The traditional view is that cancer cells predominately produce ATP by glycolysis, rather than by oxidation of energy-providing substrates. Mitochondrial uncoupling -the continuing reduction of oxygen without ATP synthesis -has recently been shown in leukemia cells to circumvent the ability of oxygen to inhibit glycolysis, and may promote the metabolic preference for glycolysis by shifting from pyruvate oxidation to fatty acid oxidation (FAO). Here we have demonstrated that pharmacologic inhibition of FAO with etomoxir or ranolazine inhibited proliferation and sensitized human leukemia cells -cultured alone or on bone marrow stromal cells -to apoptosis induction by ABT-737, a molecule that releases proapoptotic Bcl-2 proteins such as Bak from antiapoptotic family members. Likewise, treatment with the fatty acid synthase/lipolysis inhibitor orlistat also sensitized leukemia cells to ABT-737, which supports the notion that fatty acids promote cell survival. Mechanistically, we generated evidence suggesting that FAO regulates the activity of Bak-dependent mitochondrial permeability transition. Importantly, etomoxir decreased the number of quiescent leukemia progenitor cells in approximately 50% of primary human acute myeloid leukemia samples and, when combined with either ABT-737 or cytosine arabinoside, provided substantial therapeutic benefit in a murine model of leukemia. The results support the concept of FAO inhibitors as a therapeutic strategy in hematological malignancies. IntroductionMore than half a century ago, Otto Warburg proposed that the origin of cancer cells was closely linked to a permanent respiratory defect that circumvents the Pasteur effect, i.e., the inhibition of anaerobic fermentation by oxygen (1). However, we have recently demonstrated that in leukemia cells, mitochondrial uncoupling - the continuing reduction of oxygen without the synthesis of ATP - could mimic the Warburg effect in the absence of permanent, transmissible alterations to the oxidative capacity of cells (2). This metabolic pattern was observed when leukemia cells were cultured on feeder layers of bone marrow-derived mesenchymal stromal cells (MSCs). MSCs have previously been reported to support both normal and malignant hematopoiesis (reviewed in refs. 3-5) and have become an important component in the in vitro modeling of the bone marrow microenvironment. Leukemia cells cultured on MSC feeder layers demonstrated increased lactate generation, and, most curiously, decreased mitochondrial membrane potential in the presence of a transient (6-8 hour) increase in oxygen consumption. Additionally, this uncoupled phenotype appeared to be associated with the antiapoptotic effect of MSC feeder layers, and we hypothesized a shift away from the complete oxidation of glucose. This concept has already been alluded to by Lynen (6), and by Ronzoni and Ehrenfest in experiments using the prototypical protonophore 2,4-dinitrophenol, and suggests a metabolic shift to fatty acid oxidation (FAO) rather than pyruvate oxidation (2, 7). Although i...

Recently, strategies for AML therapy have been developed that target anti-apoptotic BCL2 family members using BH3 mimetic drugs such as ABT-737. Though effective against BCL2 and BCL-XL, ABT-737 poorly inhibits MCL-1. Here we report that, unexpectedly, ABT-737 induces activation of ERK and induction of MCL-1 in AML cells. MEK inhibitors such as PD0325901 and CI-1040 have been used successfully to suppress MCL-1. We report that PD0325901 blocked ABT-737 –induced MCL-1 expression and when combined with ABT-737 resulted in potent synergistic killing of AML derived cell lines, primary AML blast and CD34+38−123+ progenitor/stem cells. Finally, we tested the combination of ABT-737 and CI-1040 in a murine xenograft model using MOLM-13 human leukemia cells. While control and CI-1040 treated mice exhibited progressive leukemia growth, ABT-737 and, to a significantly greater extent, ABT-737 + CI-1040 exerted major anti-leukemia activity. Collectively, results demonstrate unexpected anti-apoptotic interaction between the BCL2 family-targeted BH3 mimetic ABT-737 and MAPK signaling in AML cells: the BH3 mimetic is not only restrained in its activity by MCL-1, but also induces it’s expression. However, concomitant inhibition by BH3 mimetics and MEK inhibitors could abrogate this effect and may be developed into a novel and effective therapeutic strategy for patients with AML.

Purpose:We examined a cohort of patients with alveolar soft part sarcoma (ASPS) treated at our institution and showed the characteristic ASPSCR1-TFE3 fusion transcript in their tumors. Investigation of potential angiogenesis-modulating molecular determinants provided mechanistic and potentially therapeutically relevant insight into the enhanced vascularity characteristic of this unusual tumor. Experimental Design: Medical records of 71patients with ASPS presenting at the University of Texas M.D. Anderson Cancer Center (1986-2005 were reviewed to isolate 33 patients with formalin-fixed paraffin-embedded material available for study. RNA extracted from available fresh-frozen and formalin-fixed paraffin-embedded human ASPS tumors were analyzed for ASPSCR1-TFE3 fusion transcript expression using reverse transcription-PCR and by angiogenesis oligomicroarrays with immunohistochemical confirmation. Results: Similar to previous studies, actuarial 5-and 10-year survival rates were 74% and 51%, respectively, despite frequent metastasis. ASPSCR1-TFE3 fusion transcripts were identified in 16 of 18 ASPS samples. In the three frozen samples subjected to an angiogenesis oligoarray, 18 angiogenesis-related genes were up-regulated in tumor over adjacent normal tissue. Immunohistochemistry for jag-1, midkine, and angiogenin in 33 human ASPS samples confirmed these results. Comparison with other sarcomas indicates that the ASPS angiogenic signature is unique. Conclusion: ASPS is a highly vascular and metastatic tumor with a surprisingly favorable outcome; therapeutically resistant metastases drive mortality. Future molecular therapies targeting overexpressed angiogenesis-promoting proteins (such as those identified here) could benefit patients with ASPS. Alveolar soft part sarcoma (ASPS) is a malignancy of uncertain histogenesis, first described in 1952 (1). A rare soft tissue sarcoma (STS) subtype, it has a uniquely indolent growth pattern combined with unexpectedly high metastatic (particularly pulmonary) activity. Unlike other STS, ASPS also metastasizes to the brain (2 -7). Interestingly, whereas the metastatic rates in this disease are high, prior large series indicate prolonged survival with metastatic disease. Surgery is the therapeutic mainstay for localized and metastatic disease; chemoresistance frequently precludes meaningful systemic intervention. A characteristic translocation in ASPS results in a novel fusion of the ASPSCR1 (previously designated ASPL) and TFE3 genes (chromosomes 17q25 and Xp11.2, respectively), resulting in a functional transcription factor with altered target gene activation (8). This creates a novel ASPSCR1-TFE3 fusion protein that seems to acts as an aberrant transcription factor that induces unregulated transcription of TFE3-regulated genes.Histologically, ASPS has a distinctive appearance, usually consisting of nests of epithelioid to polygonal cells arranged in nests or bundles, sometimes with a central lack of cohesion that is reminiscent of pulmonary alveolar structures. Intracellul...

Human sarcomas are rare but diverse malignant tumors derived from mesenchymal tissue. Clinical response to therapy is currently determined by the modified World Health Organization (WHO) criteria or the Response Evaluation Criteria in Solid Tumors (RECIST), but these standards correlate poorly with sarcoma patient outcome. We introduced ligand-directed particles with elements of AAV and phage (AAVP) to enable integration of tumor targeting to molecular imaging. We report drug-response monitoring and prediction in a nude rat model of human sarcoma by AAVP imaging. As a proof-of-concept, we imaged Herpes simplex thymidine kinase in a clinic-ready setting with PET to show that one can a priori predict tumor response to a systemic cytotoxic. Given the target expression in patient-derived sarcomas, this platform may be translated in clinical applications. Sarcoma-specific ligands and promoters may ultimately lead to an imaging transcriptome.H uman sarcomas are rare yet heterogeneous malignant tumors from mesenchymal tissues (1). Monitoring drug responses in soft-tissue sarcoma has long been clinically problematic. Currently, responses are determined by the modified World Health Organization (WHO) criteria (2-4) or the Response Evaluation Criteria in Solid Tumors (RECIST) (5-7), which require marked tumor size decrease for patients to be considered responding to therapy. A major assumption of these criteria is that a solid tumor volume is directly proportional to the cancer cell number. However, in soft-tissue sarcomas, there are reasons to challenge such an assumption (8). First, in addition to tumor cells, sarcomas contain nonmalignant stromal cells and extracellular matrix (ECM) that do not disappear, even if the malignant component is treated. Moreover, when cytotoxics are used against sarcomas, there is often associated necrosis resulting in reduced total cell number but not necessarily overall tumor size changes. Finally, even if a soft-tissue sarcoma is predominantly or entirely composed of cancer cells, its remnant composition may not be fully eliminated when tumor cells are destroyed by therapy, because myxoid-type degeneration is not always promptly removed. Ultimately, the modified WHO criteria and RECIST correlate poorly with drug response and outcome in patients with soft-tissue sarcomas; validation in this setting is sporadic and restricted. In another level of complexity, drug responses in patients with soft-tissue sarcoma are determined through standard methods, such as CT, MRI, or PET scans. However, because systematic quantitative measurements have not been established because of the rarity and diversity of soft-tissue sarcomas, decreases in tumor size and/or density are not accepted as unequivocal evidence of response. Consequently, many conventional soft-tissue sarcoma responses in individual patients are evaluated qualitatively. Thus, new or alternative quantitative imaging criteria to improve management and follow-up of responses were proposed in ref. 9.We have introduced a hybrid vector that ena...

IRF8 is a transcription factor that was originally identified in myeloid cells. Mice with a null mutation of IRF8 exhibit uncontrolled expansion of myeloid cells that progress into a phenotype resembling human chronic myelogenous leukemia (CML). In human patients with CML, IRF8 transcript levels are frequently diminished. Here, we report that disruption of IRF8 function diminished Fas‐mediated apoptosis in solid tumor cells. Furthermore, it was found that constitutively expressed IRF8 is associated with the Fas promoter to activate Fas transcription. In addition, disruption of constitutively expressed IRF8 function diminished JAK1 expression and thereby inhibited IFNγ‐initiated induction of STAT1 phosphorylation. Interestingly, the constitutively expressed IRF8 was also essential for TNF‐α‐sensitization of Fas‐mediated apoptosis since disruption of IRF8 function also inhibited TNFα‐sensitized and Fas‐mediated apoptosis. Analysis of IRF8‐deficient dendritic cells (DC) generated from IRF8 null mice revealed that Fas expression level is also down‐regulated as compared to the wild type mice. In addition, IRF8‐deficient DCs also exhibited higher level of FLIPs protein as compared to the control wt DCs. Taken together, our data suggest that IRF8 is an essential regulator of Fas‐mediated apoptosis and that IRF8 mediates apoptosis through regulation of Fas and FLIP.

The AKT signaling pathway is activated in soft tissue sarcoma (STS). However, AKT blockade has not yet been studied as a potential targeted therapeutic approach. Here, we examined the in vitro and in vivo effects of AKT inhibition in STS cells. Western blot analysis was used to evaluate the expression of AKT pathway components and the effect of AKT stimulation and inhibition on their phosphorylation. Cell culture assays were used to assess the effect of AKT blockade (using a phosphatidylinositol 3-kinase inhibitor and a specific AKT inhibitor) on STS cell growth, cell cycle, and apoptosis. Oligoarrays were used to determine gene expression changes in response to AKT inhibition. Reverse transcription-PCR was used for array validation. Specific small inhibitory RNA was used to knockdown GADD45A. Human STS xenografts in nude mice were used for in vivo studies, and immunohistochemistry was used to assess the effect of treatment on GADD45A expression, proliferation, and apoptosis. Multiple STS cell lines expressed activated AKT. AKT inhibition decreased STS downstream target phosphorylation and growth in vitro; G 2 cell cycle arrest and apoptosis were also observed. AKT inhibition induced GADD45a mRNA and protein expression in all STS cells treated independent of p53 mutational status. GADD45A knockdown attenuated the G 2 arrest induced by AKT inhibition. In vivo, AKT inhibition led to decreased STS xenograft growth. AKT plays a critical role in survival and proliferation of STS cells. Modulation of AKT kinase activity may provide a novel molecularly based strategy for STStargeted therapies. [Cancer Res 2008;68(8):2895-903]

The mutations of testicular insulin-like 3 (INSL3) hormone or its receptor RXFP2 cause cryptorchidism in male mice. Here we have examined Rxfp2 gene expression at different stages of embryonic and postnatal mouse development in male reproductive tissues employing quantitative RT-PCR and several RXFP2-specific antibodies directed toward different parts of the RXFP2 protein. Receptor expression was markedly increased after birth and was readily detectable in the epididymis, Leydig cells, and germ cells of the testis. The strongest expression was detected in adult mouse cremaster muscle. INSL3 treatment increased cell proliferation of embryonic gubernacular and TM3 embryonic Leydig cells, implicating active INSL3-mediated autocrine signaling in these cells and identifying TM3 as a novel in vitro model to study the effects of RXFP2 signaling. We generated Tg(Rxfp2-cre)Aia (Rxfp2-iCre) transgenic mice expressing improved Cre recombinase (iCre) under the control of the 2.4-kb mouse Rxfp2 promoter. The iCre was expressed in the gubernacular ligament at E14.5, indicating that this promoter is able to drive Rxfp2 gene expression during transabdominal testis descent. We demonstrated that the transcription factor Sox9, a known male sex determination factor, is expressed in mouse embryonic gubernacula and upregulated human, but not mouse, promoter luciferase reporter constructs. In conclusion, we have determined the developmental expression profile of INSL3 receptor employing newly characterized RXFP2 antisera and a novel Rxfp2-iCre transgenic mouse model. We determined the promoter region capable of providing the gubernacular-specific expression of Rxfp2. Analysis of RXFP2 promoter identified SOX9 as a new transcriptional enhancer of human gene expression.

Purpose: The epidermal growth factor receptor (EGFR) is highly expressed in many human soft tissue sarcomas (STS). However, EGFR blockade has not apparently been used for human STS therapy; therefore, we examined the in vitro and in vivo effects and the underlying mechanisms before considering EGFR blockade as a therapy for STS patients. Experimental Design: Human STS tissues and cell lines were used to study EGFR expression and activation. Western blot analysis was used to evaluate effects of EGFR activation on downstream signaling. Cell culture assays were used to assess the effect of EGF stimulation as well as EGFR blockade (using an EGFR tyrosine kinase inhibitor, Iressa; AstraZeneca) on STS cell growth, apoptosis, and chemosensitivity. An in vivo study (HT1080 human fibrosarcoma cell line in nude/nude mice: Iressa, doxorubicin, Iressa + doxorubicin, vehicle) was used to examine tumor growth; pEGFR, proliferating cell nuclear antigen, and terminal deoxyribonucleotide transferasem ediated nick-end labeling staining helped assess the effect of therapy in vivo on STS EGFR activation, proliferation, and apoptosis. Results: EGFR was expressed and activated in STS cell lines and tumors, probably due to ligand binding rather than EGFR mutation. Stimulation caused activation of AKTand mitogen-activated protein kinase pathways. EGFR blockade inhibited these effects and also caused increased apoptosis, a p53-independent G 0 -G 1 cell cycle arrest, and decreased cyclin D1 expression. In vivo, Iressa + doxorubicin had markedly synergistic anti-STS effects. Conclusion: EGFR blockade combined with conventional chemotherapy results in anti-human STS activity in vitro and in vivo, suggesting the possibility that combining these synergistic treatments will improve anti-STS therapy.