Xiaoyang Ling

Mutant FLT3: A Direct Target of Sorafenib in Acute Myelogenous Leukemia

BACKGROUND Internal tandem duplication (ITD) mutations in the juxtamembrane domain-coding sequence of the Fms-like tyrosine kinase 3 (FLT3) gene have been identified in 30% of acute myeloid leukemia (AML) patients and are associated with a poor prognosis. The kinase inhibitor sorafenib induces growth arrest and apoptosis at much lower concentrations in AML cell lines that harbor FLT3-ITD mutations than in AML cell lines with wild-type FLT3. METHODS The antileukemic activity of sorafenib was investigated in isogenic murine Ba/F3 AML cell lines that expressed mutant (ITD, D835G, and D835Y) or wild-type human FLT3, in primary human AML cells, and in a mouse leukemia xenograft model. Effects of sorafenib on apoptosis and signaling in AML cell lines were investigated by flow cytometry and immunoblot analysis, respectively, and the in vivo effects were determined by monitoring the survival of leukemia xenograft-bearing mice treated with sorafenib (groups of 15 mice). In a phase 1 clinical trial, 16 patients with refractory or relapsed AML were treated with sorafenib on different dose schedules. We determined their FLT3 mutation status by a polymerase chain reaction assay and analyzed clinical responses by standard criteria. All statistical tests were two-sided. RESULTS Sorafenib was 1000- to 3000-fold more effective in inducing growth arrest and apoptosis in Ba/F3 cells with FLT3-ITD or D835G mutations than in Ba/F3 cells with FLT3-D835Y mutant or wild-type FLT3 and inhibited the phosphorylation of tyrosine residues in ITD mutant but not wild-type FLT3 protein. In a mouse model, sorafenib decreased the leukemia burden and prolonged survival (median survival in the sorafenib-treated group vs the vehicle-treated group = 36.5 vs 16 days, difference = 20.5 days, 95% confidence interval = 20.3 to 21.3 days; P = .0018). Sorafenib reduced the percentage of leukemia blasts in the peripheral blood and the bone marrow of AML patients with FLT3-ITD (median percentages before and after sorafenib: 81% vs 7.5% [P = .016] and 75.5% vs 34% [P = .05], respectively) but not in patients without this mutation. CONCLUSION Sorafenib may have therapeutic efficacy in AML patients whose cells harbor FLT3-ITD mutations.

Knockdown of STAT3 Expression by RNA Interference Inhibits the Induction of Breast Tumors in Immunocompetent Mice

Constitutively activated STAT3 is involved in the formation of multiple types of tumors including breast cancer. We examined the effects of Stat3 protein knockdown by RNA interference using a dicistronic lentivirus small hairpin (shRNA) delivery system on the growth of mammary tumors in BALB/c mice induced by the 4T1 cell line. A single exposure of 4T1 cells to shRNA/STAT3 lentivirus transduced 75% of the cells with green fluorescent protein (GFP) within 96 hours. In cells selected for GFP expression, neither Stat3 protein nor phosphotyrosine Stat3 was detected. Tumor formation induced by injecting 4T1 cells into the mammary fat pad was blocked by expression of the shRNA for STAT3 whereas all mice injected with 4T1 cells expressing only GFP efficiently formed tumors. c-Myc expression was reduced 75% in cells expressing greatly reduced levels of Stat3 compared with the GFP control. Of interest, the level of activated Src, which is known to activate Stat3, was virtually eliminated but the level of the Src protein itself remained the same. Importantly, expression of Twist protein, a metastatic regulator, was eliminated in STAT3 knockdown cells. Invasion activity of STAT3 knockdown cells was strongly inhibited. However, the proliferation rate of cells in Stat3 knockdown cells was similar to that of the GFP control; the cell cycle was also not affected. We conclude from these studies that activated Stat3 protein plays a critical role in the induction of breast tumors induced by 4T1 cells by enhancing the expression of several important genes including c-Myc and the metastatic regulator Twist. These studies suggest that stable expression of small interfering RNA for STAT3 has potential as a therapeutic strategy for breast cancer.

The novel triterpenoid C-28 methyl ester of 2-cyano-3, 12-dioxoolen-1, 9-dien-28-oic acid inhibits metastatic murine breast tumor growth through inactivation of STAT3 signaling

We and others have reported that C-28 methyl ester of 2-cyano-3, 12-dioxoolen-1, 9-dien-28-oic acid (CDDO-Me) effectively inhibits the growth of multiple cancer cell types. Our previous studies indicated that prolonged CDDO-Me treatment inactivated extracellular signal-regulated kinase signaling in acute myelogenous leukemia cells. Whether treatment with CDDO-Me has an earlier effect on other proteins that are important for either signal transduction or oncogenesis is unknown. Constitutively activated signal transducer and activator of transcription 3 (STAT3) is frequently found in human breast cancer samples. Constitutively activated STAT3 was shown to up-regulate c-Myc in several types of cancer and has a feedback effect on Src and Akt. To examine the effects of CDDO-Me on STAT3 signaling in breast cancer, we used the murine 4T1 breast tumor model, which is largely resistant to chemotherapy. In vitro, after treatment of 4T1 cells with 500 nmol/L CDDO-Me for 2 h, we found (a) inactivation of STAT3, (b) inactivation of Src and Akt, (c) 4-fold reduction of c-Myc mRNA levels, (d) accumulation of cells in G(2)-M cell cycle phase, (e) abrogation of invasive growth of 4T1 cells, and (f) lack of apoptosis induction. In in vivo studies, CDDO-Me completely eliminated 4T1 breast cancer growth and lung metastases induced by 4T1 cells in mice when treatment started 1 day after tumor implantation and significantly inhibited tumor growth when started after 5 days. In vivo studies also indicated that splenic mature dendritic cells were restored after CDDO-Me treatment. In summary, these data suggest that CDDO-Me may have therapeutic potential in breast cancer therapy, in part, through inactivation of STAT3.

Bcr-Abl-mediated suppression of normal hematopoiesis in leukemia

A variety of experimental evidence including findings in various mouse models indicates that the BCR-ABL oncogene is the cause of chronic myeloid leukemia (CML). Since normal hematopoietic cells in marrow and spleen are replaced with proliferating leukemic blasts, we determined whether this is an active process mediated by the leukemia cells. The lipocalin 24p3 was reported to be secreted by mouse hematopoietic cells deprived of IL-3, resulting in apoptosis induction in a variety of hematopoietic cells including bone marrow cells. Here, we show that BCR-ABL+ mouse hematopoietic cells induced persistent expression and secretion of 24p3. Importantly, BCR-ABL+ hematopoietic cells were resistant to the apoptotic effects of 24p3. The expression of the Bcr-Abl oncoprotein and its tyrosine kinase were required for induction of 24p3 expression. Co-culture studies showed that BCR-ABL+ cells induced apoptosis in BCR-ABL negative cells. Antisense 24p3/siRNA expression reduced the level of 24p3 protein in both BCR-ABL+ cells and in conditioned medium (CM) obtained from these cells. CM from BCR-ABL+ cells expressing antisense 24p3/siRNA had reduced apoptotic activity for target cells; 24p3 antibody also reduced the apoptotic activity of the CM. Leukemic mice induced by BCR-ABL+ cells expressing either antisense 24p3 or 24p3 siRNA had increased levels of normal hematopoiesis and reduced invasion of leukemia cells in marrow and spleen tissues. These findings indicate that suppression of normal hematopoiesis in BCR-ABL-induced leukemia is an active process involving secretion of the cell death-inducing factor 24p3 by mouse leukemia cells, raising the possibility that similar factors are involved in BCR-ABL+ CML.Oncogene advance online publication, 28 February 2005; doi:10.1038/sj.onc.1208500

MRx102, a triptolide derivative, has potent antileukemic activity in vitro and in a murine model of AML

Triptolide, isolated from the herb Tripterygium wilfordii, has been shown to potently induce apoptosis in various malignant cells by inhibiting RNA synthesis and nuclear factor-κB activity. Previously, we showed that triptolide promotes apoptosis in acute myeloid leukemia (AML) cells via the mitochondria-mediated pathway, in part, by decreasing levels of the anti-apoptotic proteins XIAP and Mcl-1. MRx102 is a triptolide derivative, currently in preclinical development. Here we show that MRx102 potently promoted apoptosis in AML cell lines, with EC50 values of 14.5±0.6 nM and 37.0±0.9 nM at 48 h for OCI-AML3 and MV4–11 cells, respectively. MRx102, at low nanomolar concentrations, also induced apoptosis in bulk, CD34+ progenitor, and more importantly, CD34+CD38− stem/progenitor cells from AML patients, even when they were protected by coculture with bone marrow derived mesenchymal stromal cells. MRx102 decreased XIAP and Mcl-1 protein levels and inhibited RNA synthesis in OCI-AML3 cells. In vivo, MRx102 greatly decreased leukemia burden and increased survival time in non-obese diabetic/severe combined immunodeficiency mice harboring Ba/F3-ITD cells. Collectively, we demonstrated that MRx102 has potent antileukemic activity both in vitro and in vivo, has the potential to eliminate AML stem/progenitor cells and overcome microenvironmental protection of leukemic cells, and warrants clinical investigation.

AML-induced osteogenic differentiation in mesenchymal stromal cells supports leukemia growth

Genotypic and phenotypic alterations in the bone marrow (BM) microenvironment, in particular in osteoprogenitor cells, have been shown to support leukemogenesis. However, it is unclear how leukemia cells alter the BM microenvironment to create a hospitable niche. Here, we report that acute myeloid leukemia (AML) cells, but not normal CD34+ or CD33+ cells, induce osteogenic differentiation in mesenchymal stromal cells (MSCs). In addition, AML cells inhibited adipogenic differentiation of MSCs. Mechanistic studies identified that AML-derived BMPs activate Smad1/5 signaling to induce osteogenic differentiation in MSCs. Gene expression array analysis revealed that AML cells induce connective tissue growth factor (CTGF) expression in BM-MSCs irrespective of AML type. Overexpression of CTGF in a transgenic mouse model greatly enhanced leukemia engraftment in vivo. Together, our data suggest that AML cells induce a preosteoblast-rich niche in the BM that in turn enhances AML expansion.

Osteopontin mediates glioblastoma-associated macrophage infiltration and is a therapeutic target

Glioblastoma is highly enriched with macrophages, and osteopontin (OPN) expression levels correlate with glioma grade and the degree of macrophage infiltration; thus, we studied whether OPN plays a crucial role in immune modulation. Quantitative PCR, immunoblotting, and ELISA were used to determine OPN expression. Knockdown of OPN was achieved using complementary siRNA, shRNA, and CRISPR/Cas9 techniques, followed by a series of in vitro functional migration and immunological assays. OPN gene–deficient mice were used to examine the roles of non-tumor-derived OPN on survival of mice harboring intracranial gliomas. Patients with mesenchymal glioblastoma multiforme (GBM) show high OPN expression, a negative survival prognosticator. OPN is a potent chemokine for macrophages, and its blockade significantly impaired the ability of glioma cells to recruit macrophages. Integrin &agr;v&bgr;5 (ITG&agr;v&bgr;5) is highly expressed on glioblastoma-infiltrating macrophages and constitutes a major OPN receptor. OPN maintains the M2 macrophage gene signature and phenotype. Both tumor-derived and host-derived OPN were critical for glioma development. OPN deficiency in either innate immune or glioma cells resulted in a marked reduction in M2 macrophages and elevated T cell effector activity infiltrating the glioma. Furthermore, OPN deficiency in the glioma cells sensitized them to direct CD8+ T cell cytotoxicity. Systemic administration in mice of 4-1BB–OPN bispecific aptamers was efficacious, increasing median survival time by 68% (P < 0.05). OPN is thus an important chemokine for recruiting macrophages to glioblastoma, mediates crosstalk between tumor cells and the innate immune system, and has the potential to be exploited as a therapeutic target.

Abstract 4291: An optimized therapeutic nanoparticle delivery platform of miRNA in preclinical murine models of malignancy

INTRODUCTION: We have previously shown robust therapeutic efficacy of miRNAs in preclinical murine models of glioblastoma and were one of the first groups to deliver therapeutic miRNAs intravenously. However a major hurdle to clinical translation is a scalable formulation that affords protection against circulatory RNAses. Nanoparticles can encapsulate and protect the miRNA from degradation and enhance delivery into the immune cell compartment facilitating antitumor effects, in part through the reversal of tumor-mediate immune suppression and increased expression of effector cytokines - thus, overcoming the need for direct tumor delivery of the therapeutic agent. METHODS: FDA acceptable lipid nanoparticles were devised to enhance delivery of miRNA into the peripheral blood mononuclear cells (PBMCs) and verified by in vivo compartmental pharmacokinetic analysis and functional immune monitoring. Nanoparticle test articles contain an active immune modulatory agent - miR-124, which inhibits the signal transducer and activator of transcript 3 (STAT3) pathway. The lead candidate was designated LUNAR-301, and further refinements included unlocking the nucleic acids (LUNAR-302) to enhance efficacy. Nanoparticle formulations were tested in multiple murine models of malignancy including established intracerebral gliomas. RESULTS: In non-tumor bearing mice dosed with intravenous LUNAR-301, miR-124 was delivered to the peripheral blood mononuclear cells (PBMCs) with no clinical signs of toxicity or organ damage on histopathologic exam. In an intracerebral GL261 model, lower pSTAT3 expression was observed in mice treated with LUNAR-301 compared to both empty nanoparticle treated mice or untreated mice, p = 0.0081 and p = 0.0001 respectively. Similarly, lower Foxp3 expression was observed in the LUNAR-301 treated mice, p = 0.0057 and p = 0.0223 respectively. Median survival time for mice treated with LUNAR-301 exceeded 70 days, compared to only 32.5 days for mice treated with the previous gold-standard, miR-124 + lipofectamine. The cure rate difference between LUNAR-301 (9 out of 15 mice) and LUNAR-302 (2 out of 10 mice) was 40% (P = 0.0576); the difference in cure rates between LUNAR-301 and miR-124 + lipofectamine (4 out of 16 mice) was 35% (P = 0.0532). In a subcutaneous murine model of melanoma, tumor growth rate per day without treatment was 44% (i.e., tumor volume was expected to increase 44% cumulatively on a daily basis), while it was reduced to 26.1% in the LUNAR-301-treated group (P = 0.007), and to 16.2% in the LUNAR-302-treated group (P CONCLUSIONS: Nanoparticle delivery of miR-124 has a favorable safety and efficacy profile to justify implementation in client-owned canines or human clinical trials for the treatment of gliomas. Citation Format: Nasser K. Yaghi, Jun Wei, Ling-Yuan Kong, Yuuri Hashimoto, Edjah K. Nduom, Neal Huang, Xiaoyang Ling, Shouhao Zhou, Jonathan M. Levine, Virginia R. Fajt, Kiyoshi Tachikawa, Padmanabh Chivukula, David C. Webb, Joseph E. Payne, Amy B. Heimberger. An optimized therapeutic nanoparticle delivery platform of miRNA in preclinical murine models of malignancy. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4291. doi:10.1158/1538-7445.AM2015-4291

Abstract LB-95: Inhibition of CXCR4/Stat3 signaling by AMD3465 exerts major tumor- and metastasis-suppressive effects in syngenic 4T1 breast cancer model

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL Stromal-derived factor-1 α (SDF-1α, also termed CXCL12) and its receptor CXCR4 have been shown to play important roles in hematopoietic stem cell homing, and high expression of CXCR4 has been associated with poor prognosis in hematologic malignancies. The CXCR4/SDF-1α axis is also functional in solid tumors, including breast cancer. It has been postulated that Stat3 may promote metastases in breast cancer through direct interaction with CXCR4, promoting tumor growth and survival. Phosphorylated Stat3 was found to trigger overexpression or activation of several potent oncoproteins. In order to evaluate the role of CXCR4 in metastases, we used the specific CXCR4 inhibitor, AMD3465, to block CXCR4 in the syngeneic 4T1 breast cancer model. Stat3, Akt, Erk1/2, Jak2 and MMP9 activity was found to be almost completely inhibited and expression levels of GSK-3β and cMyc were greatly reduced. Growth of 4T1 cells orthotopically implanted into the mammary fat pad as well as pulmonary and liver metastases were significantly (>95%) reduced in vivo when AMD3465 was given as continuous infusion at a dose of 428.57μg/mouse/day over 14 days. Furthermore, we observed that CXCR4 inhibition by AMD3465 significantly reduced lung metastases in three immunocompetent syngeneic mouse models after surgical resection of the primary breast tumors. This experimental design mimics current clinical treatment strategies. Kozin et al. (Cancer Research 70: 5679–85; 2010) recently suggested that CD11b+ myeloid/monocytic cells (a cell type implicated in supporting angiogenesis) may play a role in tumor re-growth in response to SDF-1 α. In support of this observation, we observed significantly reduced CD11b+ cells in lungs, livers and spleens (P<0.01 for all 3 organs) following treatment with AMD3465, as compared to PBS controls. Hence, our data suggest that pharmacological CXCR4 inhibition in breast cancer-bearing mice following primary tumor resection is well tolerated and potently controls the development of distant metastatic lesions. In conclusion, interruption of the SDF-1/CXCR4/Stat3 signaling axis by the CXCR4-specific inhibitor, AMD3465, effectively inhibits pulmonary and hepatic breast cancer metastasis via disruption of intracellular microenvironment mediated signaling in breast cancer cells and through effects on CD11b+ tumor infiltrating myeloid cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr LB-95. doi:10.1158/1538-7445.AM2011-LB-95

Mesenchymal Stem/Stromal Cells as Cellular Vehicles for Tumor Targeting

Data published over the last 10 years suggest that mesenchymal stem/stromal cells (MSC) possess the innate capacity to home to sites of inflammation, including tumors and wounding microenvironments. Evidence suggests that the increased production of inflammatory mediators found at these sites is potential attractants for recruitment and engraftment. This innate homing response can be exploited by using MSC as a cellular delivery vehicle to deliver anticancer agents directly to tumors. The high-level intratumoral production of these agents controls tumor growth and prolongs survival in numerous animal models. In this review, we examine the ability of MSC to selectively home to and engraft within the tumor microenvironment and the multiple gene products delivered by MSC when used as delivery vehicles for anticancer therapies.