Borys Korchin

Pharmacologic inhibition of fatty acid oxidation sensitizes human leukemia cells to apoptosis induction

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.

MEK Inhibition Enhances ABT-737-Induced Leukemia Cell Apoptosis via Prevention of ERK activated MCL-1 induction and Modulation of MCL-1/BIM Complex

Recently, strategies for acute myeloid leukemia (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 the extracellular receptor activated kinase 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.Whereas control mice 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 demonstrated unexpected anti-apoptotic interaction between the BCL2 family-targeted BH3-mimetic ABT-737 and mitogen-activated protein kinase signaling in AML cells: the BH3 mimetic is not only restrained in its activity by MCL-1, but also induces its 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.

Angiogenesis-Promoting Gene Patterns in Alveolar Soft Part Sarcoma

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 71 patients 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.

Inhibition of mitochondrial metabolism by methyl-2-cyano-3,12-dioxooleana-1,9-diene-28-oate induces apoptotic or autophagic cell death in chronic myeloid leukemia cells

The initial success of the first synthetic bcr-abl kinase inhibitor imatinib has been dampened by the emergence of imatinib-resistant disease in blast crisis chronic myeloid leukemia. Here, we report that the novel triterpenoid methyl-2-cyano-3,12-dioxooleana-1,9-diene-28-oate (CDDO-Me) potently induced cytotoxicity in imatinib-resistant KBM5 cells expressing the T315I mutation of bcr-abl (24-h EC50, 540 nmol/L). In long-term culture, CDDO-Me abrogated the growth of human parental KBM5 and KBM5-STI cells with 96-h IC50 of 205 and 221 nmol/L, respectively. In addition, CDDO-Me rapidly decreased the viability of murine lymphoid Ba/F3 cells expressing wild-type p210 as well as the imatinib-resistant E255K and T315I mutations of bcr-abl. The low-dose effects of CDDO-Me are associated with inhibition of mitochondrial oxygen consumption, whereas the cytotoxic effects appear to be mediated by a rapid and selective depletion of mitochondrial glutathione that accompanies the increased generation of reactive oxygen species and mitochondrial dysfunction. Interestingly, the mitochondriotoxic effects of CDDO-Me are followed by the rapid autophagocytosis of intracellular organelles or the externalization of phosphatidylserine in different cell types. We conclude that alterations in mitochondrial function by CDDO-Me can result in autophagy or apoptosis of chronic myeloid leukemia cells regardless of the mutational status of bcr-abl. CDDO-Me is in clinical trials and shows signs of clinical activity, with minimal side effects and complete lack of cardiotoxicity. Studies in leukemias are in preparation. [Mol Cancer Ther 2008;7(5):1130–9]

A preclinical model for predicting drug response in soft-tissue sarcoma with targeted AAVP molecular imaging

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.

Soft Tissue Sarcoma Cells Are Highly Sensitive to AKT Blockade: A Role for p53-Independent Up-regulation of GADD45α

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 GADD45 alpha. Human STS xenografts in nude mice were used for in vivo studies, and immunohistochemistry was used to assess the effect of treatment on GADD45 alpha 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 GADD45 alpha mRNA and protein expression in all STS cells treated independent of p53 mutational status. GADD45 alpha 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 STS-targeted therapies.

IFN Regulatory Factor 8 Mediates Apoptosis in Nonhemopoietic Tumor Cells via Regulation of Fas Expression

IFN regulatory factor 8 (IRF8) is a transcription factor that was originally identified in myeloid cells and has been shown to be essential for differentiation and function of hemopoietic cells. Mice with a null mutation of IRF8 exhibit uncontrolled expansion of the granulocytic and monocytic lineages that progress into a phenotype resembling human chronic myelogenous leukemia. In human patients with chronic myelogenous leukemia, IRF8 transcript levels are frequently diminished. Therefore, IRF8 is a key regulator of myeloid tumor development. In this study, we report that IRF8 is a critical regulator of apoptosis in nonhemopoietic tumor cells. Disruption of IRF8 function with IRF8 dominant-negative mutants diminished Fas-mediated apoptosis in sarcoma tumor cells. Both constitutively expressed and IFN-γ-activated IRF8 were involved in regulation of apoptosis. 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 IFN-γ-initiated induction of STAT1 phosphorylation, which in turn, blocked IFN-γ-induced Fas up-regulation. Interestingly, the constitutively expressed IRF8 was also essential for TNF-α sensitization of Fas-mediated apoptosis because disruption of IRF8 function also inhibited TNF-α-sensitized and Fas-mediated apoptosis. Taken together, our data suggest that IRF8 is an essential mediator of Fas-mediated apoptosis and that IRF8 mediates apoptosis through regulation of Fas expression in nonhemopoietic tumor cells.

Midkine Enhances Soft-Tissue Sarcoma Growth: A Possible Novel TherapeuticTarget

Purpose: New therapeutic targets for soft-tissue sarcoma (STS) treatment are critically needed. Midkine (MK), a multifunctional cytokine, is expressed during midgestation but is highly restricted in normal adult tissues. Renewed MK expression was shown in several malignancies where protumorigenic properties were described. We evaluated the expression and function of MK in STS. Experimental Design: Immunohistochemistry, reverse transcription-PCR, and Western blotting (WB) evaluated MK expression in human STS tissues and cell lines. WB and flow cytometry analyzed MK receptor expression. Cell growth assays evaluated the effect of MK on STS cell growth, and WB assessed MK downstream signaling. MK knock-in and knockout experiments further evaluated MK function. The growth of parental versus MK-transfected human fibrosarcoma cells was studied in vivo. Results: MK was found to be overexpressed in a variety of human STS histologies. Using a rhabdomyosarcoma (RMS) tissue microarray, cytoplasmic and nuclear MK was identified; nuclear MK expression was significantly increased in metastases. Similarly, several STS cell lines expressed and secreted MK; RMS cells exhibited nuclear MK. STS cells also expressed the MK receptors protein tyrosine phosphatase ζ and lipoprotein receptor-related protein. MK significantly enhanced STS cell growth potentially via the Src and extracellular signal-regulated kinase pathways. STS cells stably transfected with MK exhibited increased growth in vitro and in vivo. MK-expressing human STS xenografts showed increased tumor-associated vasculature. Furthermore, MK knockdown resulted in decreased STS cell growth, especially in RMS cells. Conclusion: MK enhances STS tumor growth; our results support further investigation of MK and its receptors as therapeutic targets for human STS.

Epidermal growth factor receptor blockade in combination with conventional chemotherapy inhibits soft tissue sarcoma cell growth in vitro and in vivo

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 transferase–mediated 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 AKT and mitogen-activated protein kinase pathways. EGFR blockade inhibited these effects and also caused increased apoptosis, a p53-independent G0-G1 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.

High prevalence of p53 exon 4 mutations in soft tissue sarcoma

p53 is the most commonly mutated gene in cancer, including soft tissue sarcoma (STS). The authors characterized p53 alterations (protein accumulation and gene mutation) in STS to evaluate possible associations with patient outcomes.