DJ4 Targets Rho-associated Protein Kinase Pathway and Attenuates Disease Progression in Pre-clinical Murine Models of Acute Myeloid Leukemia

Introduction: The poor prognosis of acute myeloid leukemia (AML) and the highly heterogenous nature of the disease motivates targeted gene therapeutic investigations. Rho-associated protein kinases (ROCKs) are crucial for various actin cytoskeletal changes, which have established malignant consequences in various cancers, yet are still not being successfully utilized clinically towards cancer treatment. ROCK 1 and 2 overexpression has been linked to AML cell lines and overall survival of AML patients. This work reports the considerable therapeutic efficacy of the ROCK inhibitor DJ4 in both in vitro and in vivo preclinical models of AML to highlight the potential of this class of inhibitors. Experimental Design: The cytotoxic and pro-apoptotic activities of DJ4 for primary human AML samples and human AML cell lines was determined by cell viability (MTS), colony forming assays, and Annexin V assays. Immunoblot analysis was used to detect phosphorylation of downstream substrates of ROCK. To assess the preclinical therapeutic efficacy, the luciferase-expressing human AML cell line OCIAML-3-YFP-Luc was injected subcutaneously (SC) and intravenously (IV) into NRG-S mice. Mice were treated through an intraperitoneal (IP) injection with either vehicle control or DJ4 (10 mg/kg) for 3 weeks. Modified AML cell lines, OCI-AML3-YFP-Luc and MV4-11-Luc2-EGFP, were also treated with their respective IC50 dose of DJ4 or with vehicle control for 24 h and then administered via IV into NRG-S mice and the survival advantage was monitored over time without further treatment. Disease progression was tracked in mice studies by flow cytometry analysis and examining the survival, bioluminescent signal, tumor volume, and tumor weights of the animals over time. Results: DJ4 induced cytotoxic and pro-apoptotic effects, within the micromolar range and in a dose-dependent manner, in human AML cell lines (IC50: 0.05-1.68 μM) and primary patient cells (IC50: 0.264-13.43 μM) that harbored various mutations; however, normal hematopoietic cells are largely spared (Figures A-C). Treatment of DJ4 demonstrated ~5-fold selectivity towards AML patient samples relative to the CB-MNCs of healthy donors (IC50= 25 μM, Figure B). Representative flow cytometry plots of the Annexin V assay with AML primary cells (Figure C) depicted the increase in the apoptotic populations as a result of treatment with increasing concentrations of DJ4. ROCK inhibition by DJ4 disrupts the phosphorylation of downstream targets, myosin light chain (MLC2), and myosin binding subunit of MLC phosphatase (MYPT) in OCI-AML3 and MV4-11, yielding a potent yet selective treatment response at micromolar concentrations, 0.02 to 1 μM (Figure D). Analysis of mice treated with DJ4 via IP for 2.5 weeks indicated that DJ4 was well-tolerated with comparable CBC with differential and chemistry values and tissue morphology from cross-sections of the spleen, liver, lung, and kidney relative to the vehicle treated group. Mice that were IV or SC injected with OCIAML3-YFP-Luc and treated via IP with DJ4 exhibited an increase in overall survival and reduction in disease progression relative to the vehicle treated control mice (Figure E). Towards the end of the study, the OCIAML3-YFP-Luc SC injected mice exhibited at least a 3-fold reduction in the bioluminescent signal, ~4-fold decrease in tumor volume, and the tumor weight was significantly reduced by ~3-fold among the DJ4 treated group relative to the control (Figure E). Mice that were IV administered DJ4 pretreated AML cells also exhibited a decreased bioluminescent signal, increased survival of 10 and 20 days, and a significant reduction in the percent of human CD45 cells in the bone marrow or spleen, indicating a diminished tumor burden (Figure F). Conclusion: The observed potency of DJ4 towards various AML cell lines and primary samples with a diverse set of mutations suggests that this is a promising candidate to be incorporated into the standard AML regimen to help many different subsets of AML patients. Therapeutics that induce apoptosis have been shown to be promising candidates toward overcoming chemoresistance and to work well with appropriate combinations of standard of care drugs. This work highlights the potential of targeting the Rho-ROCK pathway to improve the prognosis of AML and the need for the future development of chemotherapeutics that are both less toxic and more effective. Figure 1 Figure 1. Disclosures Claxton: Astellas: Other: Clinical Trial; Novartis: Research Funding; Astex: Research Funding; Cyclacel: Research Funding; Daiichi Sankyo: Research Funding; Incyte: Research Funding.

Quantification of bacteria by in vivo bioluminescence imaging in comparison with standard spread plate method and reverse transcription quantitative PCR (RT-qPCR)

In vivo bioluminescence imaging (BLI) offers a unique opportunity to analyze ongoing bacterial infections qualitatively and quantitatively in intact animals over time, leading to a reduction in the number of animals needed for a study. Since accurate determination of the bacterial burden plays an essential role in microbiological research, the present study aimed to evaluate the ability to quantify bacteria by non-invasive BLI technique in comparison to standard spread plate method and reverse transcription quantitative PCR (RT-qPCR). For this purpose, BALB/c mice were intranasally infected with 1 × 105 CFU of bioluminescent Streptococcus pneumoniae A66.1. At day 1 post-infection, the presence of S. pneumoniae in lungs was demonstrated by spread plate method and RT-qPCR, but not by in vivo BLI. However, on the second day p.i., the bioluminescent signal was already detectable, and the photon flux values positively correlated with CFU counts and RT-qPCR data within days 2–6. Though in vivo BLI is valuable research tool allowing the continuous monitoring and quantification of pneumococcal infection in living mice, it should be kept in mind that early in the infection, depending on the infective dose, the bioluminescent signal may be below the detection limit.

Penentuan Cepat Toksisitas Logam Perak Menggunakan Bioluminesen Bakteri Laut Aliivibrio fischeri, Beijerinck, 1889 (Gammaproteobacteria: Vibrionaceae)

Rapid determination of toxicity based on changes in the bioluminescent signal of marine bacterium Aliivibrio fischeri (A. fischeri) to evaluate toxicity of Ag(I) has been successfully developed. Assessment of toxicity was designed using inhibition of bioluminescent signal from A. fischeri bacteria, which was exposed with toxic material of Ag(I). This metal ion was utilized as a model of toxic material to evaluate the effects of cytotoxicity on bacteria cell. Measurement of bioluminescent were taken based on differences in bacterial cell signals before and after exposure to Ag(I) ion at an emission wavelength of 488±2 nm. The concentration of bacterial cell was used to assess the toxicity of Ag(I) at optical density (OD600 nm) of 0.78 Abs. The results found that the linear response of Ag toxicity was in the range of 0.05–10 mg/L, with EC50% of 8.42 mg/L for 4 minutes. The repeatability value within the relative standard deviation (RSD) was 2.5-4.7% (n=8). The results demonstrated that the marine bacteria of A. fischeri have good potential to evaluate toxicity of toxic material in environmental samples. Penentuan cepat toksisitas berdasarkan perubahan sinyal bioluminesen bakteri laut Aliivibrio fischeri (A. fischeri) untuk mengevaluasi toksisitas logam perak (Ag(I)) telah sukses dikembangkan. Penilaian toksisitas didesain berdasarkan penghambatan sinyal bioluminesen bakteri A. fischeri oleh bahan toksik. Ion logam Ag(I) digunakan sebagai model bahan toksik untuk menilai efek sitotiksisitas pada sel bakteri. Pengukuran bioluminesen diambil berdasarkan perbedaan sinyal sel bakteri sebelum dan sesudah diekspos pada ion logam Ag(I) pada panjang gelombang emisi 488±2 nm. Konsentrasi sel bakteri yang digunakan untuk menilai toksisitas Ag(I) pada optikal densitas 600 (OD 600 nm) = 0.78 Abs. Hasil kajian ditemukan bahwa respons linear toksisitas Ag(I) pada rentang 0.05–10 mg/L, dengan nilai EC50% sebesar 8.42 mg/L pada waktu respons 4 menit dan nilai repeatibilitas toksisitas diperoleh sebesar 2.5-4.7 % RSD (relatif standar deviasi, n=8). Hasil ini menunjukkan bahwa bakteri laut A. fischeri memiliki potensi yang baik untuk menilai toksisitas bahan toksik dalam sampel lingkungan.

Bread Feeding Is a Robust and More Physiological Enteropathogen Administration Method Compared to Oral Gavage

ABSTRACT Oral administration is a preferred model for studying infection by bacterial enteropathogens such as Yersinia spp. In the mouse model, the most frequent method for oral infection consists of oral gavage with a feeding needle directly introduced in the animal stomach via the esophagus. In this study, we compared needle gavage to bread feeding as an alternative mode of bacterial administration. Using bioluminescence-expressing strains of Yersinia pseudotuberculosis and Yersinia enterocolitica, we detected very early upon needle gavage a bioluminescent signal in the neck area together with a signal in the abdominal region, highlighting the presence of two independent sites of bacterial colonization and multiplication. Bacteria were often detected in the esophagus and trachea, as well as in the lymph nodes draining the salivary glands, suggesting that lesions made during needle introduction into the animal oral cavity lead to rapid bacterial draining to proximal lymph nodes. We then tested an alternative mode of bacterial administration using pieces of bread containing bacteria. Upon bread feeding infection, mice exhibited a stronger bioluminescent signal in the abdominal region than with needle gavage, and no signal was detected in the neck area. Moreover, Y. pseudotuberculosis incorporated in the bread is less susceptible to the acidic environment of the stomach and is therefore more efficient in causing intestinal infections. Based on our observations, bread feeding constitutes a natural and more efficient administration method which does not require specialized skills, is less traumatic for the animal, and results in diseases that more closely mimic foodborne intestinal infection.

Bread feeding is a robust and more physiological enteropathogen administration method compared to oral gavage

Oral administration is a preferred model for studying infection by bacterial enteropathogens such as Yersinia. In the mouse model, the most frequent method for oral infection consists of oral gavage with a feeding needle directly introduced in the animal stomach via the esophagus. In this study, we compared needle gavage to bread feeding as an alternative mode of bacterial administration. Using a bioluminescence-expressing strain of Yersinia pseudotuberculosis, we detected very early upon needle gavage a bioluminescent signal in the neck area together with a signal in the abdominal region, highlighting the presence of two independent sites of bacterial colonization and multiplication. Bacteria were often detected in the esophagus and trachea, as well as in the lymph nodes draining the salivary glands, suggesting that lesions made during needle introduction into the animal oral cavity lead to rapid bacterial draining to proximal lymph nodes. We then tested an alternative mode of bacterial administration using small pieces of white bread containing bacteria. Upon bread feeding infection, mice exhibited a stronger bioluminescent signal in the abdominal region as compared to needle gavage, and no signal was detected in the neck area. Moreover, Y. pseudotuberculosis incorporated in the bread is less susceptible to the acidic environment of the stomach and is therefore more efficient in causing intestinal infections. Based on our observations, bread feeding constitutes a natural and more efficient administration method which does not require specialized skills, is less traumatic for the animal, and results in diseases that more closely mimic food-borne intestinal infection.

Bioluminescent Ross River Virus Allows Live Monitoring of Acute and Long-Term Alphaviral Infection by In Vivo Imaging

Arboviruses like chikungunya and Ross River (RRV) are responsible for massive outbreaks of viral polyarthritis. There is no effective treatment or vaccine available against these viruses that induce prolonged and disabling arthritis. To explore the physiopathological mechanisms of alphaviral arthritis, we engineered a recombinant RRV expressing a NanoLuc reporter (RRV-NLuc), which exhibited high stability, near native replication kinetics and allowed real time monitoring of viral spread in an albino mouse strain. During the acute phase of the disease, we observed a high bioluminescent signal reflecting viral replication and dissemination in the infected mice. Using Bindarit, an anti-inflammatory drug that inhibits monocyte recruitment, we observed a reduction in viral dissemination demonstrating the important role of monocytes in the propagation of the virus and the adaptation of this model to the in vivo evaluation of treatment strategies. After resolution of the acute symptoms, we observed an increase in the bioluminescent signal in mice subjected to an immunosuppressive treatment 30 days post infection, thus showing active in vivo replication of remnant virus. We show here that this novel reporter virus is suitable to study the alphaviral disease up to the chronic phase, opening new perspectives for the evaluation of therapeutic interventions.

Whole-mouse in vivo bioluminescence imaging applied to drug screening against Leishmania infantum: a reliable method to evaluate efficacy and optimize treatment regimens

ABSTRACTLeishmaniasis is an important vector-borne neglected tropical disease caused by Leishmania parasites. Current anti-Leishmania chemotherapy is unsatisfactory, justifying the continued search for alternative treatment options. Herein, we propose the use of a minimally invasive bioluminescence-based murine model for preliminary in vivo screening of compounds against visceral infection by Leishmania infantum. We demonstrate that luciferase-expressing axenic amastigotes, unlike promastigotes, are highly infectious to BALB/c mice and generate a robust bioluminescent signal in the main target organs, such as the liver and spleen. Finally, we validate the use of this technique to evaluate in vivo treatment efficacy using reference drugs amphotericin B and miltefosine.

BioluminescentFrancisella tularensisSCHU-S4 enables non-invasive tracking of bacterial dissemination and the evaluation of antibiotics in an inhalational mouse model of tularemia

Bioluminescence imaging (BLI) enables real-time, non-invasive tracking of infectionin vivoand longitudinal infection studies. In this study, a bioluminescentF. tularensisSCHU S4-luxstrain was used to develop an inhalational infection model in BALB/c mice. Mice were infected intranasally and the progression of infection was monitored in real time using BLI. A bioluminescent signal was detectable from 3 days post-infection (dpi), initially in the spleen, then in the liver and lymph nodes before finally becoming systemic. The level of bioluminescent signal correlated with bacterial numbersin vivoenabling non-invasive quantification of bacterial burden in tissues. Treatment with levofloxacin (commencing at 4 dpi) significantly reduced the BLI signal. Furthermore, BLI was able to non-invasively distinguish between different levofloxacin treatment regimens and identify sites of relapse following treatment cessation. These data demonstrate that BLI and SCHU S4-luxare suitable for the study ofF. tularensispathogenesis and the evaluation of therapeutics for tularemia.

Screening of anti-mycobacterial compounds in a naturally infected zebrafish embryo model

Mycobacterium tuberculosisis a deadly human pathogen that latently infects a third of the worlds population, resulting in approximately 1.5 million deaths per year. Due to the difficulties and expense of carrying out animal drug trials using M. tuberculosis and rodents, infections of the zebrafishDanio reriowithM. marinumhave been used as a surrogate. However the methods so far described require specialised equipment and a high level of operator expertise. We investigated a natural infection model where zebrafish embryos are infected through incubation in media containingM. marinum. Using bioluminescently labelledM. marinum, we have characterised the nature of infection and established a model for interventional drug therapy. We have used a selection of traditional and experimental compounds to validate this model for anti-mycobacterial drug discovery. We observed that only three of the six treatments tested (Delamonid, SN30527 and rifampicin) retarded the growth ofM. marinumin vitro. In contrast, five of the six treatments (Pretomanid, Delamanid, SN30488, SN30527 and rifampicin) retarded the growth ofM. tuberculosisin vitro. Importantly, these same five treatments significantly reduced the bioluminescent signal from naturally infected zebrafish embryos. Overall this study has demonstrated that zebrafish embryos naturally infected with bioluminescentM. marinumM can be used for the rapid screening of anti-mycobacterial compounds with readily available equipment and limited expertise. The result is an assay that can be carried out by a wide variety of laboratories for minimal cost and without high levels of zebrafish expertise.

Development of in Vivo Models of Paediatric Burkitt Lymphoma Allowing Therapeutic Target Analysis and Drug Testing

Childhood sporadic Burkitt lymphoma (BL) is a highly aggressive mature B-cell lymphoma. Patients require intensive chemotherapy which results in prolonged periods of hospitalisation for distressing and dangerous side effects. Additionally those with refractory disease or those that relapse have an extremely poor prognosis. As a result, there is an urgent need to develop new targeted treatments that have undergone rigorous pre-clinical evaluation. The aim of this project is to develop improved models for target selection and pre-clinical validation to reduce the failure of translation of novel agents. Two models are being developed. Firstly, xenotransplantation of BL cell lines is being used to optimise transplantation protocols and to provide an orthotopic assay for in vivo functional genomic (RNAi) screening. This is currently being used in genome-wide target identification screens. Secondly, cryopreservation of viable BL blasts is allowing development of a patient-derived xenograft model, suitable for validation of candidate targets by both RNAi and pharmacological inhibition and for testing novel therapeutic agents. The sporadic BL cell lines CA46 and RAMOS have been transplanted into NOD/LtSz-scid IL-2Rg-/- (NSG) and RAG2-/-gc-/- (RAG2) mice at reducing cell doses (105, 104 and 103 cells/mouse). This approach will identify the optimal conditions for orthotopic engraftment. Prior to transplantation, cells were transduced with the lentiviral vector pSLIEW expressing green fluorescent protein (analysis and cell sorting) and firefly luciferase (in vivo bioluminescent imaging). Engraftment was then monitored in real-time using the Caliper in vivo imaging system (IVIS). CA46 and RAMOS cell lines have been transduced with SLIEW, sorted to high purity (>90%) and engrafted in both NGS and RAG2 mice. IVIS imaging of mice transplanted with CA46 cells showed that rate of engraftment was relative to cell dose. All six mice showed a bioluminescent signal over the femurs by 3 weeks post transplantation, indicative of bone marrow (BM) engraftment. Analysis of BM by flow cytometry confirmed engraftment in the two mice analysed, as determined by GFP positivity and presence of the human B-cell markers CD10, CD19 and CD20. Renal enlargement was observed in all mice analysed, with dense infiltration by mature lymphoid blasts evident microscopically. Immunohistochemistry confirmed infiltration with human CD20 positive cells. Splenic enlargement was observed in five out of six mice and engraftment was again confirmed histologically. Onset of disease was rapid. Mice were killed when they showed signs of ill health (see table). A similar pattern of engraftment was observed in NSG and RAG2 mice transplanted with RAMOS cells, although signal intensity was not relative to transplanted cell dose. Bioluminescent signal over the femur was detected in four out of six mice by 3 weeks post transplantation. NSG and RAG2 mice receiving 103 cells, with no IVIS signal, were negative for BM engraftment by flow cytometry. Renal enlargement was not observed, however focal renal lesions were evident macroscopically in three out of six mice. These lesions were composed of human CD20 positive cells. Splenic enlargement was not observed, although a degree of splenic engraftment was identified histologically. Onset of disease was also rapid. Following the initial cell line model development, we are now able to transplant the first patient-derived BL sample. If successful, primograft material will be labelled with luciferase (pSLIEW) and re-transplanted, allowing in vivo imaging. When combined with in vivo RNAi screening, this advanced pre-clinical model for target selection and validation may help reduce target drop-out during early phase clinical trials. This is a critical step, not only to avoid exposing children to ineffectual and potentially toxic drugs, but also to avoid delay in the development of new effective therapies. Abstract 5501. TableCA46DoseSpleenKidneyBMSurvival (days)RAMOSDoseSpleenKidneyBMSurvival (days) RAG2105+++++na29 RAG2105+++++31104+++++++38104-/+-/++29103+++na38103--/+-39 NSG105++na23 NSG105-+++37104++++++31104-/+-/+na30103+++++na38103-++-39 CD20 positivity for spleen and kidney; none (-), < 10% (-/+), 10 - 50% (+), > 50% (++), 100% (+++). For BM engraftment (CD10, CD19 and CD20 positive); no engraftment (-), engrafted (+), not assessed (na). Disclosures No relevant conflicts of interest to declare.