cross-linked anti-CD7, anti-CD33 Bi-Fabs were produced using click chemistry via an individual cysteine residue in each CH1 domain. Bi-Fabs were conjugated to valine-citrulline-monomethylauristatin E (vcMMAE) and maleimidocaproyl monomethylauristatin F (mcMMAF) at native cysteine residues. Bi-Fab drug conjugates were assessed for cytotoxicity against HNT-34 and Kasumi-3 cells (CD7+CD33+), and assessed for selectivity against primary human T cells and myeloid progenitor cells, using monospecific Bi-Fab and gemtuzumab drug conjugates with equivalent drug-antibody ratios as controls. The activity of the lead Bi-Fab, BVX130-mcMMAF, was assessed in a subcutaneous (s.c.) xenograft model in SCID mice implanted with HNT-34 cells. Animals (n=10/group) received BVX-130-mcMMAF (10mg/kg twice weekly i.v. for 4 weeks), cytarabine at its maximum tolerated dose (20mg/kg, 5 days on 2 days off i.p. for 3 weeks), or vehicle control for 4 weeks. The wild-type Fabs had Kd values of 3.6nM (anti-CD7 Fab) and 1.6 nM (anti-CD33 Fab) for their respective antigens. The resulting anti-CD7, anti-CD33 Bi-Fab MMAE conjugate (BVX100-vcMMAE) was highly cytotoxic to HNT-34 and Kasumi-3 cells with IC50 values of 0.20±0.04nM and 0.20±0.03nM respectively, compared to IC50 values of 1.3nM and 1.2nM for gemtuzumab MMAE. BVX100-vcMMAE had 5-fold lower cytotoxicity towards Jurkat cells (CD7+CD33−) and >10-fold margins relative to MV4.11 and SHI-1 (CD7−CD33+) cells whereas corresponding homo-dimeric Bi-Fabs were non-selective. Affinity reduction of the CD7 Fab resulted in Bi-Fab drug conjugates with similar activity and improved selectivity. One such Bi-Fab, BVX130mcMMAF, had no measurable cytotoxicity to resting T-cells, and did not inhibit colony formation in a GM-CFU assay, at concentrations up to 10nM. Treatment of mice carrying s.c. HNT-34 tumors with BVX130-mcMMAF resulted in Tumor Growth Inhibition (TGI) values of 100% (98% regression, p<0.001) and 100% (97% regression, p<0.001) at days 21 and 28 respectively, with three animals tumor-free at the end of dosing. Treatment with cytarabine resulted in TGI of 100% (47% regression, p<0.001) at day 21. No adverse effects were observed in the BVX130-mcMMAF treatment group. Anti-CD7, anti-CD33 Bi-Fab drug conjugates are highly cytotoxic to CD7+CD33+ cells in vitro and in vivo, and have substantially greater selectivity than monospecific drug conjugates for such cells, supporting the development of bispecific antibody drug conjugates targeting CD7 and CD33 for the treatment of CD7+/CD33+ AML. Citation Format: Richard Bethell, Cath Eberlein, Victoria Pollard, Theonie Georgiou, George Orphanides, Lorraine Mooney, Jane Kendrew, Tiffany Daniels. Bispecific antibody drug conjugates targeting CD7 and CD33 for the treatment of acute myeloid leukemia [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2887.

Abstract 2645: Obesity promotes tumor growth, reduces breast tumor latency and correlates with neovascularization: Therapeutic and diagnostic implications

Abstract Obesity is associated with an increased risk of, and a poor prognosis for, postmenopausal (PM) breast cancer (BC). The goal of this study was to determine whether diet-induced obesity (DIO) promotes (i) shorter tumor latency, (ii) escape from tumor dormancy, and (iii) acceleration of tumor growth, angiogenesis and metastasis and to elucidate the underlying mechanism/s. Two different BC models, an orthotopic model (luciferase/MDA-MB-436 injected into the mammary fat pad (MFP) of SCID mice) and a transgenic model (doxycycline-driven conditional MMTV/TWNT/luciferase) were utilized. To model the effect of obesity on PM BC, mice were ovariectomized (OVX) before being placed on a DIO (obese) or normal chow (NC; lean) regimen. In both the orthotopic and transgenic BC models, obese mice had significantly higher tumor frequency, higher tumor volume, and lower overall survival compared to lean mice. Breast tumors in OVX/DIO mice had significantly higher microvessel density (MVD), SMA+ mature vessels and increased aggressive local invasion into the surrounding fat pad and muscle. OVX/DIO TWNT mice displayed a significantly shorter tumor latency period (7 vs. >24 wk, P=0.003) compared to OVX/NC mice. Levels of VEGF, IL-6, bFGF, Lcn-2 and MMP-9 were higher, whereas Tsp-1 levels were lower, in the sera/urine of DIO mice during tumor progression, suggesting that these circulating factors could serve as surrogate biomarkers of BC development. OVX/DIO tumors had increased expression of the angiogenic factors VEGF and bFGF and higher pVEGFR2. Systematic time-course analysis of MFP in the TWNT BC model indicated that DIO MFP achieved advanced tumor proliferative and angiogenic status at significantly earlier time points compared to NC MFP. DIO MFP also exhibited an increased incidence of extensive hyperplasia and carcinoma in situ at earlier time points, suggesting that obesity promotes tumor initiation as well as progression. Hyperplastic ducts and microscopic neoplastic lesions (≤1mm) in DIO MFP in the early phases of tumor development displayed increased Ki67 staining and were associated with significantly higher numbers of blood vessels. Collectively, our data suggest that PM obesity reduces breast tumor latency, promotes escape from tumor dormancy and initiates aggressive tumor growth via increased angiogenesis. Consistent with these findings, targeting angiogenesis via a small molecule inhibitor resulted in delayed tumor formation compared to vehicle-treated mice in the OVX/DIO TWNT BC model, suggesting that targeting angiogenesis may be a useful strategy in obesity-driven breast tumor initiation and progression. Ongoing studies in our laboratory are investigating the mechanism(s) by which an obesogenic microenvironment mediates these effects. [This work was supported by NIH RO1CA185530, the Karp Family Foundation and the Breast Cancer Research Foundation] Citation Format: Roopali Roy, Jiang Yang, Lauren Merritt, Justine Alluine, Lewis A. Chodosh, Marsha A. Moses. Obesity promotes tumor growth, reduces breast tumor latency and correlates with neovascularization: Therapeutic and diagnostic implications [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2645.

Abstract 73: Development of APG-3526 as a novel and highly efficacious MCL-1 inhibitor

Abstract Background MCL-1 is an important anti-death BCL-2 family protein and plays a key role in blocking apoptosis in cancer cells. MCL-1 gene is located in one of the most frequently amplified locus in various hematologic malignancies and solid tumors, including prostate, lung, pancreatic, breast, ovarian and cervical cancers, as well as melanoma, B-cell chronic lymphocytic leukemia (B-CLL), acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL)); moreover, MCL-1 overexpression is implicated as a resistance factor for multiple therapies including widely prescribed microtubule-targeted agents for breast cancers. Therefore, MCL-1 is an attractive therapeutic target for the treatment of cancers. In this study, we discovered the lead preclinical compound APG-3526, from its chemical synthesis optimization to potent antiproliferative and antitumor activity using multiple in vitro and in vivo xenograft models, respectively. Methods and Experiments APG-3526 was chemically synthesized by using one convergent multistep synthesis in gram scale. The binding affinity of APG-3526 was determined by a FP assay using a fluorescein labelled peptide (FAM-Bid) which binds to the MCL-1 protein leading to an increased anisotropy measured in milli-polarization (mP) values. The effect of APG-3526 on cell viability was determined using CCK-8 assay and its antitumor activities were tested in NCI-H929 and OPM-2 multiple myeloma (MM) xenograft SCID mice model (dosed i.v. at 50 mg/kg and p.o. at 25 mg/kg). Results Our biochemical study has demonstrated that APG-3526 binds to MCL-1 with a nanomolar affinity (IC50 = 7 nM). It is highly potent in inhibition of cell growth in MCL-1-dependent multiple myeloma cell lines (NCI-H929 cells: IC50 = 14 nM; OPM-2 cells: IC50 = 62 nM). Administration of APG-3526 (p.o. at 25 mg/kg, QDx14D, or i.v. at 50 mg/kg, once) achieved complete tumor regression in both NCI-929 and OPM-2 multiple myeloma xenograft models. The pharmacodynamics (PD) study using tumor samples further revealed caspase 3 activation and PARP cleavage triggered by APG-3526. MCL-1 complex MSD assay confirmed that APG-3526 disrupted MCL-1:BIM binding thus freeing BIM to initiate the apoptotic cascade. In conclusion, we have developed a novel and highly potent MCL-1 inhibitor, APG-3526, which displays clinically relevant pharmacokinetic properties and elicits potent antiproliferative and antitumor activities via disrupting MCL-1 complex and triggering caspase activation, especially in MCL-1 driven MM models. These results support APG-3526 as a promising MCL-1inhibitor for further clinical development. Citation Format: Jianyong Chen, Chengzhe Wu, Lingling Jiao, Leilei Zhao, Yunlong Zhou, Dongbo Li, Guozhi Tang, Shoulai Gu, Jing Deng, Guangfeng Wang, Douglas D. Fang, Shaomeng Wang, Dajun Yang, Yifan Zhai. Development of APG-3526 as a novel and highly efficacious MCL-1 inhibitor [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 73.

Non‑invasive monitoring of paclitaxel and lenvatinib efficacy against anaplastic thyroid cancer in orthotopic SCID mouse models using small‑animal FDG‑PET/CT.

Anaplastic thyroid carcinoma (ATC) is a rare type of thyroid carcinoma with a poor prognosis. Thus, suitable preclinical tumor models are required for the development of new ATC therapies. In the present study, orthotopic tumor xenograft models were established using ATC cell lines and SCID mice, and tumor invasion and the effects of anticancer drugs were evaluated using positron emission tomography/computed tomography (PET/CT) to repeatedly and non‑invasively monitor these models. Three ATC cell lines (8305c, 8505c, and ACT‑1) were used. Their sensitivities to two anticancer drugs (paclitaxel and lenvatinib) were investigated. The 8505c cell line was orthotopically implanted into SCID mice, which were then divided into three groups: No chemotherapy, paclitaxel (5mg/kg, administered intraperitoneally, every week), and lenvatinib (5mg/kg, oral route, every day) groups. PET/CT was performed and tumor growth and the effects of anticancer drugs based on tumor volume and fludeoxyglucose (FDG) uptake were evaluated. 8505c cells exhibited the highest sensitivity to the anticancer drugs. In mice implanted with 8505c cells, continuous increases in FDG uptake associated with tumor growth were detected on PET/CT in the group that received no chemotherapy. The tumor volume and FDG uptake increased by 91.5‑ and 2.4‑fold, respectively, within 2weeks. The increase observed in tumor volume was 26.9‑ and 12.2‑fold in the paclitaxel and lenvatinib groups, respectively, within 2weeks. Furthermore, the increase in FDG uptake was 1.8 and 1.6‑fold in the paclitaxel and lenvatinib groups, respectively, within 2weeks. In our orthotopic SCID mouse model, tumor growth and the effects of anticancer drugs were repeatedly and non‑invasively monitored using PET/CT. The present method is useful for the development of new ATC treatments.

The Antitumor Effect of Gene-Engineered Exosomes in the Treatment of Brain Metastasis of Breast Cancer.

Strategies for treating brain metastases of breast cancer have demonstrated limited efficacy due to the blood–brain barrier (BBB). Gene therapy could improve the efficacy of chemotherapeutic drugs. Exosomes derived from the mesenchymal stem cells (MSCs) are small membrane-based gene vectors that can pass through the BBB. CXCR4 is the most commonly found chemokine receptor in human cancer cells. Furthermore, the SDF-1/CXCR4 axis plays an important role in the homing of MSCs for tumor cell diffusion and metastasis. TRAIL can selectively induce apoptosis in transformed cells without significant toxic side effects in normal tissues. In this study, exosomes were isolated from MSCCXCR4+TRAIL transduced with CXCR4 and TRAIL using a lentiviral vector. Synergistic antitumor study showed that exosomeCXCR4+TRAIL exerted significant activity as a cooperative agent with carboplatin in an MDA-MB-231Br SCID mouse model, potentially engendering a novel strategy for advancing the treatment of brain metastases of breast cancer. Based on this study, further investigation of the effect of the vector on BBB and inducing apoptosis of brain tumors is warranted. In addition, the safety of the vector in animals during the treatment needs to be evaluated.

Modification of PLGA Scaffold by MSC-Derived Extracellular Matrix Combats Macrophage Inflammation to Initiate Bone Regeneration via TGF-β-Induced Protein.

The immunologic response toward chronic inflammation or bone regeneration via the accumulation of M1 or M2 macrophages after injury could determine the fate of biomaterial. Human umbilical cord mesenchymal stem cells (hUCMSCs) have a pivotal immunomodulatory property on directing macrophage behaviors. Herein, for the first time, 3D-printed poly(lactide-co-glycolide) (PLGA) scaffolds modified with hUCMSC-derived extracellular matrix (PLGA-ECM) are prepared by a facile tissue engineering technique with physical decellularization and 2.44±0.29mg cm-3 proteins immobilized on the PLGA-ECM contain multiple soluble cytokines with a sustainable release profile. The PLGA-ECM not only attenuates the foreign body response, but also improves bone regeneration by increasing the accumulation of M2 macrophages in an improved heterotopic transplantation model of SCID mice. Furthermore, the PLGA-ECM scaffolds with the knockdown of transforming growth factor-β-induced protein (TGFβI/βig-H3) demonstrate that M2 macrophage accumulation improved by the PLGA-ECM could be attributed to increasing the migration of M2 macrophages and the repolarization of M1 macrophages to M2 phenotype, which are mediated by multiple integrin signaling pathways involving in integrin β7, integrin α9, and integrin β1 in a TGFβI-dependent manner. This study presents an effective surface modification strategy of polymeric scaffolds to initiate tissue regeneration and combat inflammatory response by increasing M2 macrophage accumulation.

Accelerator-based boron neutron capture therapy for malignant glioma: a pilot neutron irradiation study using boron phenylalanine, sodium borocaptate and liposomal borocaptate with a heterotopic U87 glioblastoma model in SCID mice

Purpose: To evaluate the efficacy of boron neutron capture therapy (BNCT) for a heterotopic U87 glioblastoma model in SCID mice using boron phenylalanine (BPA), sodium borocaptate (BSH) and liposomal BSH as boron compounds at a unique, accelerator-based neutron source.Materials and methods: Glioblastoma models were obtained by subcutaneous implantation of U87 cells in the right thighs of SCID mice before administration of 350 mg/kg of BPA (BPA-group), 100 mg/kg of BSH (BSH-group) or 100 mg/kg of BSH in PEGylated liposomes (liposomal BSH-group) into the retroorbital sinus. Liposomes were prepared by reverse-phase evaporation. Neutron irradiation was carried out at a proton accelerator with a lithium target developed for BNCT at the Budker Institute of Nuclear Physics, Novosibirsk, Russian Federation. A proton beam current integral of 3 mA/h and energy of 2.05 MeV were used for neutron generation.Results: Boron compound accumulation in tumor tissues at the beginning of irradiation was higher in the BPA group, followed by the Liposomal BSH and BSH groups. Tumor growth was significantly slower in all irradiated mice from the 7th day after BNCT compared to untreated controls (p < .05). Tumor growth in all treated groups showed no large variation, apart from the Irradiation only group and the BPA group on the 7th day after BNCT. The overall trend of tumor growth was clear and the differences between treatment groups became significant from the 50th day after BNCT. Tumor growth was significantly slower in the Liposomal BSH group compared to the Irradiation only group on the 50th (p = .012), 53rd (p = .005), and the 57th (p = .021) days after treatment. Tumor growth in the Liposomal BSH group was significantly different from that in the BPA group on the 53rd day after BNCT (p = .021) and in the BSH group on the 50th (p = .024), 53rd (p = .015), and 57th (p = .038) days after BNCT. Skin reactions in the form of erosions and ulcers in the tumor area developed in treated as well as untreated animals with further formation of fistulas and necrotic decay cavities in most irradiated mice.Conclusions: We observed a tendency of BNCT at the accelerator-based neutron source to reduce or suspend the growth of human glioblastoma in immunodeficient animals. Liposomal BSH showed better long-term results compared to BPA and non-liposomal BSH. Further modifications in liposomal boron delivery are being studied to improve treatment outcomes.

In vivo anti-MUC1+ tumor activity and sequences of high-affinity anti-MUC1-SEA antibodies.

Cleavage of the MUC1 glycoprotein yields two subunits, an extracellular alpha-subunit bound to a smaller transmembrane beta-subunit. Monoclonal antibodies (mAbs) directed against the MUC1 alpha-beta junction comprising the SEA domain, a stable cell-surface moiety, were generated. Sequencing of all seven anti-SEA domain mAbs showed that they clustered into four groups and sequences of all groups are presented here. mAb DMB5F3 with picomolar affinity for the MUC1 SEA target was selected for further evaluation. Immunohistochemical staining of a series of malignancies with DMB5F3 including lung, prostate, breast, colon, and pancreatic carcinomas revealed qualitative and qualitative differences between MUC1 expression on normal versus malignant cells: DMB5F3 strongly stained malignant cells in a near-circumferential pattern, whereas MUC1 in normal pancreatic and breast tissue showed only weak apical positivity of ductal/acinar cells. Humanized chimeric DMB5F3 linked to ZZ-PE38 (ZZ IgG-binding protein fused to Pseudomonas exotoxin) induced vigorous cytotoxicity of MUC1+ malignant cells in vitro. The intensity of cell killing correlated with the level of MUC1 expression by the target cell, suggesting a MUC1 expression threshold for cell killing. MUC1+ Colo357 pancreatic cancer cells xenotransplanted into nude and SCID mice models were treated with the chDMB5F3:ZZ-PE38 immunocomplex. In both transplant models, chDMB5F3:ZZ-PE38 exhibited significant in vivo anti-tumor activity, suppressing up to 90% of tumor volume in the SCID model compared with concomitant controls. The efficacy of chDMB5F3:ZZ-PE38 immunotoxin in mediating tumor killing both in vitro and in vivo strongly suggests a clinical role for anti-MUC1 SEA antibody in the treatment of MUC1-expressing malignancies.

Abstract B14: Natural killer immune response is promoted by the treatment of B-cell lymphoma cancer cells with membrane ionophores

Abstract Introduction: Over the last two decades, very compelling clinical and experimental evidences have accelerated the use of natural killer (NK) cells’ properties for the recognition and eradication of hematologic malignancies. NK cells mediate antitumor killing responses via antibody-dependent cell-mediated cytotoxicity (ADCC). In order to accelerate and sustain NK antitumor immunity, antibodies targeting antigens expressed on the membrane surface of cancer cells are used as therapeutic interventions. Here, we investigated the antitumor benefit of the combination of the therapeutic anti-CD20 antibody, rituximab, with shortlisted membrane ionophores to overcome the resistance of B-cell lymphomas toward anti-CD20 targeted monoclonal antibodies. Methods: Flow cytometry was used to quantify cell surface protein levels and the rate of tumor cell death. Serum and PBMCs of human healthy donors were used as source of complement and natural killer cells, respectively. ADCC, complement-dependent cytotoxicity (CDC), and degranulation assays were used to assess the sensitivity of NHL cell lines and primary cancer cells upon treatment with ionophores plus rituximab. RNA-seq and qPCR were used to identify and confirm the deferentially expressed genes upon ionophores treatment. GSEA, an online resource of the Broad Institute (Boston, MA), was used to screen the differentially expressed genes and major signaling pathways. Results: Our data show that the sublethal doses of these ionophores (&amp;lt; 0.5uM) greatly increased surface CD20 (protein target for rituximab therapy) levels onB-cell lymphoma (Burkitt and DLBCL) cell lines. B-cell lymphoma cell death induced by rituximab, either mediated by complement or NK cell cytotoxicities, was significantly increased upon treatment with ionophores. These in vitro effects have been confirmed in xenograft model in SCID mice. While rituximab treatment alone slightly delayed tumor growth, the combination of rituximab with ionophores caused a dramatic decrease (up to 3x) of tumor size. The anticipated signaling pathways influencing NK cell activity are currently being interrogated; transcriptomic analysis combined with GSEA will allow to identify markedly altered pathways that are potentially closely involved in enhancing the sensitivity of rituximab-treated B-cell lymphoma cells. Summary: The data indicate that ionophores are promising therapeutic agents that could strongly enhance CD20 expression on the surface of target tumor cells. When combined with therapeutic anti-CD20 monoclonal antibodies, impressive high NK cell-mediated cytotoxicity is demonstrated that potentially offsets the B lymphoma resistance to R-CHOP regimen. These results also support the clinical development of these molecules as adjuvant to existing cancer immunotherapies. Support: National Science Centre (NCN, Poland), ID: 2016/23/B/NZ5/02622; Ministry of Science and Higher Education in Poland, ID: DI2014007344 and iONKO grant. Citation Format: Abdessamad Zerrouqi, Anna Torun, Nina Miazek, Zofia Pilch, Jakub Golab, Beata Pyrzynska. Natural killer immune response is promoted by the treatment of B-cell lymphoma cancer cells with membrane ionophores [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2019 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(3 Suppl):Abstract nr B14.

Coxiella effector protein CvpF subverts RAB26-dependent autophagy to promote vacuole biogenesis and virulence

ABSTRACT Coxiella burnetii, the etiological agent of the zoonosis Q fever, replicates inside host cells within a large vacuole displaying autolysosomal characteristics. The development of this compartment is mediated by bacterial effectors, which interfere with a number of host membrane trafficking pathways. By screening a Coxiella transposon mutant library, we observed that transposon insertions in cbu0626 led to intracellular replication and vacuole biogenesis defects. Here, we demonstrate that CBU0626 is a novel member of the Coxiella vacuolar protein (Cvp) family of effector proteins, which is translocated by the Dot/Icm secretion system and localizes to vesicles with autolysosomal features as well as Coxiella-containing vacuoles (CCVs). We thus renamed this effector CvpF for Coxiella vacuolar protein F. CvpF specifically interacts with the host small GTPase RAB26, leading to the recruitment of the autophagosomal marker MAP1LC3B/LC3B (microtubule associated protein 1 light chain 3 beta) to CCVs. Importantly, cvpF::Tn mutants were highly attenuated compared to wild-type bacteria in the SCID mouse model of infection, highlighting the importance of CvpF for Coxiella virulence. These results suggest that CvpF manipulates endosomal trafficking and macroautophagy/autophagy induction for optimal C. burnetii vacuole biogenesis. Abbreviations: ACCM: acidified citrate cystein medium; AP: adaptor related protein complex; CCV: Coxiella-containing vacuole; Cvp: Coxiella vacuolar protein; GDI: guanosine nucleotide dissociation inhibitor; GDF: GDI dissociation factor; GEF: guanine exchange factor; LAMP1: lysosomal associated membrane protein 1; MAP1LC3B/LC3B: microtubule associated protein 1 light chain 3 beta; MTORC1: mechanistic target of rapamycin kinase MTOR complex 1; PBS: phosphate-buffered saline; PMA: phorbol myristate acetate; SQSTM1/p62: sequestosome 1; WT: wild-type.

Inhibition of growth of Asian keloid cells with human umbilical cord Wharton\u2019s jelly stem cell-conditioned medium

BackgroundKeloid formation occurs in Caucasian, African, and Asian populations and is a severe psychosocial burden on patients. There is no permanent treatment for this problem as its pathogenesis is not properly understood. Furthermore, differences in keloid behavior between ethnic groups are not known. It has been hypothesized that keloids behave like benign tumors because of their uncontrolled growth. The present study evaluated the tumoricidal properties of human Wharton’s jelly stem cell-conditioned medium (hWJSC-CM) on fresh Asian keloid cells (AKCs).MethodsHuman Wharton’s jelly stem cells (hWJSCs) and AKCs were isolated based on our previous methods. hWJSCs and human skin fibroblasts (HSF) (controls) were used to collect hWJSC-CM and HSF-conditioned medium (HSF-CM). AKCs were treated with hWJSC-CM and HSF-CM in vitro and in vivo in a human keloid xenograft SCID mouse model. The inhibitory effect of hWJSC-CM on AKCs was tested in vitro using various assays and in vivo for attenuation/abrogation of AKC tumors created in a xenograft mouse model.ResultsqRT-PCR analysis showed that the genes FN1, MMP1, and VCAN were significantly upregulated in AKCs and ANXA1, ASPN, IGFBP7, LGALS1, and PTN downregulated. AKCs exposed to hWJSC-CM in vitro showed significant decreases in cell viability and proliferation, increases in Annexin V-FITC+ cell numbers, interruptions of the cell cycle at Sub-G1 and G2/M phases, altered CD marker expression, downregulated anti-apoptotic-related genes, and upregulated pro-apoptotic and autophagy-related genes compared to controls. When AKCs were administered together with hWJSC-CM into immunodeficient mice there were no keloid tumors formed in 7 mice (n = 10) compared to the untreated control mice. When hWJSC-CM was injected directly into keloid tumors created in mice there were significant reductions in keloid tumor volumes and weights in 30 days.ConclusionshWJSC-CM inhibited the growth of AKCs in vitro and in xenograft mice, and it may be a potential novel treatment for keloids in the human. The specific molecule(s) in hWJSC-CM that induce the anti-keloid effect need to be identified, characterized, and tested separately in larger preclinical and clinical studies.

An Efficient Photodynamic Therapy Treatment for Human Pancreatic Adenocarcinoma.

To date, pancreatic adenocarcinoma (ADKP) is a devastating disease for which the incidence rate is close to the mortality rate. The survival rate has evolved only 2–5% in 45 years, highlighting the failure of current therapies. Otherwise, the use of photodynamic therapy (PDT), based on the use of an adapted photosensitizer (PS) has already proved its worth and has prompted a growing interest in the field of oncology. We have developed a new photosensitizer (PS-FOL/PS2), protected by a recently published patent (WO2019 016397-A1, 24 January 2019). This photosensitizer is associated with an addressing molecule (folic acid) targeting the folate receptor 1 (FOLR1) with a high affinity. Folate binds to FOLR1, in a specific way, expressed in 100% of ADKP or over-expressed in 30% of cases. The first objective of this study is to evaluate the effectiveness of this PS2-PDT in four ADKP cell lines: Capan-1, Capan-2, MiapaCa-2, and Panc-1. For this purpose, we first evaluated the gene and protein expression of FOLR1 on four ADKP cell lines. Subsequently, we evaluated PS2’s efficacy in our cell lines and we assessed the impact of PDT on the secretome of cancer cells and its impact on the immune system. Finally, we evaluate the PDT efficacy on a humanized SCID mouse model of pancreatic cancer. In a very interesting way, we observed a significant increase in the proliferation of activated-human PBMC when cultured with conditioned media of ADKP cancer cells subjected to PDT. Furthermore, to evaluate in vivo the impact of this new PS, we analyzed the tumor growth in a humanized SCID mice model of pancreatic cancer. Four conditions were tested: Untreated, mice (nontreated), mice with PS (PS2), mice subjected to illumination (Light only), and mice subjected to illumination in the presence of PS (PDT). We noticed that the mice subjected to PDT presented a strong decrease in the growth of the tumor over time after illumination. Our investigations have not only suggested that PS2-PDT is an effective therapy in the treatment of PDAC but also that it activates the immune system and could be considered as a real adjuvant for anti-cancer vaccination. Thus, this new study provides new treatment options for patients in a therapeutic impasse and will provide a new arsenal in the fight against PDAC.

Targeting MAP3K19 prevents human lung myofibroblast activation both in vitro and in a humanized SCID model of idiopathic pulmonary fibrosis

Idiopathic Pulmonary Fibrosis (IPF) is a disease with a devastating prognosis characterized by unrelenting lung scarring. Aberrant activation of lung fibroblasts is a key feature of this disease, yet the key pathways responsible for this are poorly understood. Mitogen-activated protein kinase, kinase, kinase- 19 (MAP3K19) was recently shown to be upregulated in IPF and this MAPK has a key role in target gene transcription in the TGF-β pathway. Herein, we further investigate the role of MAP3K19 in cultured normal and IPF fibroblasts and in a humanized SCID mouse model of IPF employing both short interfering (si) RNA and novel small-molecule inhibitors directed at this kinase. Targeting MAP3K19 had significant inhibitory effects on the expression of both alpha smooth muscle actin and extracellular matrix in cultured human IPF fibroblasts. Quantitative protein and biochemical assays, as well as histological analysis, showed that MAP3K19 was required for the development of lung fibrosis in SCID mice humanized with IPF lung fibroblasts. MAP3K19 was required for IPF myofibroblast differentiation, and targeting its activity attenuated the profibrotic activity of these cells both in vitro and in an adoptive transfer SCID model of pulmonary fibrosis.

Targeting Myeloma Cell Metabolism Via Disruption of the Lnc-17-92 Transcriptional Program: Druggable New Vulnerability in Multiple Myeloma

Long noncoding RNAs (lncRNA) are major regulators of chromatin dynamics and gene expression. We have recently performed deep RNA sequencing of CD138+ cells from 360 uniformly-treated, newly-diagnosed multiple myeloma (MM) patients (IFM/DFCI 2009) and described the lncRNA landscape and their role as independent risk predictors for clinical outcome in MM. Moreover, we have identified one of these lncRNAs - lnc-17-92 - as an independent risk predictor highly correlating with EFS and OS in newly-diagnosed MM providing rationale to define its molecular role in MM. Lnc-17-92 is generated at MIR17HG gene locus and is known for being involved in the biogenesis of miR-17-92 cluster of microRNAs. We here establish, for the first time, role of this transcript as a lncRNA with microRNA-independent function and molecular and biological implications in MM. Having confirmed its expression in MM cell lines and primary MM cells, we have utilized antisense oligonucleotides (n=3) to suppress lnc-17-92 expression in large panel of human MM cell lines (HMCLs) (n=12) and primary patient MM cells (n=13). Lnc-17-92 inhibition impaired MM cell proliferation leading to apoptotic cell death. This inhibitory effect was not rescued by ectopic expression of miR-17-92 microRNAs, confirming independent activity of lnc-17-92 on MM cell growth and viability. The microRNA-independent role of lnc-17-92 in transcriptional control was further confirmed using DROSHAKOcells. Analysis of transcriptomic changes after lnc-17-92 modulation in HMCLs and primary MM cells identified bona fide transcriptional targets of lnc-17-92. Using two independent MM RNA-seq datasets, we observed high correlation (R&amp;gt; 0.4) between lnc-17-92 expression and the expression of 12 of the transcriptional targets identified above. Interestingly, these genes were significantly enriched within metabolic pathways, suggesting an unexplored role for lnc-17-92 in MM cell metabolism. Further analysis using an RNAi-based loss-of-function screening in 3 HMCLs revealed Acetyl-CoA Carboxylase Alpha (ACC1) as a novel myeloma vulnerability. ACC1 encodes the limiting enzyme in the de novo lipogenesis pathway. Analysis of incorporation of C14-radiolabeled glucose into lipids in MM cells revealed that inhibition of ACC1 or lnc-17-92 strongly inhibited de novo lipogenesis in HMCLs and in primary MM cells. We have used ACC1 conditional KD MM cells expressing IPTG-inducible ACC1 shRNAs and confirmed significant role of ACC1 in MM cell growth and survival, both in vitro and in vivo in SCID mice model. Importantly, supplementation of palmitate, the main downstream product of ACC1 activity, significantly reverses the growth inhibitory effect of either ACC1 or lnc-17-92 suppression in MM cells. These data suggest an important role for lipogenesis pathway on lnc-17-92-promoted MM cell growth. We have further investigated mechanism by which lnc-17-92 may exert its transcriptional control. Protein-RNA pulldown assay established MYC as interacting partner of lnc-17-92. This interaction was confirmed by immunoprecipitation of MYC-bound RNA followed by qRT-PCR with specific primers for detection of lnc-17-92. ChIP-seq analysis revealed a direct binding of MYC at regulatory regions of ACC1 in MM.1S cells; these data were corroborated by the decreased ACC1 expression observed in MYC KD MM cells. Taken together, these data suggest that lnc-17-92 may function as a scaffold between MYC and the E-box motifs present on ACC1 intronic sequences, facilitating MYC binding and its transcriptional activity on ACC1. Finally, for translational application, we have pre-clinically investigated ND-646, a clinically applicable small molecule inhibitor of ACC1. Analysis of incorporation of C14-radiolabeled glucose into lipids confirmed its effect on lipogenesis in MM, which was associated with a significant in vitro growth inhibitory activity in large panel of HMCLs and primary patient MM cells. In vivo studies in murine model of human MM, using this oral agent, are ongoing and will be presented. In conclusion, we here report for the first time the microRNA-independent role of lnc-17-92 in MM pathobiology with direct impact on transcriptional control of lipogenesis. The availability of oral inhibitor of this pathway may allow the clinical application of this unique targeted therapy in MM. Disclosures Anderson: Janssen: Other: Advisory Board; Gilead Sciences: Other: Advisory Board; OncoPep: Other: Scientific founder ; Sanofi-Aventis: Other: Advisory Board; C4 Therapeutics: Other: Scientific founder . Munshi:Abbvie: Consultancy; Adaptive: Consultancy; Amgen: Consultancy; Celgene: Consultancy; Janssen: Consultancy; Adaptive: Consultancy; Oncopep: Consultancy; Celgene: Consultancy; Takeda: Consultancy; Janssen: Consultancy; Oncopep: Consultancy; Takeda: Consultancy; Amgen: Consultancy; Abbvie: Consultancy.

Development of an Immunotherapeutic Monoclonal Antibody Recognizing DKK1-HLA-A2 Complex to Treat Human Hematologic Malignancies

Immunotherapy is a promising option for cancer treatment. Our previous studies demonstrated that DKK1 is widely expressed by various tumor cells including multiple myeloma (MM) and other hematological malignancies but not normal tissues, and DKK1 peptide (such as P20 and P66v, which bind with HLA-A2 molecule) specific cytotoxic T cells specifically kill myeloma and other cancer cells that express DKK1 and HLA-A2, but not HLA-A2+ normal cells, indicating that DKK1+ tumor cells naturally express these peptides, in the context of HLA-A2 molecules, on their surface. To develop cancer therapeutic antibodies, DKK1 peptide P20-HLA-A2 monomer was synthesized and used to immunize mice. Hybridomas secreting monoclonal antibodies (mAbs) recognizing soluble and cell surface-expressed DKKl P20-HLA-A2 complex were obtained and analyzed. The mAbs bind specifically with DKK1-expressing, HLAA2+ cancer cells but not DKK1-expressing, HLA-A2- cancer cells or DKK1- HLA-A2+ normal blood cells. The mAbs exhibited potent in vitro tumoricidal activity on HLA-A2+DKK1+ U266 multiple myeloma cells, HLA-A2+DKK1+ PC-3 prostate cancer cells and T2 cells loaded with DKK1-P20 peptides. Our results also showed that the anti-DKK1-HLA-A2 mAbs effectively lysed cancer cells via antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC), which were correlated with and dependent on the surface expression of DKK1-HLA-A2 complex on cancer cells. Furthermore, anti-DKK1/HLA-A2 mAbs were also active and therapeutic in vivo. In MM xenografted SCID mouse model, the mAbs were able to eradicate U266 MM cells and more than 60% of mAb-treated mice survived for 3 months, while control mice all died within 2 months. Toxicity and safety assay in MM xenografted A2-SCID mouse model showed that the mAbs had no significant negative effects on different normal tissues. Therefore, these results support clinical development of anti- DKK1-HLA-A2 mAbs as therapeutic agents to treat hematological malignancies and possibly solid tumors that express surface DKK1-HLA-A2. Disclosures No relevant conflicts of interest to declare.

STEM-21. INVESTIGATING DOT1L AS AN EPIGENETIC VULNERABILITY IN BRAIN TUMOR STEM CELLS

Abstract Glioblastoma (GBM), the most common and aggressive primary adult brain cancer, is thought to be driven by a small subpopulation of brain tumor stem cells (BTSCs). BTSCs exhibit shared properties with normal stem cells such as self-renewal and multilineage differentiation. These stem cell properties have been proposed to underlie GBM tumorigenicity, treatment evasion and contribute to tumor heterogeneity. To investigate the biology underlying the stem cell properties of GBM, we compared gene essentiality profiles for a panel of BTSCs, fetal neural stem cells and non-GBM cell lines using a CRISPR Cas9 knockout library. Interestingly, from these screens, we identified the histone methyltransferase disrupter of telomeric silencing-1-like (DOT1L) as an essential gene for the growth of BTSCs and fetal neural stem cells but not for non-GBM cell lines. DOT1L is the only known histone methyltransferase responsible for histone 3 lysine 79 methylation, an epigenetic mark associated with active gene transcription. The role of this epigenetic regulator in BTSCs was investigated in depth using EPZ-5676, a clinically relevant small molecule inhibitor. Short-term treatment with EPZ-5676 in BTSCs showed minimal effects on cell viability but led to striking morphological changes, increased neuronal and astrocytic differentiation and a reduction in self-renewal. Longer treatment periods with EPZ-5676 led to a decrease in BTSC proliferation and an increase in apoptosis. Furthermore, BTSCs pretreated with EPZ-5676 led to slowed orthotopic tumor growth and improved overall survival in a SCID mouse model. Overall, these findings suggest DOT1L epigenetically regulates GBM stem cell properties and tumor growth. We are further investigating the mechanisms underlying DOT1L regulation of gene expression in BTSCs with the goal of improving the field’s understanding of epigenetics and the therapeutic implications of targeting epigenetic processes in GBM.

Prohibitin (PHB) expression is associated with aggressiveness in DLBCL and flavagline-mediated inhibition of cytoplasmic PHB functions induces anti-tumor effects

BackgroundDiffuse large B-cell lymphomas (DLBCLs) are aggressive lymphomas accounting for approximately a third of non-Hodgkin lymphomas. Prohibitin 1 (PHB1) and prohibitin 2 (PHB2) are scaffold proteins that promote mitochondria homeostasis and consequently cell survival, but biological functions of cytoplasmic PHBs remain largely unknown in DLBCL.MethodsPHB expression was analyzed in 82 DLBCL biopsies and five DLBCL cell lines by immunohistochemistry (IHC) and Western blotting. Pharmacological inhibition of PHB using the synthetic flavagline FL3 was realized in vitro to gain insight PHB cellular functions. Effects of FL3 on DLBCL cell line viability, apoptosis, C-Raf-ERK–MNK–eIF4E signaling pathway and eIF4F complex formation and activity were evaluated by XTT assay, annexin V-FITC/PI dual staining and Western blotting respectively. Subcutaneous DLBCL xenograft model in SCID mice was also performed to determine in vivo FL3 effect.ResultsAs in DLBCL cell lines, PHB1 and PHB2 were expressed in germinal center B-cell–like (GCB) and activated B-cell–like (ABC) subtypes. In patient samples, high PHB levels were associated with higher serum LDH (PHB1 and PHB2), IPIaa (PHB2), and Ki-67 (PHB2) expression. Higher PHB1 expression tends to be associated with shorter event-free survival (EFS) in patients, especially in male patients. FL3 induced apoptosis of DLBCL cell lines that was associated with inhibition of the ERK-MNK-eIF4E signaling pathway, including aggressive double/triple-hit DLBCL cell lines. This resulted in altered eIF4F complex formation and activity leading to a reduction of Bcl-2 and c-Myc expression levels. Moreover, FL3 strongly downregulated DLBCL cellular levels of Akt protein and AKT mRNA. FL3 antitumor activity was also confirmed in vivo in a murine xenograft model.ConclusionOur data indicate that PHB overexpression is associated with markers of tumor aggressiveness in DLBCL, and that targeting PHBs may be a therapeutic option, notably in aggressive subtypes.

Baricitinib reverses HIV-associated neurocognitive disorders in a SCID mouse model and reservoir seeding in vitro

BackgroundSince HIV-associated neurocognitive disorders (HANDs) occur in up to half of HIV-positive individuals, even with combined antiretroviral therapy (cART), adjunctive therapies are needed. Chronic CNS inflammation contributes to HAND and HIV encephalitis (HIVE). Baricitinib is a JAK 1/2 inhibitor approved in the USA, EU, and Japan for rheumatoid arthritis, demonstrating potent inhibition of IL-6, D-dimer, CRP, TNF-α, IFN-α/β, and other pro-inflammatory cytokines.MethodsOur modified murine HAND model was used to evaluate the ability of baricitinib to cross the blood-brain barrier (BBB) and modulate monocyte/macrophage-driven HAND. Severity of HAND was measured by assessing cognitive performance of low- and high-dose baricitinib treated versus untreated HAND mice. The severity of brain neuroinflammation was evaluated in these mouse groups after flow cytometric analyses. We also assessed the ability of baricitinib to block events in myeloid and lymphoid cells in vitro that may undergird the persistence of HIV in the central nervous system (CNS) in primary human macrophages (Mϕ) and lymphocytes including HIV replication, HIV-induced activation, reservoir expansion, and reservoir maintenance.ResultsIn vivo, both doses of 10 and 50 mg/kg qd baricitinib crossed the BBB and reversed behavioral abnormalities conferred by HIV infection. Moreover, baricitinib significantly reduced HIV-induced neuroinflammation marked by glial activation: activated microglia (MHCII+/CD45+) and astrogliosis (GFAP). Baricitinib also significantly reduced the percentage of p24+ human macrophages in mouse brains (p < 0.05 versus HAND mice; t test). In vitro, baricitinib significantly reduced markers of persistence, reservoir size, and reseeding in Mϕ.ConclusionThese results show that blocking the JAK/STAT pathway reverses cognitive deficits and curtails inflammatory markers in HAND in mice. Our group recently reported safety and tolerability of ruxolitinib in HIV-infected individuals (Marconi et al., Safety, tolerability and immunologic activity of ruxolitinib added to suppressive ART, 2019), underscoring potential safety and utility of JAK inhibitors for additional human trials. The data reported herein coupled with our recent human trial with JAK inhibitors provide compelling preclinical data and impetus for considering a trial of baricitinib in HAND individuals treated with cART to reverse cognitive deficits and key events driving viral persistence.

Astrocytes enhance glioblastoma growth.

Glioblastoma (GBM) is a deadly disease with a need for deeper understanding and new therapeutic approaches. The microenvironment of glioblastoma has previously been shown to guide glioblastoma progression. In this study, astrocytes were investigated with regard to their effect on glioblastoma proliferation through correlative analyses of clinical samples and experimental in vitro and in vivo studies. Co-culture techniques were used to investigate the GBM growth enhancing potential of astrocytes. Cell sorting and RNA sequencing were used to generate a GBM-associated astrocyte signature and to investigate astrocyte-induced GBM genes. A NOD scid GBM mouse model was used for in vivo studies. A gene signature reflecting GBM-activated astrocytes was associated with poor prognosis in the TCGA GBM dataset. Two genes, periostin and serglycin, induced in GBM cells upon exposure to astrocytes were expressed at higher levels in cases with high "astrocyte signature score". Astrocytes were shown to enhance glioblastoma cell growth in cell lines and in a patient-derived culture, in a manner dependent on cell-cell contact and involving increased cell proliferation. Furthermore, co-injection of astrocytes with glioblastoma cells reduced survival in an orthotopic GBM model in NOD scid mice. In conclusion, this study suggests that astrocytes contribute to glioblastoma growth and implies this crosstalk as a candidate target for novel therapies.