Therapeutic efficacy of RAS inhibitor trametinib using a juvenile myelomonocytic leukemia patient-derived xenograft model.

Juvenile myelomonocytic leukemia (JMML) is an aggressive pediatric leukemia with few effective treatments and poor outcomes even after stem cell transplantation, the only current curative treatment. We developed a JMML patient-derived xenograft (PDX) mouse model and demonstrated the in vivo therapeutic efficacy and confirmed the target of trametinib, a RAS-RAF-MEK-ERK pathway inhibitor, in this model. A PDX model was created through transplantation of patient JMML cells into mice, up to the second generation, and successful engraftment was confirmed using flow cytometry. JMML PDX mice were treated with trametinib versus vehicle control, with a median survival of 194 days in the treatment group versus 124 days in the control group (p = 0.02). Trametinib's target as a RAS pathway inhibitor was verified by showing inhibition of ERK phosphorylation using immunoblot assays. In conclusion, trametinib monotherapy significantly prolongs survival in our JMML PDX model by inhibiting the RAS pathway. Our model can be effectively used for assessment of novel targeted treatments, including potential combination therapies, to improve JMML outcomes.

Cmpd10357 to treat B-cell acute lymphoblastic leukemia.

B-cell acute lymphoblastic leukemia (B-ALL) is the most common type of cancer found in children. Although the overall survival rates are now >80%, 15%-20% of pediatric patients relapse, with survival rates subsequently dropping to 5%-10%. Cmpd10357, 3-amino-5-arylamino-6-chloro-N- (diaminomethylene) pyrazine-2-carboximide, is a highly potent, cell-permeant compound recently shown to have cytotoxic effects on solid tumors, including human breast cancer and high-grade gliomas, independent of their proliferative status. Cmpd10357 demonstrated concentration-dependent cytotoxicity in two human B-ALL cell lines, JM1 and Reh, at half-maximal inhibitory concentrations (IC50) of 3.2 and 3.3 µM, respectively. Cmpd10357, at a dose of 5 mg/kg, significantly prolonged survival in our B-ALL xenograft mouse model, with a median survival time of 49.0 days compared with 45.5 days in the control group (p < 0.05). The cytotoxicity of Cmpd10357 demonstrated caspase-independent, nonapoptotic cancer cell demise associated with the nuclear translocation of apoptosis-inducing factor (AIF). The cytotoxicity of Cmpd10357 in B-ALL cells was inhibited by Necrostatin-1 but not by Necrosulfonamide. These studies suggest that an AIF-mediated, caspase-independent necrosis mechanism of Cmpd10357 in B-ALL could be used in combination with traditional apoptotic chemotherapeutic agents.

A distinct subpopulation of leukemia initiating cells in acute precursor B lymphoblastic leukemia: quiescent phenotype and unique transcriptomic profile.

In leukemia, a distinct subpopulation of cancer-initiating cells called leukemia stem cells (LSCs) is believed to drive population expansion and tumor growth. Failing to eliminate LSCs may result in disease relapse regardless of the amount of non-LSCs destroyed. The first step in targeting and eliminating LSCs is to identify and characterize them. Acute precursor B lymphoblastic leukemia (B-ALL) cells derived from patients were incubated with fluorescent glucose analog 2-(N-(7-Nitrobenz-2-oxa-1, 3-diazol-4-yl) Amino)-2-Deoxyglucose (NBDG) and sorted based on NBDG uptake. Cell subpopulations defined by glucose uptake were then serially transplanted into mice and evaluated for leukemia initiating capacity. Gene expression profiles of these cells were characterized using RNA-Sequencing (RNA-Seq). A distinct population of NBDG-low cells was identified in patient B-ALL samples. These cells are a small population (1.92% of the entire leukemia population), have lower HLA expression, and are smaller in size (4.0 to 7.0 µm) than the rest of the leukemia population. All mice transplanted with NBDG-low cells developed leukemia between 5 and 14 weeks, while those transplanted with NBDG-high cells did not develop leukemia (p ≤ 0.0001-0.002). Serial transplantation of the NBDG-low mouse model resulted in successful leukemia development. NBDG-medium (NBDG-med) populations also developed leukemia. Interestingly, comprehensive molecular characterization of NBDG-low and NBDG-med cells from patient-derived xenograft (PDX) models using RNA-Seq revealed a distinct profile of 2,162 differentially-expressed transcripts (DETs) (p<0.05) with 70.6% down-regulated in NBDG-low cells. Hierarchical clustering of DETs showed distinct segregation of NBDG-low from NBDG-med and NBDG-high groups with marked transcription expression alterations in the NBDG-low group consistent with cancer survival. In conclusion, A unique subpopulation of cells with low glucose uptake (NBDG-low) in B-ALL was discovered. These cells, despite their quiescence characteristics, once transplanted in mice, showed potent leukemia initiating capacity. Although NBDG-med cells also initiated leukemia, gene expression profiling revealed a distinct signature that clearly distinguishes NBDG-low cells from NBDG-med and the rest of the leukemia populations. These results suggest that NBDG-low cells may represent quiescent LSCs. These cells can be activated in the appropriate environment in vivo, showing leukemia initiating capacity. Our study provides insight into the biologic mechanisms of B-ALL initiation and survival.

Novel Patient Metastatic Pleural Effusion-Derived Xenograft Model of Renal Medullary Carcinoma Demonstrates Therapeutic Efficacy of Sunitinib.

BACKGROUND: Renal medullary carcinoma (RMC) is a rare but aggressive tumor often complicated by early lung metastasis with few treatment options and very poor outcomes. There are currently no verified RMC patient-derived xenograft (PDX) mouse models established from metastatic pleural effusion (PE) available to study RMC and evaluate new therapeutic options. METHODS: Renal tumor tissue and malignant PE cells from an RMC patient were successfully engrafted into 20 NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG) mice. We evaluated the histopathological similarity of the renal tumor and PE PDXs with the original patient renal tumor and PE, respectively. We then evaluated the molecular integrity of the renal tumor PDXs between passages, as well as the PE PDX compared to two generations of renal tumor PDXs, by microarray analysis. The therapeutic efficacy of sunitinib and temsirolimus was tested in a serially-transplanted generation of 27 PE PDX mice. RESULTS: The pathologic characteristics of the patient renal tumor and patient PE were retained in the PDXs. Gene expression profiling revealed high concordance between the two generations of renal tumor PDXs (RMC-P0 vs. RMC-P1, r=0.865), as well as between the first generation PE PDX and each generation of the renal tumor PDX (PE-P0 vs. RMC-P0, r=0.919 and PE-P0 vs. RMC-P1, r=0.843). A low number (626) of differentially-expressed genes (DEGs) was seen between the first generation PE PDX and the first generation renal tumor PDX. In the PE-P1 xenograft, sunitinib significantly reduced tumor growth (p<0.001) and prolonged survival (p=0.004) compared to the vehicle control. CONCLUSIONS: A metastatic PE-derived RMC PDX model was established and shown to maintain histologic features of the patient cancer. Molecular integrity of the PDX models was well maintained between renal tumor and PE PDX as well as between two successive renal tumor PDX generations. Using the PE PDX model, sunitinib demonstrated therapeutic efficacy for RMC. This model can serve as a foundation for future mechanistic and therapeutic studies for primary and metastatic RMC.

MXD3 antisense oligonucleotide with superparamagnetic iron oxide nanoparticles: A new targeted approach for neuroblastoma.

Neuroblastoma (NB) is the most common extracranial solid tumor in children. The outcomes for aggressive forms of NB remain poor. The aim of this study was to develop a new molecular-targeted therapy for NB using an antisense oligonucleotide (ASO) and superparamagnetic iron oxide (SPIO) nanoparticles (NPs), as a delivery vehicle, targeting the transcription regulator MAX dimerization protein 3 (MXD3). We previously discovered that MXD3 was highly expressed in high-risk NB, acting as an anti-apoptotic factor; therefore, it can be a good therapeutic target. In this study, we developed two ASO-NP complexes using electrostatic conjugation to polyethylenimine-coated SPIO NPs and chemical conjugation to amphiphilic polymers on amine-functionalized SPIO NPs. Both ASO-NP complexes demonstrated MXD3 knockdown, which resulted in apoptosis in NB cells. ASO chemically-conjugated NP complexes have the potential to be used in the clinic as they showed great efficacy with minimum NP-associated cytotoxicity.

Novel targeted therapy for neuroblastoma: silencing the MXD3 gene using siRNA.

BackgroundNeuroblastoma is the second most common extracranial cancer in children. Current therapies for neuroblastoma, which use a combination of chemotherapy drugs, have limitations for high-risk subtypes and can cause significant long-term adverse effects in young patients. Therefore, a new therapy is needed. In this study, we investigated the transcription factor MXD3 as a potential therapeutic target in neuroblastoma.MethodsMXD3 expression was analyzed in five neuroblastoma cell lines by immunocytochemistry and quantitative real-time reverse transcription PCR, and in 18 primary patient tumor samples by immunohistochemistry. We developed nanocomplexes using siRNA and superparamagnetic iron oxide nanoparticles to target MXD3 in neuroblastoma cell lines in vitro as a single-agent therapeutic and in combination with doxorubicin, vincristine, cisplatin, or maphosphamide-common drugs used in current neuroblastoma treatment.ResultsMXD3 was highly expressed in neuroblastoma cell lines and in patient tumors that had high-risk features. Neuroblastoma cells treated in vitro with the MXD3 siRNA nanocomplexes showed MXD3 protein knockdown and resulted in cell apoptosis. Furthermore, on combining MXD3 siRNA nanocomplexes with each of the four drugs, all showed additive efficacy.ConclusionThese results indicate that MXD3 is a potential new target and that the use of MXD3 siRNA nanocomplexes is a novel therapeutic approach for neuroblastoma.

Novel Targeted Therapy for Precursor B Cell Acute Lymphoblastic Leukemia: anti-CD22 Antibody-MXD3 Antisense Oligonucleotide Conjugate.

The exponential rise in molecular and genomic data has generated a vast array of therapeutic targets. Oligonucleotide-based technologies to down regulate these molecular targets have promising therapeutic efficacy. However, there is relatively limited success in translating this into effective in vivo cancer therapeutics. The primary challenge is the lack of effective cancer cell-targeted delivery methods, particularly for a systemic disease such as leukemia. We developed a novel leukemia-targeting compound composed of a monoclonal antibody directly conjugated to an antisense oligonucleotide (ASO). Our compound uses an ASO that specifically targets the transcription factor MAX dimerization protein 3 (MXD3), which was previously identified to be critical for precursor B cell (preB) acute lymphoblastic leukemia (ALL) cell survival. The MXD3 ASO was conjugated to an anti-CD22 antibody (αCD22 Ab) that specifically targets most preB ALL. We demonstrated that the αCD22 Ab-ASO conjugate treatment showed MXD3 protein knockdown and leukemia cell apoptosis in vitro. We also demonstrated that the conjugate treatment showed cytotoxicity in normal B cells, but not in other hematopoietic cells, including hematopoietic stem cells. Furthermore, the conjugate treatment at the lowest dose tested (0.2mg/kg Ab for 6 doses - twice a week for 3 weeks) more than doubled the mouse survival time in both Reh (median survival time 20.5 vs. 42.5 days, p<0.001) and primary preB ALL (median survival time 29.3 vs. 63 days, p<0.001) xenograft models. Our conjugate that uses αCD22 Ab to target the novel molecule MXD3, which is highly expressed in preB ALL cells, appears to be a promising novel therapeutic approach.

Loss of MXD3 induces apoptosis of Reh human precursor B acute lymphoblastic leukemia cells.

MXD3 is a transcription factor that plays an important role in proliferation of human DAOY medulloblastoma cells. Here, we demonstrate that MXD3 is highly enriched in human precursor B acute lymphoblastic leukemia (preB ALL) samples compared to mobilized peripheral blood mononuclear cells, bone marrow, or hematopoietic stem cells from healthy donors. MXD3 knock-down in the preB ALL cell line Reh resulted in decreased cell numbers with no change in G0/G1, S or G2/M populations but increased apoptosis compared to control cells. Our results suggest that MXD3 is important for survival of Reh preB ALL cells, possibly as an anti-apoptotic factor.

Targeted therapy with MXD3 siRNA, anti-CD22 antibody and nanoparticles for precursor B-cell acute lymphoblastic leukaemia.

Conventional chemotherapy for precursor B-cell (preB) acute lymphoblastic leukaemia (ALL) has limitations that could be overcome by targeted therapy. Previously, we discovered a potential therapeutic molecular target, MDX3 (MAX dimerization protein 3), in preB ALL. In this study, we hypothesize that an effective siRNA therapy for preB ALL can be developed using antiCD22 antibody (αCD22 Ab) and nanoparticles. We composed nanocomplexes with super paramagnetic iron oxide nanoparticles (SPIO NPs), αCD22 Abs and MXD3 siRNA molecules based on physical interactions between the molecules. We demonstrated that the MXD3 siRNA-αCD22 Ab-SPIO NP complexes entered leukaemia cells and knocked down MXD3, leading the cells to undergo apoptosis and resulting in decreased live cell counts in the cell line Reh and in primary preB ALL samples in vitro. Furthermore, the cytotoxic effects of the MXD3 siRNA-αCD22 Ab-SPIO NP complexes were significantly enhanced by addition of the chemotherapy drugs vincristine or doxorubicin. We also ruled out potential cytotoxic effects of the MXD3 siRNA-αCD22 Ab-SPIO NP complexes on normal primary haematopoietic cells. Normal B cells were affected while CD34-positive haematopoietic stem cells and non-B cells were not. These data suggest that MXD3 siRNA-αCD22 Ab-SPIO NP complexes have the potential to be a new targeted therapy for preB ALL.

Novel single-cell functional analysis of red blood cells using laser tweezers Raman spectroscopy: application for sickle cell disease.

Laser tweezers Raman spectroscopy was used to characterize the oxygenation response of single normal adult, sickle, and cord blood red blood cells (RBCs) to an applied mechanical force. Individual cells were subjected to different forces by varying the laser power of a single-beam optical trap, and the intensities of several oxygenation-specific Raman spectral peaks were monitored to determine the oxygenation state of the cells. For all three cell types, an increase in laser power (or mechanical force) induced a greater deoxygenation of the cell. However, sickle RBCs deoxygenated more readily than normal RBCs when subjected to the same optical forces. Conversely, cord blood RBCs were able to maintain their oxygenation better than normal RBCs. These results suggest that differences in the chemical or mechanical properties of fetal, normal, and sickle cells affect the degree to which applied mechanical forces can deoxygenate the cell. Populations of normal, sickle, and cord RBCs were identified and discriminated based on this mechanochemical phenomenon. This study demonstrates the potential application of laser tweezers Raman spectroscopy as a single-cell, label-free analytical tool to characterize the functional (e.g., mechanical deformability, oxygen binding) properties of normal and diseased RBCs.

Efficacy of a CD22-targeted antibody-saporin conjugate in a xenograft model of precursor-B cell acute lymphoblastic leukemia.

Targeted therapies, such as those using imatinib and rituximab, have revolutionized the treatment of Philadelphia chromosome-positive and CD20-positive acute lymphoblastic leukemia (ALL) respectively, yet these therapies are effective in only a subset of patients and remission is generally not durable. The next generation of targeted therapies includes the use of antibodies conjugated to potent cytotoxic agents and are classified as antibody drug conjugates (ADC). For B-lineage ALL, CD22 is an ideal target for ADC therapy because it is expressed on the majority of B-lineage ALL cells and because antibody binding mediates receptor internalization. HB22.7-SAP is a conjugate of our anti-CD22 monoclonal antibody (mAb), HB22.7, and the ribosome inhibiting protein, saporin (SAP). In vitro, HB22.7-SAP effectively bound to CD22 on the surface of pre-B ALL cell lines and exhibited potent and specific cytotoxicity. In a NOD/SCID xenograft mouse model of pre-B ALL, when compared to the vehicle-treated control, HB22.7-SAP increased the median survival time from 20 days to over 50 days without significant toxicity.

Thrombotic thrombocytopenic purpura in a child with systemic lupus erythematosus.

We report a child with thrombotic thrombocytopenic purpura (TTP) secondary to systemic lupus erythematosus. The diagnosis was confirmed by low ADAMTS13 activity (<5%) along with the presence of a low titer inhibitor. Her clinical course was complicated by systemic lupus erythematosus, immunosuppressant therapy, and septic shock. She responded to plasma exchange and ADAMTS13 activity levels recovered. This case illustrates the heterogeneity of TTP and the difficulty of making a diagnosis of TTP. ADAMTS13 activity assay can be useful in the differential diagnosis of diseases with clinical features of thrombotic microangiopathy in pediatric patients. However, treatment needs to be decided carefully case-by-case.

Diagnostic challenges in native valve fungal endocarditis producing a massive septic pulmonary embolus.

Diagnosis and treatment of Candida albicans endocarditis can be difficult. We report a case of this rare condition in which a patient on oral fluconazole presented with septic pulmonary emboli without initial echocardiographic evidence of vegetation. Rapid attainment of a tissue diagnosis, along with combined medical surgical treatment proved to be effective for this patient.

Neuroblastoma of unknown primary site with periorbital bone metastasis in a child.

Neuroblastoma is the second most common solid tumor in children. Most tumors arise in the adrenal glands or paravertebral region. Rarely, patients present with metastatic disease but no primary site can be found despite extensive imaging. We report here a patient with a large periorbital bone metastasis and bone marrow involvement but with no known primary site.

STX11 mutations and clinical phenotypes of familial hemophagocytic lymphohistiocytosis in North America.

BACKGROUND: Mutations in STX11 are responsible for Familial Hemophagocytic Lymphohistiocytosis (FHLH) type 4, a rare primary immunodeficiency which has previously been observed only in patients of Kurdish, Turkish, and Lebanese ethnic background. METHODS: We reviewed our experience with STX11 mutations among North American patients and studied the impact of patient mutations upon syntaxin 11 expression and NK cell function. RESULTS: Between 2007 and 2008, 243 patients with HLH (lacking disease-causing mutations in PRF1 and UNC13D) were referred for STX11 mutational analysis. We observed 1 novel homozygous nonsense mutation, 73 G > T (E25X), occurring in Hispanic siblings, and 2 novel biallelic heterozygous missense mutations, 106G > C (E36Q) and 616G > A (E206K), occurring in 1 Caucasian patient. The N-terminal nonsense mutation resulted in absence of detectable syntaxin 11 and abrogation of in vitro NK cell degranulation and function, while the biallelic heterozygous missense mutations resulted in detectable mutated syntaxin 11 and preservation of in vitro NK cell degranulation and cytotoxicity. The two sibling patients with the nonsense mutations presented with HLH during infancy, whereas the patient with biallelic heterozygous missense mutations presented at 5 years of age. CONCLUSION: We conclude that mutations in STX11 are responsible for HLH in approximately 1% of North American patients and can cause variable defects in syntaxin 11 expression and function with resultant impact on clinical phenotype.

Facilitating action of asiaticoside at low doses on burn wound repair and its mechanism.

Some reports published from 1967 to 1999 describe the use of ointments containing high doses (0.1 to 0.2%, w/w) C. asiataica herb extracts to enhance wound repair. Lower doses at which burn wound repair is enhanced by such topical applications have not been established yet. We found that the application of asiaticoside at low doses of 10(-8) to 10(-12)% (w/w) facilitated burn wound repair. To clarify the accelerating mechanisms of asiaticoside on burn wound repair, we examined the effects of asiaticoside on the levels of various cytokines produced at the site of the burn wound. The topical application of a low dose (10 pg, 1 ng, or 100 ng/wound area) of asiaticoside increased monocyte chemoattractant protein-1 (MCP-1), vascular endothelial growth factor (VEGF), and interleukin (IL)-1beta levels in burn wound exudates. Asiaticoside (10 pg to 100 ng/ml) enhanced MCP-1 production in HaCaT cells, but it had no direct effect on VEGF production. Furthermore, asiaticoside (10 pg to 100 ng/ml) increased the IL-1beta production in THP-1 macrophages with MCP-1, but it had no effect on IL-1beta production without MCP-1 or with lipopolysaccharide (LPS). These findings suggest that the enhancement of burn wound healing by asiaticoside might be due to the promotion of angiogenesis during skin wound repair as a result of the stimulation of VEGF production caused by the increase in MCP-1 expression in keratinocytes and the increase in IL-1beta expression in macrophages induced cooperatively by asiaticoside plus MCP-1.

Treatment of the mouse model of mucopolysaccharidosis I with retrovirally transduced bone marrow.

Mucopolysaccharidosis I is a lysosomal storage disorder caused by mutations in the IDUA gene, resulting in deficiency of alpha-L-iduronidase and accumulation of glycosaminoglycans. Bone marrow transplantation has been the only available therapy, soon to be joined by enzyme replacement. We have tested retroviral gene therapy in a knockout mouse model of the disease. Bone marrow from Idua-/- male donor mice was transduced with human IDUA cDNA in an MND vector and transplanted into 6-8-week-old, lethally irradiated female Idua-/- mice. Sham-treated mice received Idua-/- bone marrow that was either unmodified or transduced with eGFP. Unmodified Idua+/+ (wild type) bone marrow was transplanted for comparison. Recipient mice were sacrificed 2-6 months after transplantation. Three biochemical parameters were used to gauge therapeutic success: appearance of alpha-L-iduronidase activity, reduction of beta-hexosaminidase activity and reduction of soluble glycosaminoglycan accumulation. Transplantation of unmodified +/+ bone marrow was effective in reducing storage in liver and spleen, but not in kidney or brain. The level of alpha-L-iduronidase activity achieved by transplantation of IDUA-transduced bone marrow varied greatly between experiments. But even modest activity resulted in correction of pathology of kidney, bladder epithelium, fibrocartilage, choroid plexus, and thalamus, as seen by light microscopy, while electron microscopy showed the presence of some normal neurons in the cortex. The partial correction of brain pathology is attributed to migration of donor hematopoietic cells, demonstrated by the presence of the Y chromosome and of normal microglia in the brain of mice receiving IDUA cDNA.