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Gene targeting has been used to create a variety of lines of mice with Pkd1 mutations that share many common features. Homozygous Pkd1 mutants invariably develop pancreatic and renal cysts if they survive to day 15.5 post coitum and die in either the fetal or the perinatal period. In contrast, mice with heterozygous mutations of Pkd1 are generally normal and have few if any renal cysts. These features have limited the utility of these models as tools to study the pathogenesis of cyst formation and the effect of various therapeutic interventions on disease progression. This report describes a new line of mice with a floxed allele of Pkd1 (Pkd1(cond)) that has an FRT-flanked neomycin cassette inserted into intron 1 and lox P sites inserted into intron 1 and intron 4. The Pkd1(cond) allele is fully functional, and homozygotes are viable and healthy. It is shown that the lox P and FRT sites can be selectively induced to recombine to produce two new alleles, Pkd1(del2-4) and Pkd1(cond-Deltaneo), by crossing to animals that express either the cre or FLPe recombinase, respectively. It is found that Pkd1(del2-4) allele functions as a true null, whereas presence or absence of the neomycin gene has no functional effects. It also is shown that somatic loss of Pkd1 results in renal and hepatic cysts. This new line of mice will be invaluable in the study of Pkd1 biology and serve as a powerful new tool that can be used to study the pathogenesis of autosomal dominant polycystic kidney disease.

When spores of the anaerobic bacteriumClostridium novyi-NTare systemically injected into animals, they germinate exclusively within the hypoxic regions of cancers. The germinated bacteria destroy adjacent tumor cells but spare a rim of well oxygenated tumor cells that subsequently expand. Surprisingly, we found that ≈30% of mice treated with such spores were cured of their cancers despite the viable tumor rim initially remaining after spore germination. The mechanism underlying this effect was shown to be immune-mediated, because cured animals rejected a subsequent challenge of the same tumor. Similar effects were observed in rabbits with intrahepatic tumors. It was particularly notable that the induced immune response, when combined with the bacteriolytic effects ofC. novyi-NT, could eradicate large established tumors.

Imperfect maintenance of genome integrity has been postulated to be an important cause of aging. Here we provide support for this hypothesis by demonstrating that the disruption of PASG (lsh), a SNF2-like factor that facilitates DNA methylation, causes global hypomethylation, developmental growth retardation and a premature aging phenotype. PASG mutant mice display signs of growth retardation and premature aging, including low birth weight, failure to thrive, graying and loss of hair, reduced skin fat deposition, osteoporosis, kyphosis, cachexia, and premature death. Fibroblasts derived from PASG mutant embryos show a replicative senescence phenotype. Both PASG mutant mice and fibroblasts demonstrate a markedly increased expression of senescence-associated tumor suppressor genes, such as p16INK4a, that is independent of promoter methylation, but, instead, is associated with down-regulation of bmi-1, a negative regulator of p16INK4a. These studies show that PASG is essential for properly maintaining DNA methylation and gene expression patterns that are required for normal growth and longevity. PASG mutant mice provide a useful model for the study of aging as well as the mechanisms regulating epigenetic patterning during development and postnatal life.

Current approaches for treating cancer are limited, in part, by the inability of drugs to affect the poorly vascularized regions of tumors. We have found that C. novyi-NT in combination with anti-microtubule agents can cause the destruction of both the vascular and avascular compartments of tumors. The two classes of microtubule inhibitors were found to exert markedly different effects. Some agents that inhibited microtubule synthesis, such as HTI-286 and vinorelbine, caused rapid, massive hemorrhagic necrosis when used in combination with C. novyi-NT. In contrast, agents that stabilized microtubules, such as the taxanes docetaxel and MAC-321, resulted in slow tumor regressions that killed most neoplastic cells. Remaining cells in the poorly perfused regions of tumors could be eradicated by C. novyi-NT. Mechanistic studies showed that the microtubule destabilizers, but not the microtubule stabilizers, radically reduced blood flow to tumors, thereby enlarging the hypoxic niche in which C. novyi-NT spores could germinate. A single intravenous injection of C. novyi-NT plus selected anti-microtubule agents was able to cause regressions of several human tumor xenografts in nude mice in the absence of excessive toxicity.

To examine the role of ataxia-telangiectasia mutated (Atm) in telomere function, we generated Atm and telomerase null mice (Atm(-/-) mTR(-/-) iG6 mice). These mice exhibited increased germ cell death and chromosome fusions compared with either Atm(-/-) or mTR(-/-) iG6 mice. Furthermore, the Atm(-/-) mTR(--) iG6 mice had a delayed onset and reduced incidence of thymic lymphoma compared with Atm(-/-) mice. The tumors in the Atm(-/-) mTR(-/-) iG6 mice showed increased apoptosis and anaphase bridges. Finally, lymphomas from Atm(-/-) mTR(-/-) iG6 mice were derived from CD8 immature, single-positive T cells, whereas Atm(-/-) lymphomas were from CD4(+)CD8(+) double-positive T cells. We propose that Atm protects short telomeres and that Atm deficiency cooperates with short telomeres, leading to increased cell death, decreased tumorigenesis, and increased overall survival.

Variation in the effects of selective estrogen receptor modulators (SERMs) on the estrous cycle and reproductive organs during aging could play an important role in the observed heterogeneity of tamoxifen chemoprevention efficacy against breast cancer.Of the 1,022 female Sprague Dawley rats enrolled in a long-term tamoxifen chemoprevention study, 87 were randomly chosen from four groups (irradiated, irradiated and tamoxifen treated, tamoxifen treated, and control). Vaginal smears were evaluated for determination of cycle stage, and vaginal pathologic changes. Correlation with the histologic features of reproductive tissues in 43 animals was made.More tamoxifen-treated (21.9%; 7/32) rats had irregular cycling than did control (9%; 3/23) rats. Ovarian granulosa cell hyperplasia was present in 50% (3/6) of tamoxifen-treated rats, and 20% (2/10) of control rats. Endometrial-type cells (ETCs) were present only in tamoxifen-treated (tamoxifen alone 6.25% [2/32]) and tamoxifen/ radiation-treated (28.6% [4/14]) rats.The modified Papanicolaou stain used here provided excellent morphologic detail for evaluating the estrous cycle in rodents. Tamoxifen altered vaginal cytologic and ovarian histologic features during aging. Results indicated that tamoxifen had direct and indirect effects on the reproductive tract, causing disturbance of the estrous cycle, shedding of ETCs, and promoting granulosa cell hyperplasia. Understanding of the heterogeneous response to tamoxifen chemoprevention during aging in rodents may provide important insights into the basis for tamoxifen chemoprevention failures in humans.

Marfan syndrome is a connective tissue disorder caused by mutations in the gene encoding fibrillin-1 (FBN1). A dominant-negative mechanism has been inferred based upon dominant inheritance, mulitimerization of monomers to form microfibrils, and the dramatic paucity of matrix-incorporated fibrillin-1 seen in heterozygous patient samples. Yeast artificial chromosome-based transgenesis was used to overexpress a disease-associated mutant form of human fibrillin-1 (C1663R) on a normal mouse background. Remarkably, these mice failed to show any abnormalities of cellular or clinical phenotype despite regulated overexpression of mutant protein in relevant tissues and developmental stages and direct evidence that mouse and human fibrillin-1 interact with high efficiency. Immunostaining with a human-specific mAb provides what we believe to be the first demonstration that mutant fibrillin-1 can participate in productive microfibrillar assembly. Informatively, use of homologous recombination to generate mice heterozygous for a comparable missense mutation (C1039G) revealed impaired microfibrillar deposition, skeletal deformity, and progressive deterioration of aortic wall architecture, comparable to characteristics of the human condition. These data are consistent with a model that invokes haploinsufficiency for WT fibrillin-1, rather than production of mutant protein, as the primary determinant of failed microfibrillar assembly. In keeping with this model, introduction of a WT FBN1 transgene on a heterozygous C1039G background rescues aortic phenotype.

An important process in the generation of tolerance to peripheral self-Ags is the induction of unresponsiveness in mature specific T cells. Although the end stage of this process, termed anergy, is well defined, the pathway by which naive T cells become anergic remains to be elucidated. Using an in vivo self-tolerance model, we demonstrate that CD4+ T cells pass through a significant effector stage on their way to an anergic state. This stage is characterized by production of effector cytokines, provision of help for CD8+ T cells, and induction of in vivo pathology within organs that express cognate Ag. These results suggest that the initial activation stage in T cell tolerance is similar to that seen in memory induction. They also suggest that autoimmune pathology can result during the natural process of tolerance induction rather than requiring that tolerance be broken.

Background Medulloblastomas, embryonal tumors arising in the cerebellum, commonly contain mutations that activate Wnt signaling. To determine whether increased Wnt signaling in the adult CNS is sufficient to induce tumor formation, we created transgenic mice expressing either wild-type or activated β-catenin in the brain. Methods Wild-type and mutant human β-catenin transgenes were expressed under the control of a murine PrP promoter fragment that drives high level postnatal expression in the CNS. Mutant β-catenin was stabilized by a serine to phenylalanine alteration in codon 37. Results Expression of the mutant transgene resulted in an approximately two-fold increase in β-catenin protein levels in the cortex and cerebellum of adult animals. Immunohistochemical analysis revealed nuclear β-catenin in hippocampal, cortical and cerebellar neurons of transgenic animals but not in non-transgenic controls. Tail kinking was observed in some transgenic animals, but no CNS malformations or tumors were detected. Conclusions No tumors or morphologic alterations were detected in the brains of transgenic mice expressing stabilized β-catenin, suggesting that postnatal Wnt signaling in differentiated neurons may not be sufficient to induce CNS tumorigenesis.

Cytoplasmic nucleophosmin (NPMc + ) mutations and FMS-like tyrosine kinase 3 ( FLT3 ) internal tandem duplication (ITD) mutations are two of the most common known molecular alterations in acute myeloid leukemia (AML); they frequently occur together, suggesting cooperative leukemogenesis. To explore the specific relationship between NPMc+ and FLT3/ITD in vivo, we crossed Flt3/ITD knock-in mice with transgenic NPMc+ mice. Mice with both mutations develop a transplantable leukemia of either myeloid or lymphoid lineage, definitively demonstrating cooperation between Flt3/ITD and NPMc+. In mice with myeloid leukemia, functionally significant loss of heterozygosity of the wild-type Flt3 allele is common, similar to what is observed in human FLT3/ITD+ AML, providing further in vivo evidence of the importance of loss of wild-type FLT3 in leukemic initiation and progression. Additionally, in vitro clonogenic assays reveal that the combination of Flt3/ITD and NPMc+ mutations causes a profound monocytic expansion, in excess of that seen with either mutation alone consistent with the predominance of myelomonocytic phenotype in human FLT3/ITD+/NPMc+ AML. This in vivo model of Flt3/ITD+/NPMc+ leukemia closely recapitulates human disease and will therefore serve as a tool for the investigation of the biology of this common disease entity.

Current chemotherapeutic approaches for cancer are in part limited by the inability of drugs to destroy neoplastic cells within poorly vascularized compartments of tumors. We have here systematically assessed anaerobic bacteria for their capacity to grow expansively within avascular compartments of transplanted tumors. Among 26 different strains tested, one ( Clostridium novyi ) appeared particularly promising. We created a strain of C. novyi devoid of its lethal toxin ( C. novyi-NT ) and showed that intravenously injected C. novyi-NT spores germinated within the avascular regions of tumors in mice and destroyed surrounding viable tumor cells. When C. novyi-NT spores were administered together with conventional chemotherapeutic drugs, extensive hemorrhagic necrosis of tumors often developed within 24 h, resulting in significant and prolonged antitumor effects. This strategy, called co mbination ba cterio l ytic t herapy (COBALT), has the potential to add a new dimension to the treatment of cancer.

We report, to our knowledge, the first HIV type 1 (HIV-1) transgenic (Tg) rat. Expression of the transgene, consisting of an HIV-1 provirus with a functional deletion of gag and pol , is regulated by the viral long terminal repeat. Spliced and unspliced viral transcripts were expressed in lymph nodes, thymus, liver, kidney, and spleen, suggesting that Tat and Rev are functional. Viral proteins were identified in spleen tissue sections by immunohistochemistry and gp120 was present in splenic macrophages, T and B cells, and in serum. Clinical signs included wasting, mild to severe skin lesions, opaque cataracts, neurological signs, and respiratory difficulty. Histopathology included a selective loss of splenocytes within the periarterial lymphoid sheath, increased apoptosis of endothelial cells and splenocytes, follicular hyperplasia of the spleen, lymphocyte depletion of mesenteric lymph nodes, interstitial pneumonia, psoriatic skin lesions, and neurological, cardiac, and renal pathologies. Immunologically, delayed-type hypersensitivity response to keyhole limpet hemocyanin was diminished. By contrast, Ab titers and proliferative response to recall antigen (keyhole limpet hemocyanin) were normal. The HIV-1 Tg rat thus has many similarities to humans infected with HIV-1 in expression of viral genes, immune-response alterations, and pathologies resulting from infection. The HIV-1 Tg rat may provide a valuable model for some of the pathogenic manifestations of chronic HIV-1 diseases and could be useful in testing therapeutic regimens targeted to stages of viral replication subsequent to proviral integration.

To evaluate renal cryosurgery by studying the feasibility of laparoscopic delivery and the radiographic characteristics and histopathologic effects in a porcine model using different freeze cycles. On the basis of the results, a clinical trial of laparoscopic cryosurgical ablation in select patients with clinical stage T1 renal tumors was started.Twelve kidneys from six farm pigs underwent cryosurgery. Each kidney was treated with two freeze cycles to -180 degrees C. Six kidneys were retroperitonealized, and six were not. An abdominal CT scan was performed at various times to evaluate for the presence of urinoma or hematoma and to monitor lesion changes. Organs were harvested at times ranging from 24 hours to 13 weeks. Radiographic and histopathologic changes were recorded for each time period. Eight patients with small (average 2-cm) exophytic renal masses underwent laparoscopic biopsy and cryosurgical ablation using a 3- or 4.8-mm probe (Cryomedical Sciences Inc., Rockville, MD) for one 15-minute or two 5-minute freeze cycles to a temperature of -180 degrees C to extend the ice ball at least 7 mm beyond the tumor margin.Dense adhesions between the bowel and cryoablated renal tissue were encountered in all non-retroperitonealized kidneys, but no fistula formation was present. The retroperitonealized kidneys had minimal adhesion formation. None of the animals developed a urinary fistula. At 24 hours and 1 week, CT scanning demonstrated an enhancement defect corresponding to the region of the ice ball with no urinoma or hematoma. At 13 weeks, only a nonenhancing cortical defect was seen. At immediate harvest, hemorrhage was noted in the area of the ice ball with a sharp demarcation at the edge of the freeze zone. At 1 week, four distinct zones were seen: central necrosis, inflammatory infiltrate, hemorrhage, and fibrosis with regeneration. At 13 weeks, the necrotic tissue had been replaced with a circumscribed area of fibrosis. There were no intraoperative or postoperative complications in the eight patients. The estimated blood loss was 140 mL, and the mean hospital stay was 3.5 days. At a mean clinical follow-up of 7.7 (range 1-18) months and radiographic follow-up of 5 months; there have been no tumor recurrences or significant changes in the serum creatinine concentration. At 24 hours, there was an enhancement defect in the area of the ice ball. The CT images at 13 weeks showed a nonenhancing cortical defect in the area of the ice ball.Cryosurgery can be readily delivered laparoscopically, creating a discrete lesion at the time of treatment that appears to be consistent over time. In the animal studies, complete tissue necrosis developed in the freeze zone, followed by reabsorption, and by 13 weeks, fibrous tissue had replaced the defect. In the animal and human trials, there were no operative complications, urinomas, hematomas, or bowel or urinary fistulas. Follow-up imaging in human trials revealed a persistent nonenhancing defect in the area of the freeze zone. Long-term clinical follow-up will be necessary to determine the cancer-free survival rate.

NO ⋅ is a free radical that modulates heart function and metabolism. We report that a neuronal-type NO synthase (NOS) is located on cardiac sarcoplasmic reticulum (SR) membrane vesicles and that endogenous NO ⋅ produced by SR-associated NOS inhibits SR Ca 2+ uptake. Ca 2+ -dependent biochemical conversion of l -arginine to l -citrulline was observed from isolated rabbit cardiac SR vesicles in the presence of NOS substrates and cofactors. Endogenous NO ⋅ was generated from the vesicles and detected by electron paramagnetic resonance spin-trapping measurements. Immunoelectron microscopy demonstrated labeling of cardiac SR vesicles by using anti-neuronal NOS (nNOS), but not anti-endothelial NOS (eNOS) or anti-inducible NOS (iNOS) antibodies, whereas skeletal muscle SR vesicles had no nNOS immunoreactivity. The nNOS immunoreactivity also displayed a pattern consistent with SR localization in confocal micrographs of sections of human myocardium. Western blotting demonstrated that cardiac SR NOS is larger than brain NOS (160 vs. 155 kDa). No immunodetection was observed in cardiac SR vesicles from nNOS knockout mice or with an anti-nNOSμ antibody, suggesting the possibility of a new nNOS-type isoform. 45 Ca uptake by cardiac SR vesicles, catalyzed by Ca 2+ -ATPase, was inhibited by NO ⋅ produced endogenously from cardiac SR NOS, and 7-nitroindazole, a selective nNOS inhibitor, completely prevented this inhibition. These results suggest that a cardiac muscle nNOS isoform is located on SR of cardiac myocytes, where it may respond to intracellular Ca 2+ concentration and modulate SR Ca 2+ ion active transport in the heart.

While oxygen free radicals are important mediators of brain injury, questions remain regarding which cell types and enzyme pathways trigger this radical generation. Microglial cells have been hypothesized to be an important source of radical generation; however, the magnitude, kinetics, and mechanism of this process are unknown. Oxygen radical generation by stimulated primary microglia was directly measured and characterized by electron paramagnetic resonance spin trapping. Microglia, when stimulated by phorbol ester or opsonified zymosan, gave rise to EPR spectra characteristic of superoxide. Experiments performed in the presence of superoxide dismutase, catalase, deferoxamine, and dimethyl sulfoxide excluded generation of hydroxyl radicals in significant amounts. Microglial superoxide generation was blocked by the NADPH oxidase inhibitor diphenylene iodonium in a manner similar to that seen in neutrophils, suggesting that a neutrophil like NADPH oxidase was the source of superoxide production. However, microglia produced 20 to 40 times less superoxide compared to a similar number of neutrophils during the first 30 min following stimulation, indicating a marked difference in the regulation of NADPH oxidase activation. Western blots of microglia lysates demonstrated that both large (gp91-phox) and small (p22-phox) NADPH oxidase subunits are expressed in both unstimulated and stimulated microglia. Indirect immunofluorescence demonstrated localization at the membrane surfaces of activated cells. Thus, microglial cells generate superoxide via a neutrophil-like NADPH oxidase but exhibit distinctly different time course and magnitude of activation than that seen in neutrophils.

Despite advances in our ability to detect and treat breast cancer, it remains a leading cause of death in women with cancer worldwide, and the incidence is rising. Approximately 15-20% of all cases are classified as triple negative breast cancer (TNBC), a subtype that is frequently associated with rapid progression and poor outcome. TNBC refers to the lack of detectable markers for the estrogen receptor (ER), progesterone receptor (PR), and Her2/neu amplification. These tumors do not respond to our most effective and least toxic therapies, including hormonal therapy (tamoxifen) or trastuzumab. We are studying molecular pathways that lead to tumor progression in TNBC and can be targeted with novel therapies. Our focus is the high mobility group A1 (HMGA1) oncogene. HMGA1 is highly expressed during embryogenesis, with low or undetectable levels in differentiated, adult tissues. HMGA1 is enriched in virtually all high-grade (poorly differentiated) cancers studied to date, including TNBCs, and high expression portends a poor prognosis in breast and other cancers. To investigate the role of HMGA1 in tumor progression in breast cancer, we silenced HMGA1 expression in TNBC cell lines (MDA-MB-231, Hs578T) using lentiviral-mediated delivery of short hairpin RNA. Strikingly, proliferation was markedly impaired, and many cells underwent apoptotic cell death within 5 days following HMGA1 knock-down. Surprisingly, cell morphology also changed dramatically, whereby the fibroblast-like, spindle-shaped cells became cuboidal and epithelial-like, consistent with a mesenchymal-epithelial transition, or MET. E-CADHERIN mRNA was induced, while both SNAIL and VIMENTIN were repressed in the knock-down cells, also consistent with MET. In addition, silencing HMGA1 blocked migration, invasion, and the formation of tumor foci in the lungs following tail vein injection of MDA-MB-231 cells. Moreover, both primary tumorigenesis and metastatic progression to the lungs were markedly inhibited in MDA-MB-231 cells with knock-down of HMGA1 following implantation in mammary fat pads. Furthermore, silencing HMGA1 blocked primary and secondary mammosphere formation, indicating that HMGA1 is required for this stem cell property. Tumorigenesis experiments at limiting dilutions showed that silencing HMGA1 depletes the tumor initiator/cancer stem cell pool. Using global gene expression analysis, we identified an HMGA1 signature of differentially-regulated genes in the control cells compared to knock-down cells. We also found that the HMGA1 signature is highly enriched in embryonic stem cells. Together, these findings indicate that silencing HMGA1 reprograms invasive, mesenchymal TNBCs into non-invasive, epithelial-like cells with slower growth and an altered gene expression signature. Studies are now needed to determine how to target HMGA1 in therapy. Citation Format: Sandeep N. Shah, Leslie Cope, Amy Belton, Weijie Poh, Saraswati Sukumar, David Huso, Linda Resar. HMGA1 reprograms breast cancer cells by inducing transcriptional networks involved in an epithelial-mesenchymal transition, stemness, and metastatic progression. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3122. doi:10.1158/1538-7445.AM2013-3122

Abstract 2374 Background: In response to DNA double-strand breaks (DSB), mammalian cells activate the DNA Damage Response (DDR), a network of factors that promotes their detection, signaling and repair. Among DDR factors, the Ataxia-Telangiectasia Mutated (ATM) kinase and the 53BP1 adaptor localize to chromatin surrounding DSBs, where 53BP1 is thought to transduce a subset of ATM-dependent reactions. In addition, 53BP1 may have ATM-independent functions via its interactions with the MRE11/RAD50/NBS1 (MRN) complex and others to protect DNA ends from degradation and to promote synapsis of distant DSBs. Both ATM and 53BP1 are tumor suppressors in humans and mice, but whether the combined mutation may aggravate the DSB repair defect and/or accelerate oncogenesis in vivo is currently unknown. Objectives: 1) To generate a compound mouse model deficient for both ATM and 53BP1 and investigate their genetic interaction during growth and development; 2) To determine whether 53BP1 plays ATM-independent functions in DSB repair and cell cycle checkpoint activation in primary thymocytes; 3) To determine the effect of 53BP1 deficiency on the penetrance and latency of T cell lymphomas in ATM−/− mice. Design/Methods: We have generated 53BP1/ATM double knock out (DKO) mice and assessed the kinetics of DSB signaling and repair in primary thymocytes and mature B cells. We have also followed cohorts of DKO and control mice for tumor development and evaluated mechanisms of tumorigenesis using a combination of molecular, biochemical and cytogenetic approaches. Results: When compared to ATM−/− mice, DKO mice showed no differences in organismal growth and development, or in lymphoid differentiation. However, DKO mice developed T cell lymphomas with higher penetrance and at younger age than ATM−/− mice, and their survival was markedly reduced (p=0.0001; log-rank test). Like ATM−/− lymphomas, DKO lymphomas contained clonal translocations with breakpoints at the TCRα/δ locus and amplification of upstream DNA sequences, indicating that 53BP1 deficiency markedly accelerates the acquisition of oncogenic translocations in an ATM−/− background. In primary cells, loss of 53BP1 further increases the frequency of ionizing irradiation (IR)-induced chromosomal aberrations in ATM−/− cells. We will present a model for how 53BP1 actions at DSBs suppress genomic instability in ATM-deficient thymocytes, including its effects on checkpoint activation and DSB repair. Conclusion: Our studies in a mouse model of T cell lymphomagenesis suggest that mutations in 53BP1 and ATM may be nonmutually exclusive in human lymphomas. The combined mutation may define a subgroup of patients with more aggressive hematological malignancies, and may have implications for their response to therapy with DNA damaging agents. Disclosures: No relevant conflicts of interest to declare.

Our research is directed at elucidating key transcriptional networks and underlying cellular pathways regulated by the high mobility group A1 (HMGA1) gene in refractory malignancies. Work from our group and others has shown that blocking HMGA1 interferes with multiple oncogenic phenotypes, including proliferation, anchorage-independent cell growth, migration, invasion, xenograft tumorigenicity, and metastatic progression in murine models. HMGA1 was also identified as a key transcription factor enriched in poorly differentiated solid tumors, leukemic stem cells, and embryonic stem (ES) cells. Importantly, previous studies on diverse tumors show that patients with tumors overexpressing HMGA1 have poor clinical outcomes. Together, these findings suggest that HMGA1 is a major regulator in diverse, poorly differentiated tumors and embryonic stem cells, although the molecular mechanisms through which HMGA1 functions remain unclear. We have begun to identify a few downstream genes and cellular pathways activated by HMGA1 in transformation. In gene expression profile analysis, we found that HMGA1 induces genes involved in mediating inflammatory signals. We identified NF-κB as a major node and inflammation as a major pathway. In previously published studies, we showed that HMGA1 induces expression of STAT3, COX-2, and MMP-2, genes which function in transformation and inflammation. Moreover, we found that anti-inflammatory agents block tumorigenesis in our HMGA1 transgenic mice and in xenograft tumors from aggressive human cancer cells overexpressing HMGA1. Together, these findings are consistent with the hypothesis that HMGA1 functions together with NF-κB to drive inflammatory pathways, induce transformation, and promote a poorly differentiated, stem-like state. To test this hypothesis and dissect the role of NF-κB in transformation induced by HMGA1, we have begun genetic experiments with HMGA1 transgenic mice and mice deficient in the major components of NF-κB. The NF-κB transcriptional complex is comprised primarily of p65:p50 heterodimers in which p65 functions as an activator, and p50, which lacks a transcriptional activation domain, is thought to function as an inhibitor. The p50/p65 heterodimers are thought to activate transcription of canonical NF-κB genes, whereas p50 homodimers repress transcription. Indeed, mice deficient in p50 have been shown to have enhanced NF-κB activity and inflammatory responses. In our preliminary studies, we found that the lymphoid tumors are significantly larger in the mice that are transgenic for HMGA1 and deficient in the inhibitory p50 subunit. This suggests that the excess in the p65 “activator” could lead to enhanced tumorigenesis induced by HMGA1. These studies should not only enhance our understanding of pathways associated with HMGA1 in malignant transformation, but also uncover novel biomarkers and therapeutic targets. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3039. doi:1538-7445.AM2012-3039

The molecular mechanisms that enable cancer cells to metastasize are poorly understood, although emerging evidence indicates that transcriptional networks required for stem cell properties during embryogenesis are co-opted by cancer cells during metastatic progression. To elucidate the molecular underpinnings of “stemness” in cancer and normal development, we investigated transcriptional networks and epigenetic alterations during the induction of pluripotent stem cells. Our focus is the high mobility group A1 (HMGA1) gene, which encodes proteins that bind to AT-rich regions of DNA and orchestrate the assembly of transcription factor complexes to alter chromatin structure and modulate gene expression. Our group first discovered that HMGA1 functions as a potent oncogene in cultured cells and causes aggressive tumors in transgenic mice. Recent studies also identified HMGA1 as a key transcription factor enriched in human embryonic stem (hESCs) cells, induced pluripotent stem cells (iPSCs), refractory leukemia, and high-grade/poorly differentiated cancers arising from diverse tissues. Together, these findings are consistent with the hypothesis that HMGA1 drives an undifferentiated, stem cell-like state during malignant transformation and normal development. To further investigate the role of HMGA1 in the stem cell state, we assessed its function in the derivation of iPSCs using multiple approaches. Here, we demonstrate for the first time that HMGA1 significantly enhances the reprogramming of somatic cells (bone marrow-derived mesenchymal stem cells, fetal lung cells, or mononuclear blood cells) into iPSCs together with OCT4, SOX2, KLF4, and cMYC (OSKM) using a retroviral or episomal, non-integrating approach. When hESCs are induced to differentiate, HMGA1 expression falls and parallels that of other pluripotency factors, such as OCT4, NANOG, and SOX2. We also found that forced expression of HMGA1 blocks differentiation of hESCs. To determine how HMGA1 induces a stem cell state, we assessed gene expression and epigenetic changes. During the reprogramming process, HMGA1 induces the expression of pluripotency and cancer genes, including SOX2, LIN28, and cMYC, while in hESCs, knock-down of HMGA1 results in repression of these genes. In addition, NANOG and OCT4 are repressed in hESCs following knock-down of HMGA1. By chromatin immunoprecipitation, HMGA1 binds to the promoters of SOX2, LIN28, and cMYC in vivo in hESCs. Moreover, HMGA1 is associated with decreased promoter methylation of select stem cell genes early in reprogramming. These findings uncover a key role for HMGA1 as a regulator of the stem cell state through transcriptional networks and epigenetic remodeling that induce pluripotency and an undifferentiated state. Further studies are needed to determine if HMGA1 pathways could be targeted in poorly differentiated, stem-like cancers or exploited in regenerative medicine. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 429. doi:1538-7445.AM2012-429

Abstract 865 Acute myeloid leukemia (AML) with myelodysplastic related changes represents 24–35% of all cases of AML and has a poor response to chemotherapy and a dismal prognosis. Activating mutations of the FMS-like tyrosine kinase 3 (FLT3) receptor have been seen in 5% of myelodysplastic syndrome (MDS) cases, and an additional 10% of patients with MDS acquire FLT3 mutations during progression to AML. We have previously generated a knock-in mouse model in which an internal tandem duplication (ITD) mutation was inserted into the murine Flt3 gene, which induces a lethal myeloproliferative neoplasm, but not progression to overt leukemia. One mouse model of MDS involves the transgenic expression of a Nup98-HoxD13 (NHD13) fusion under the hematopoietic specific vav promoter. We bred the FLT3/ITD knock-in mice to the NHD13 transgenic mice to see if the two genetic alterations would cooperate. Strikingly, the FLT3/ITD-NHD13 mice on the FVB/N background developed acute leukemia with 100% penetrance and a mean survival of 95 ± 32 days (p Competitive repopulation experiments showed that leukemic bone marrow was able to engraft in lethally irradiated recipients, with 1:200 cells in the bulk bone marrow having the potential to generate leukemia in recipient mice. To determine the identity of the leukemic initiating cell (LIC), the bulk bone marrow was further sorted into the multipotent progenitor (MPP), common myeloid progenitor (CMP), granulocyte/macrophage progenitor (GMP), and megakaryocytic/erythroid progenitor (MEP) populations and limiting dilution transplantation was performed for each group. The highest engraftment potential was found in the MPP population (1:100 cells) with much rarer LICs from the CMP and GMP. The MEP population did not engraft in any recipient mice even at the highest cell dose used of 100,000 sorted cells. RNA was extracted from the total bone marrow of mice 3 months prior to the development of disease and showed overexpression of HoxA7, HoxA9, HoxB4, HoxB6, HoxC4, and HoxC6 in the FLT3/ITD-NHD13 mice compared to the age-matched wildtype or FLT3/ITD mice. The overexpression of these genes appeared to be primarily driven by the NHD13 transgene, although there was also an increase in HoxA7 and HoxA9 expression observed in the FLT3/ITD mice. Since Hox genes are known to play an important role in stem cell self-renewal, we isolated RNA from 2-month-old littermates and stained for the cell surface markers characterizing the KSL, MPP, ST-HSC, and LT-HSC populations. FLT3/ITD-NHD13 and FLT3/ITD mice had a significant increase in the percentage of KSLs, long term HSCs, and short term HSCs. FLT3/ITD mice showed increased numbers of MPPs with a smaller but significant increase in the FLT3/ITD-NHD13 cohort. A significant number of AML patients with FLT3/ITD mutations present with loss of the wildtype allele of FLT3 and additional patients acquire this loss at the time of relapse. Hemizygosity at the FLT3 locus in FLT3/ITD mutant AML patients is associated with an even more adverse prognosis compared to patients with an intact wild-type allele. We observed spontaneous loss of heterozygosity (LOH) occurring in 100% of the FLT3/ITD-NHD13 mice and resulting in loss of the wildtype Flt3 allele. The frequent LOH seen in the FLT3/ITD-NHD13 mice provides additional evidence that this transgenic mouse serves as an accurate model to further explore the molecular pathways that underlie the development of leukemias with activating FLT3 mutations. This mouse model is also one of the first models of MDS-related AML and should provide a means to explore the molecular pathways that underlie these devastating hematopoietic neoplasms. Disclosures: Borowitz: BD Biosciences: Research Funding.

Abstract 1371 Although the high mobility group AT-hook 1 (HMGA1) gene functions as a potent oncogene in experimental models and high expression of HMGA1 portends a poor prognosis in diverse tumors, its role in leukemogenesis has remained elusive. We showed previously that HMGA1 induces leukemic transformation in cultured cells and causes aggressive lymphoid leukemia in transgenic mice. Inhibiting HMGA1 expression blocks colony formation in human lymphoid leukemia cells in vitro. Moreover, high levels correlate with relapse in childhood acute lymphoblastic leukemia (ALL), suggesting that it plays an important role in ALL. Because HMGA1 functions as a chromatin remodeling protein that modulates gene expression, we hypothesized that it drives leukemogenesis by dysregulating specific genes and pathways. To identify genes and cellular pathways induced by HMGA1 that could be targeted in therapy, we performed global gene expression profile analysis from lymphoid cells from the HMGA1 transgenic mice at different stages in tumorigenesis. All HMGA1 transgenics succumb to lymphoid malignancy with complete penetrance by 8–12 months. Pooled RNA samples at 2 months (before tumors develop) and 12 months (after tumors are well-established) were analyzed for differential expression of >20,000 unique genes by microarray analysis (Affymetrix) using both a parametric and nonparametric approach. A subset of differentially expressed genes was confirmed using quantitative, RT-PCR. Differentially expressed genes were analyzed for cellular pathways and functions using Ingenuity Pathway Analysis (IPA; www.ingenuity.com) and Gene Set Enrichment Analysis. To determine if these genes and pathways were relevant in human ALL, we knocked down HMGA1 expression in human ALL cells and assessed expression of a subset of the differentially expressed genes. Early in leukemogenesis (at 2 months), 113 genes were differentially expressed in the HMGA1 transgenics compared to controls. In established leukemia (12 months), 715 genes were differentially expressed. In established tumors, the dysregulated genes are involved in cancer, cell cycle regulation, and cell-mediated immune response by Ingenuity Pathway Analysis. Geneset enrichment showed that embryonic stem cell genes are enriched in the established leukemic cells. At both early and late stages in leukemogenesis, differentially regulated genes are involved in cellular development, hematopoiesis, and hematologic development. Early in leukemogenesis, most of the significantly dysregulated genes are involved in the inflammatory response and included NF-kappaB as a major node. In human ALL cells, knock-down of HMGA1 also resulted in knock-down of genes identified in our transgenic model, suggesting that these HMGA1 regulated genes are also relevant to human ALL. In summary, we found that HMGA1 induces inflammatory pathways early in leukemogenesis and pathways involved in embryonic stem cells, cell cycle progression, and cancer in established tumors. HMGA1 also dysregulates genes involved in cellular development and hematopoiesis at both early and late stages of tumorigenesis. Some of these HMGA1 pathways were also present in human ALL cells. Moreover, these results provide mechanistic insight into HMGA1 function at different stages in leukemogenesis and point to cellular pathways that could serve as therapeutic targets in ALL. Disclosures: No relevant conflicts of interest to declare.

Abstract 46 In murine hematopoiesis, long-term hematopoietic stem cells (LT-HSCs) are classically defined as Fms-like tyrosine kinase (Flt3) negative with Flt3 positivity often serving as a marker for differentiation and loss of self- renewal capacity. Flt3 has previously been shown to be dispensable for HSC maintenance and myeloid development. Flt3 and Flt3-ligand knockout mice have minor defects in overall hematopoiesis. However, these studies fail to take into account the effects of overlapping or compensatory pathways that may mask important roles Flt3 may play in HSC homeostasis. To date, few studies have addressed the effects of constitutive Flt3 activity on normal hematopoiesis or stem cell function. These mutations in FLT3 are of particular importance as they comprise one of the most common molecular alterations found in human acute myeloid leukemias (AML). Internal tandem duplication (ITD) mutations in FLT3 render the receptor tyrosine kinase constitutively active and portend a poor prognosis for those patients carrying the mutation. Many of these patients respond only transiently to treatment, possibly due to a reservoir of stem-like cells that are able to escape therapy. To study the effects of expression of constitutive activation mutations of FLT3 in primitive HSC compartments, our lab has developed a murine knock-in model in which this mutation is expressed under control of the endogenous murine Flt3 promoter. Flt3-ITD mice develop a myeloproliferative neoplasm (MPN), display imbalances in hematopoietic stem and progenitor cell population distributions (of note, 2–5 fold reductions in primitive LT-HSCs in SLAM (LIN-CD150+CD48−) and KSL-SLAM (Kit+Sca+Lin-CD150+CD48−) populations) and HSC defects (as measured by significantly reduced engraftment potential). Here, we show these perturbations to be the result of a Flt3-ITD-driven, cell-autonomous, overproliferation within a compartment of normally quiescent LT-HSCs, as evidenced by increased BrdU incorporation, cell cycle entry and upregulation of genes that promote cell cycle (Pim1, Ccnd1). This rapid HSC exhaustion and depletion of the LT-HSC compartment is coupled to the activation of downstream Flt3 signaling, most prominently through Stat5 with observed increases in expression of Stat5 targets (e.g. Fos, JunB, Mkp1, Socs3). Furthermore, for the first time, we show that Flt3 is expressed at detectable (albeit low) levels in this MPN-initiating, LT-HSC cell population and capable of exerting significant functional effects in this compartment. Surprisingly, the depletion and defects in HSC function are completely reversible upon treatment with the small molecule inhibitor, sorafenib, while simultaneously leading to near complete ablation of disease. Taken together, our data implicate previously unrecognized roles for Flt3 in the extremely dynamic process of stem cell homeostasis and quiescence, which appears closely coupled to pathogenesis, as well as pointing to Flt3 as an attractive target for targeted therapy. Disclosures: No relevant conflicts of interest to declare.

Abstract 924 FLT3 is a tyrosine kinase receptor that is frequently mutated in acute myeloid leukemia (AML). The most common mutations involve either an internal tandem duplication (ITD) in the juxtamembrane domain or a point mutation in the tyrosine kinase domain (TKD). The most common TKD mutation is a D835Y mutation in which aspartic acid is mutated to tyrosine at residue 835. ITD mutations are reported in approximately 25% of adult AML and 15% of pediatric leukemia. TKD mutations occur in approximately 8–10% of both adult and pediatric leukemia. Both of these mutations constitutively activate FLT3, but only patients with an ITD mutation have a significantly poorer prognosis. Small molecule inhibitors against FLT3 are currently in clinical trials with some success, but resistance to single agent treatment frequently occurs. A better understanding of the molecular mechanisms behind the prognostic difference, between the two types of mutations and the activation, inhibition, and resistance of FLT3 will hopefully lead to more tailored treatments of leukemia. To help address some of these questions we have generated a knock-in mouse with an Aspartic acid to Tyrosine mutation at reside 838 (D838Y) which is homologous to the FLT3/D835Y mutation reported in the majority of human leukemia cases with a FLT3-TKD mutation. These mice have been compared to the mice with a FLT3-ITD mutation previously generated and characterized by the lab. Mice with either the FLT3/D838Y or FLT3-ITD mutation develop predominantly a myeloproliferative disease. Mice with the FLT3/D838Y mutation show a less aggressive phenotype when compared to mice with the FLT3/ITD mutation in terms of survival, spleen size, and histopathologic changes of the bone marrow, spleen and liver.8 weeks spleen wt (g)12 weeks spleen wt (g)wildtype0.079 g ± 0.011 (n=27)0.092 g ± 0.009 (n=5)D838Y/wt0.155 g ± 0.027 (n=21)0.184 g ± 0.027 (n=9)ITD/wt0.185 g ± 0.034 (n=12)0.267 g ± 0.052 (n=8) Flow cytometry analysis shows that bone marrow from 8–12 week old mice with the FLT3/D838Y mutation have a marginally expanded fraction of granulocytic/monocytic lineage cells in their bone marrow compared to their wildtype littermate controls, but a lower fraction when compared to age-matched FLT3/ITD mice. B cell development is significantly blocked at the late pro-B to pre-B transition in the FLT3/ITD mice, with very few cells developing into pre-B or more mature B cells (Li et al. Blood. 117(11): 3131-9). In contrast, FLT3/D838Y mice demonstrate an expansion of early and late pro-B cell populations, with a significant fraction of cells maturing beyond the pre-B stage to become mature B cells. In mice with the FLT3/D835Y mutation the long-term HSC (LT-HSC) compartment, responsible for the self-renewal and maintenance of the hematopoietic stem/progenitor cell pool, appears to have a greater than 2-fold expansion in the number of immunophenotypic LT-HSCs. This appears to be a functional expansion as the BM cells from FLT3/D835Y mice have long-term engraftment superiority compared with ITD and wildtype mice. The short term HSC and MPP compartments from both FLT3/D838Y and FLT3/ITD mice appear to be comparably expanded compared to wildtype controls. In human leukemia, FLT3/ITD signaling results in the activation of downstream targets, including STAT5, MAP kinase and AKT. Consistent with the intermediate phenotype observed, intracellular levels of phospho-Stat5 in the FLT3-D838Y mice were lower than levels measured in the FLT3-ITD mice, but higher than those of wildtype littermate controls. RT-PCR measured expression levels of FLT3 and Pim1 in lineage negative cells of D838Y mice fall between those of the wildtype and ITD mice. (Ratios: FLT3 – ITD/wt 3.7; D838Y/wt 2.5; Pim1 – ITD/wt 3.4; D838Y/wt 2.5). In summary, we have generated a knock-in mouse model with the FLT3 kinase domain D838Y mutation. The mice demonstrate a less aggressive phenotype when compared to knock-in mice with the FLT3/ITD mutation, analogous to the less aggressive AML seen in cases involving D835 mutations compared to ITD mutations. Both mouse models on the same mouse background provide a useful platform for understanding the molecular mechanisms of FLT3 mutations and for testing new therapeutic agents targeting FLT3. Disclosures: No relevant conflicts of interest to declare.

Purpose: We describe a nephron‐based dosimetrymodel for renal toxicity for alpha‐particle radipharmaceutical therapy suited to the short range and high linear energy transfer of alpha‐particle emitters. This model is applied to Ac‐225‐7.16.4 radioimmunotherapy of murine metastatic breast cancer, where renal toxicity is observed for whole‐kidney and renal cortex absorbed dose values below toxicity thresholds established by external beam and targeted radiopeptide therapy. Methods: Using GEANT4 Monte Carlosoftware an idealized nephron model is created using geometrical and anatomical parameters taken from the literature and from murine samples. These samples also provide the data for volume of occupancy of the different compartments of the nephron in the kidney: glomerulus vs. proximal tubule vs. distal tubule. Radiopharmaceutical uptake in the different components of potential toxicity from (a) bound Ac‐225 and (b) free Bi‐213 are taken from experimental data and the literature and supplemented by tissueimaging with a beta‐Imager. Monte Carlo simulations were run, the absorbed doses to the model components were calculated and the results compared to experimental observations of renal toxicity. The renal cortex and medulla absorbed doses were also calculated and the results compared to traditional absorbed fraction results. Results: Preliminary results show an absorbed dose to the proximal tubule and glomerular cells approximately 3 times greater than that to the nephron as a whole. This difference in conjunction with the relative biological effect of alpha particles (a factor of about 5) would be consistent with the toxicity observed in the murine experiments. The renal cortex and renal medulla absorbed doses are consistent with the absorbed fraction methodology. Conclusions: Alpha‐particle radipharmaceutical therapy is a promising treatment modality for cancer. The nephron model enables optimal implementation of treatment and is a critical step in understanding toxicity for human translation of alpha‐particle radiopharmaceutical therapy.

Uterine cancer is the most common cancer of the female genital tract and the fourth most frequent cause for cancer death in women in the United States. Thus, further study has the potential to significantly benefit women's health worldwide. Despite the high prevalence of this disease, the molecular events that lead to uterine cancers are poorly understood. We are studying the role of the high mobility group A1 (HMGA1) gene in the pathogenesis of uterine cancer. The HMGA1 gene encodes the HMGA1a and HMGA1b chromatin remodeling proteins, which function in modulating gene expression. In a previously published pilot study, we showed that HMGA1 is overexpressed in poorly differentiated uterine cancers, but not in normal uterine tissue, benign tumors, or most low-grade neoplasms of the uterus. We also engineered HMGA1 transgenic mice and found that all females develop uterine sarcomas by 9 months of age. In lung cancer, we reported that HMGA1 induces expression of the matrix metalloproteinase-2 (MMP-2) gene, but only in poorly differentiated tumors, suggesting that this pathway could drive tumor progression and a poorly differentiated state. Here, we investigated the role of the HMGA1-MMP-2 pathway in the pathogenesis of uterine tumorigenesis. Using a genetic approach, we crossed our HMGA1 transgenic mice with mice that are null for the MMP-2 gene. We found that the uterine tumors were significantly smaller in the HMGA1 transgenic mice that are also null for MMP-2. To determine if this pathway is involved in human uterine tumorigenesis, we assessed expression of the HMGA1 and MMP-2 genes in primary, human uterine tumors. We studied: 1.) endometrioid tumors, which are derived from the endometrial epithelium and tend to be low-grade (well-differentiated), 2.) serous tumors, which are also derived from the endometrial epithelium, but tend to be high-grade (poorly differentiated), and, 3.) carcinosarcomas (or mixed malignant, mullerian tumors), which are derived from the endometrial epithelium and stroma and tend to by high-grade. We found that HMGA1 expression was highest in the high-grade serous tumors and carcinosarcomas, with lower levels in the endometrioid tumors. Moreover, we found that there was a significant correlation between HMGA1 and MMP-2, but only in the high-grade carcinosarcomas and serous tumors. Taken together, our studies demonstrate that HMGA1 induces expression of MMP-2 during uterine tumorigenesis and suggest that this pathway is important in aggressive, poorly differentiated tumors. Although further studies are needed, our findings also suggest that the HMGA1-MMP-2 pathway could serve as a rational therapeutic target in poorly differentiated uterine cancer. 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 2905. doi:10.1158/1538-7445.AM2011-2905

The molecular mechanisms that enable colon cancer cells to evade therapy and metastasize are poorly understood and have not been effectively targeted by current therapies. Recent evidence suggests that tumor cells with stem-like properties (poor differentiation, long-term self-renewal, and a relatively quiescent state) are responsible for metastatic progression and resistance to therapy. Our laboratory is investigating molecular pathways that give rise to metastatic progression and “stemness” in refractory colon cancer with the long-term goal of identifying novel therapeutic targets. Our focus is the HMGA1 gene, which encodes the HMGA1a and HMGA1b chromatin remodeling proteins. HMGA1 is highly expressed during embryogenesis, but not in differentiated, adult tissues. Strikingly, HMGA1 is overexpressed in virtually all high-grade (poorly differentiated) cancers studied to date. To study the role of HMGA1 in tumor progression and the stem cell state, we engineered HMGA1a transgenic mice. As previously reported, these mice succumb to aggressive lymphoid malignancies. Here, we report that the HMGA1 transgenics also develop hyperproliferative changes in the intestines with hamartomatous polyps. In addition, we found that the HMGA1 mice develop an expansion in the intestinal stem cell compartment, suggesting that HMGA1 promotes the maintenance or survival of intestinal stem cells. To assess the role of HMGA1 in tumor progression and the stem cell phenotype in colon cancer, we inhibited HMGA1 expression in the poorly differentiated, HCT116 colon cancer cell line. We found that knock-down of HMGA1 blocks anchorage-independent cell growth, migration, and invasion in vitro. We also demonstrate that knock-down of HMGA1 blocks tumorigenesis and metastatic progression in vivo. Moreover, three-dimensional colonosphere formation is decreased in the knock-down cells, indicating that HMGA1 is required for this stem cell phenotype. To further assess the role of HMGA1 in colon cancer stem cells, we performed limiting dilution tumorigenesis experiments. Inhibiting HMGA1 expression blocks tumorigenesis at limiting dilutions, consistent with depletion of tumor-initiator cells in the HMGA1 knock-down cells. In HCT116 cells, we also found that HMGA1 induces expression of Vimentin and Twist1, two genes involved in embryogenesis, EMT, and tumor progression. Analysis of prior gene expression profile analyses show that HMGA1 is among the most enriched genes in colon cancer compared to normal mucosa and we confirmed this by quantitative, RT-PCR in a subset of primary, high-grade tumors. Taken together, these findings suggest that HMGA1 drives tumor progression by inducing a stem-like state. Although further studies are needed, our results also suggest that HMGA1 or downstream pathways could be rational therapeutic targets in poorly differentiated colon cancer. 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 931. doi:10.1158/1538-7445.AM2011-931

Background: Unlike normal cells, most cancer cells primarily metabolize glucose by glycolysis rather than oxidative phosphorylation (Warburg effect). The cancer cells are reprogrammed to acquire these fundamental metabolic alterations during malignant transformation via different mechanisms. These mechanisms include mitochondrial defects and malfunction, adaptation to a hypoxic tumor microenvironment, oncogenic signaling, and abnormal expression of metabolic enzymes. Cancer cells also rely on glutamine for anabolism and ATP synthesis. Recent studies have shown that inhibiting glycolytic pathways leads to a significant inhibition of growth of tumor xenografts. Our group has previously shown the MYC oncogene activates glutamine metabolism which serves as a major energy source in transformed cells. Objective: Examine the antitumor effect of a glutamine metabolic inhibitor, aminooxyacetate (AOA; aminotransferases/malate-aspartate shuttle inhibitor), alone and in combination with chemotherapy (doxorubicin and carboplatin) in a breast cancer xenograft model. Methods: Immunodeficient mice bearing MDA-MB-231 breast cancer cells were treated with AOA alone, doxorubicin or carboplatin alone, or AOA in combination with the chemotherapeutic agent for four weeks. The tumors were evaluated for histopathological and molecular alterations. Statistical analysis: Tumor growth was analyzed using a mixed-effects model for repeated measures data to account for dependence among observations within the same animal. Results: We observed a significant inhibition of tumor volume in mice treated with AOA in combination with doxorubicin or carboplatin compared to groups receiving single drugs (p Conclusion: Our results suggest that targeting the metabolic pathway results in significant tumor inhibition in preclinical models of breast cancer and potentially could serve as attractive therapeutic targets for breast cancer. 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 4088. doi:10.1158/1538-7445.AM2011-4088

It is estimated that the etiology of 20-30% of epithelial cancers is directly associated with inflammation, though the molecular events linking inflammation and the necessary carcinogenic mutations remain to be clarified. In the context of gastrointestinal disease, the bacterial pathogens Helicobacter pylori and enterotoxigenic Bacteroides fragilis (ETBF) are significant sources of chronic inflammation and have been implicated as risk factors for the development of gastric and colorectal cancer, respectively. We have previously demonstrated that H. pylori and the cytokine tumor necrosis factor-α (TNF-α) rapidly induce spermine oxidase (SMO), resulting in increased cellular reactive oxygen species (ROS) and DNA damage, in gastric and lung epithelial cells. In addition, tissue microarray studies revealed that increased SMO expression was associated with prostatic intraepithelial neoplasia and prostate cancer lesions and that benign prostate epithelial tissue from men with these diseases exhibits higher SMO expression than normal controls. We now demonstrate that purified B. fragilis toxin (BFT) upregulates SMO in HT29/c1 and T84 colonic epithelial cells, resulting in SMO-dependent induction of γ-H2A.x, a marker of DNA damage. Further, ETBF-induced colitis in wild-type or APC+/− Min C57Bl/6 mice is associated with increased SMO expression. Inhibition of SMO activity by MDL 72,527 in vivo (20 mg/kg i.p. 3 days per week) significantly reduces ETBF-induced chronic inflammation, proliferation, and induction of Th17-associated cytokines. Most importantly, in Min mice, treatment with the SMO inhibitor reduces ETBF-induced colon tumorigenesis by 69% (p Citation Information: Cancer Prev Res 2010;3(12 Suppl):A56.

Abstract 145 NPMc+ mutations occur in up to 60% of adult and 20% of childhood acute myeloid leukemia (AML) with normal karyotype. Flt3 ITD mutations occur in 25% of adult and 15% of childhood AML. Flt3 ITD mutations occur twice as frequently in patients with NPMc+ mutations compared to those who lack a NPMc+ mutation. The presence of Flt3 ITD portends a poor prognosis. NPMc+ is associated with improved outcome, but only in the absence of a concomitant Flt3 ITD mutations. Given the high frequency with which these mutations occur together it is plausible that they cooperate to cause leukemia. However, this has yet to be demonstrated experimentally. To examine this, we crossed mice expressing a knock-in of an 18-bp ITD mutation in the juxtamembrane domain of the murine Flt3 gene (Flt3 wt/ITD) with transgenic mice expressing Flag-tagged type A NPMc+ mutation driven by the myeloid-specific human MRP8 promoter. Flt3wt/ITD mice develop a fatal myeloproliferative disorder (Li L, et al. Blood. 2008;111:3849-58) and NPMc+ transgenic mice develop a non-fatal myeloproliferation (Cheng K, et al. Blood. 2010;115-18), but neither develop leukemia, suggesting that cooperating events are required. Progeny were characterized by: 1) H&E staining of peripheral blood smears, bone marrow (BM) cytospins, and spleen sections; 2) FACS analysis of BM cells, splenocytes and thymocytes to determine the disease phenotype; 3) RT PCR to examine the expression of Flt3 ITD and NPMc+; and 4) immunofluorescence (IF) using an anti-Flag antibody to determine localization of the NPMc+ protein. Mice harboring both mutations develop acute leukemia with a median onset of 285 days (Figure 1). All the leukemic mice exhibit a moribund appearance, leukocytosis (mean WBC 69.3±32.7 vs 11.7±8.3K/μ L in wt/wt mice), splenomegaly (mean weight 0.63±0.3 vs 0.12±0.02g in wt/wt mice), anemia and thrombocytopenia. Four disease phenotypes based on FACS and histologic analysis have been observed (Figure 2). Thirty-three percent develop AML with infiltration of the BM and spleen with Mac1+/Gr1+ myeloblasts. Thiry-three percent develop T cell ALL with infiltration of BM, spleen, thymuses and other organs with abnormal CD3+/CD4+/CD8+ lymphoblasts. An additional 33% develop a mixed phenotype acute T/myeloid leukemia with both Mac1+/Gr1+ myeloblasts and CD3+/CD4+/CD8+ lymphoblasts. One mouse developed an undifferentiated acute leukemia with primitive blasts expressing no markers specific for either lymphoid or myeloid lineage. RT-PCR of bulk leukemia cells demonstrates expression of NPMc+ and Flt3 ITD. IF of BM cells from leukemic mice demonstrates cytoplasmic localization of the NPMc+ protein. In summary, we have utilized a mouse model to demonstrate that NPMc+ and Flt3 ITD mutations cooperate to cause leukemia. Many of the mice with both mutations develop myeloid leukemia with features similar to the human disease. There is also a striking incidence of T cell leukemia, which is particularly interesting as the NPMc+ mutation is driven by the myeloid-specific hMRP8 promotor. It is possible that there is aberrant expression of NPMc+ in lymphoid cells due to position effects of the transgene or the activation of an endogenous leukemogenic retrovirus. Other disease models using this promoter have documented similar phenomenon (Jaiswal, et al. PNAS. 2003;100:10002-7). There is a long latency of disease onset which may be due to a relatively low level of expression of NPMc+ or because additional genetic or epigenetic events are required. Perhaps the Flt3 ITD mutation causes proliferation and NPMc+ impairs DNA repair resulting in the accumulation of additional mutations that contribute to leukemogenesis. This mouse model provides the first in vivo model of NPMc+/Flt3 ITD+ leukemia. The model will allow for the further study of this disease entity, including the examination of involved pathways and the exploration for potential therapeutic targets. Kaplan-Meier Survival Curve. Mice with both NPMc+ and Flt3 ITD mutations have a median survival of 413 days. FACS analysis of leukemic mice BM cells and thymocytes. Mice with AML have cKit+/Mac1+/Gr1+ myelobasts. Mice with T cell ALL have infiltration of the BM, spleen (not shown) and thymus with CD3+/CD4+/CD8+ lymphoblasts. Mice with T/myeloid leukemia have both Mac1+/Gr1+ myeloblasts and CD3+/CD4+/CD8+ lymphoblasts. Leukemic mice have depletion of maturing Ter119+ erythroid cells. Figure 1 Kaplan-Meier Survival Curve. Mice with both NPMc+ and Flt3 ITD mutations have a median survival of 413 days. Figure 1. Kaplan-Meier Survival Curve. Mice with both NPMc+ and Flt3 ITD mutations have a median survival of 413 days. Figure 2 FACS analysis of leukemic mice BM cells and thymocytes. Mice with AML have cKit+/Mac1+/Gr1+ myelobasts. Mice with T cell ALL have infiltration of the BM, spleen (not shown) and thymus with CD3+/CD4+/CD8+ lymphoblasts. Mice with T/myeloid leukemia have both Mac1+/Gr1+ myeloblasts and CD3+/CD4+/CD8+ lymphoblasts. Leukemic mice have depletion of maturing Ter119+ erythroid cells. Figure 2. FACS analysis of leukemic mice BM cells and thymocytes. Mice with AML have cKit+/Mac1+/Gr1+ myelobasts. Mice with T cell ALL have infiltration of the BM, spleen (not shown) and thymus with CD3+/CD4+/CD8+ lymphoblasts. Mice with T/myeloid leukemia have both Mac1+/Gr1+ myeloblasts and CD3+/CD4+/CD8+ lymphoblasts. Leukemic mice have depletion of maturing Ter119+ erythroid cells. Disclosures: No relevant conflicts of interest to declare.

Background Autosomal dominant polycystic kidney disease (ADPKD) is a common cause of inherited renal failure that results from mutations in PKD1 and PKD2. The disorder is characterized by focal cyst formation that involves somatic mutation of the wild type allele in a large fraction of cysts. Consistent with a two-hit mechanism, mice that are homozygous for inactivating mutations of either Pkd1 or Pkd2 develop cystic kidneys, edema and hemorrhage and typically die in midgestation. Cystic kidney disease is unlikely to be the cause of fetal loss since renal function is not required to complete gestation. One hypothesis is that embryonic demise is due to leaky vessels or cardiac pathology. Methodology/Principal Findings In these studies we used a series of genetically modified Pkd1 and Pkd2 murine models to investigate the cause of embryonic lethality in mutant embryos. Since placental defects are a frequent cause of fetal loss, we conducted histopathologic analyses of placentas from Pkd1 null mice and detected abnormalities of the labyrinth layer beginning at E12.5. We performed placental rescue experiments using tetraploid aggregation and conditional inactivation of Pkd1 with the Meox2 Cre recombinase. We found that both strategies improved the viability of Pkd1 null embryos. Selective inactivation of Pkd1 and Pkd2 in endothelial cells resulted in polyhydramnios and abnormalities similar to those observed in Pkd1−/− placentas. However, endothelial cell specific deletion of Pkd1 or Pkd2 did not yield the dramatic vascular phenotypes observed in null animals. Conclusions/Significance Placental abnormalities contribute to the fetal demise of Pkd−/− embryos. Endothelial cell specific deletion of Pkd1 or Pkd2 recapitulates a subset of findings seen in Pkd null animals. Our studies reveal a complex role for polycystins in maintaining vascular integrity.

Mutations in the Adenomatous Polyposis Colt (APC) gene have been implicated both in familial as well as sporadic gastrointestinal (GI) cancers. APC mutations are associated with autosomal dominant inheritance of disease in humans. Similarly, mice that contain a single mutant APC gene encoding a protein truncated at aa 716 (Ape716) develop multiple polyps throughout the GI tract as early as 4 weeks after birth. Inactivation of another tumor suppressor gene, Hypermethylated in Cancer 1 (HIC1) often occurs in human cancers, including GI, via CpG island hypermethylation. Homozygous deletion of Hic1 in mice results in major developmental defects and embryonic lethality. Hic1 heterozygotes have previously been shown to develop age dependent tumors of a variety of tissue types. We now report that loss of a single Hic1 allele can promote crypt dysplasia of the small intestine and further potentiate polyp formation in Apc716 mice. Hic1+/−, Apc716 double heterozygous mice develop increased numbers of polyps throughout the GI tract by 60 days. Hic1 expression is lost in polyps with a selective increase in DNA hypermethylation of this gene only in polyps from double-heterozygous mice. Immunohistochemical analyses of polyps show increases in markers such as -catenin, SIRT1, and Sox9, indicating aberrant Wnt and Hic1 signaling. Together, our data suggest that loss of a gene frequently silenced via epigenetic mechanisms, Hic1, can cooperate with loss of a gene often mutated in GI cancer, Apc, to promote tumorigenesis in an in-vivo model of multiple intestinal neoplasia. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr LB-92.

Although the HMGA1 gene is highly expressed in human embryonic stem cells and high-grade, poorly differentiated cancers, its function in these settings has not been clearly elucidated. The HMGA1 gene encodes the HMGA1a and HMGA1b protein isoforms, which are members of a family of non-histone, chromatin remodeling proteins. These proteins bind to the minor groove of AT-rich regions in the B-form of DNA, which leads to the recruitment of transcriptional factors that modulate gene expression. The HMGA1 gene is highly expressed during embryogenesis and in aggressive human cancers arising from different embryologic origins, including cancers of hematopoietic, lung, breast, prostate, and colon cells. Forced overexpression of HMGA1a or HMGA1b induces a transformed phenotype in cultured cells and inhibiting HMGA1 blocks transformation in human cancer cell lines. Because HMGA1 functions in transcriptional regulation, we hypothesize that it induces neoplastic transformation by dysregulating specific molecular pathways. To investigate the pathways disrupted by HMGA1, we are studying our HMGA1a transgenic mice as a model system. As previously reported, these mice develop aggressive T-cell leukemia with complete penetrance and females also develop uterine cancer. At necropsy, we also discovered polyps involving the small and large intestines. Histopathologically, the transgenic intestines are characterized by a thickened mucosal surface, hyperproliferative changes, and hamartomatous polyps. Immunohistochemical analysis reveals an increase in the population of intestinal cells expressing the colon stem cell marker leucine-rich repeat-containing G protein-coupled receptor 5 (Lgr-5) in transgenic compared to wildtype mice. These data support our observation of an expansion in the crypt progenitor/intestinal stem cell populations. Our preliminary results also show that inhibiting HMGA1 expression in colon cancer cell lines interferes with anchorage-independent cell growth and tumorigenesis, indicating that HMGA1 is required for these transformation phenotypes in colon cancer. In addition, we found that HMGA1 is enriched in >70% of primary tumors with high-grade colon cancer. These findings suggest that HMGA1 is important in maintaining the stem cell niche in the intestine. Moreover, HMGA1 could promote neoplastic transformation and tumor progression in the intestine by driving a stem-like phenotype in colonic cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4243.

Pancreatic ductal adenocarcinoma is a highly lethal cancer accounting for over 160,000 deaths worldwide each year. Despite the high prevalence of pancreatic cancer, the molecular events that lead to tumor progression are poorly understood. The high-mobility group A1 gene (HMGA1) is enriched in diverse, high-grade cancers and embryonic stem cells. This gene encodes the HMGA1a and HMGA1b chromatin remodeling proteins, which function in regulating gene expression. We previously showed that HMGA1 induces oncogenic transformation in cultured cells and causes aggressive cancers in transgenic mice. Moreover, we found that high HMGA1 protein levels correlate with poor differentiation status and decreased survival in a study of > 100 primary pancreatic ductal adenocarcinomas. These findings suggest that HMGA1 could drive a poorly differentiated, stem cell-like state and tumor progression in pancreatic ductal adenocarcinoma. In cultured, human pancreatic cells that overexpress K-RAS (HPNE-K-RAS cells), forced overexpression of HMGA1a leads to increased migration, invasion, and a transformed phenotype with anchorage-independent cell growth in soft agar. Because cyclooxygenase-2 (COX-2) is a downstream gene directly activated by HMGA1a in uterine cancers, we investigated the role of the HMGA1-COX-2 pathway in pancreatic adenocarcinoma. Our preliminary data suggest that HMGA1a and COX-2 mRNA levels are positively correlated in a pancreatic cancer cell lines. By chromatin immunoprecipitation, we found that HMGA1a binds to the COX-2 promoter in human pancreatic adenocarcinoma cells. Moreover, COX-2 inhibitors block tumorigenesis in human pancreatic cancer xenografts in nude mice. Taken together, our studies suggest that HMGA1a promotes tumor progression through up-regulation of COX-2 and this pathway could be targeted to treat or prevent human pancreatic adenocarcinoma. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4098.

Abstract 2961 Poster Board II-937 Constitutive activation of FMS-like tyrosine kinase 3 (FLT3) by internal tandem duplication (ITD) is one of the most common molecular alterations in acute myeloid leukemia (AML), and provides a proliferative and survival advantage to leukemic cells. We have previously generated a knock-in mouse in which an 18-bp ITD mutation, isolated from a patient with AML, was inserted into the juxtamembrane domain of the murine FLT3 gene. Heterozygous FLT3WT/ITD mice develop a myeloproliferative disease, which progresses to mortality within 6 to 20 months. However, no sign of acute leukemia is observed over the lifetime of these mice, indicating that additional cooperating genetic events are required for leukemic progression. FLT3 activating mutations have been seen in MDS in about 5% of cases and an additional 5% of patients with MDS acquire FLT3 mutations as they progress to AML. One model of MDS has been developed by transgenic expression of the NUP98-HOXD13 (NHD13) fusion under the vav promoter. Mice expressing the NUP98-HOXD13 (NHD13) transgene develop a highly penetrant myelodysplastic syndrome (MDS) with about 50% eventually progressing to acute leukemia by 14 months. We wanted to generate a mouse model to see if FLT3/ITD mutations would cooperate with the NHD13 fusion to progress to overt leukemia. Double mutant mice (FLT3/ITD/NHD13) were generated by crossing heterozygous FLT3WT/ITD mice with mice expressing the NHD13 transgene. Strikingly, FLT3/ITD/NHD13 offspring (n=40) developed an acute leukemia with 100% penetrance and a median survival of 97 days. In contrast, NHD13 (n=20) and FLT3WT/ITD (n=20) littermates had median survivals of 385 and 410 days, respectively (Figure 1). Differential cell counts at the time of sacrifice showed elevated peripheral white blood cell counts in the double mutant mice (161.0±50.6 k/μl) compared to three-month-old wildtype (12.5 ±5.0 k/μl), NHD13(3.2 ±1.0 k/μl), or FLT3WT/ITD (7.9 ±3.5 k/μl) littermates. Organ sectioning and histological staining of double mutant mice showed leukemic infiltration of the spleen, liver, and brain. FLT3/ITD/NHD13 offspring developed a heterogeneous group of acute leukemias, with 46% developing ALL, 39% biphenotypic leukemia, and 15% AML as diagnosed by flow cytometry of the bone marrow and spleen. Transplantation experiments showed that the leukemia was able to engraft in lethally irradiated recipients, with disease occuring within 30 days post-transplantation. The disease was transferrable with as few as 1000 cells, and this ability was restricted to the B220+Mac-1−Gr-1- population. In order to identify the changes in gene expression responsible for leukemic transformation, RNA was isolated from the total bone marrow of young FLT3/ITD/NHD13, wildtype, NHD13, and FLT3WT/ITD littermates prior to the development of discernable disease and probed for over 43,000 coding and non-coding mouse sequences by Agilent 44K array. The rapid onset of acute leukemia in this model indicates the collaboration between hox gene dysregulation by a chromosomal translocation and altered signaling through the FLT3 receptor. In addition, since resistance to FLT3 inhibitors alone remains an important clinical issue, gene expression profiling of leukemic cells may help identify new molecular targets in collaborative signaling pathways. Disclosures: No relevant conflicts of interest to declare.

Abstract 1960 Poster Board I-983 FLT3 is a receptor tyrosine kinase that plays an important role in hematopoiesis and is one of the most mutated genes in acute myeloid leukemia (AML). The internal tandem duplication (ITD) mutation, which causes autophosphorylation and constitutive activation of the receptor, has been found in up to 35% of AML and 3% of ALL patients. Our lab has created and described a FLT3ITD/wt knock-in mouse with the mutation expressed under the endogenous promoter. These mice develop myeloproliferative disorder (MPD) with a median time to disease of 52 weeks, but do not progress to acute leukemia, indicating that an additional genetic event is required for leukemogenesis. We have used the MOL4070LTR retrovirus in a retroviral insertional mutagenesis (RIM) screen to identify genes that can cooperate with the FLT3/ITD to cause leukemia. FLT3ITD/wt mice were injected with the retrovirus at birth and 78% developed leukemia with a median time to disease of 18 weeks (50% B-ALL/lymphoma, 18% AML, 10% biphenotypic leukemia). By comparison, retrovirus-injected FLT3wt/wt mice developed leukemias with a much longer median time to disease of 69 weeks. The decreased latency in the retrovirally-infected FLT3ITD/wt mice implies that the viral integrations have created mutations that collaborate with FLT3/ITD signaling to generate leukemia. To identify the genes responsible, we cloned the insertion sites using an inverse PCR technique. 343 integration sites were cloned from 25 FLT3ITD/wt mice that developed leukemia, 29 of which were found in two or more mice. These common insertion sites (CIS) are considered putative cooperating genes. The most common insertion, found in 5 mice, involved the Evi1 gene, with the virus inserting approximately 17kb upstream of the gene in three of the mice or approximately 100kb upstream of the gene in the other two mice. All of the mice showed overexpression of Evi1 by quantitative RT-PCR. This is an interesting collaborating gene because its overexpression has been described in AML and predicts a poor prognosis in patients, but mouse models have shown that, like FLT3/ITD, it is not leukemogenic on its own. Though singularly insufficient, together these two mutations may cooperate to cause leukemia. Another putative cooperating gene found in the screen is Ring1a. Insertions upstream of the gene (22kb, 96kb and 209kb) were found in 3 mice displaying myeloid-associated disease, and in all cases caused overexpression of Ring1a, as seen by quantitative RT-PCR. Strong expression of Ring1a, part of the PRC1 polycomb repressor complex, has been observed in several cancer types but not in myeloid leukemia; however, another member of PRC1, Bmi-1, seems to play an extensive role in myeloid leukemia. Integrations near Ring1a have not previously been described in the Retrovirus Tagged Cancer Gene Database (RTCGD mm8), indicating a specific cooperation with FLT3/ITD and a role in the disease. With these studies we have shown that additional mutations cooperate with the FLT3/ITD to cause leukemia and we have identified several possible collaborating genes, including Evi1 and Ring1a. Disclosures: No relevant conflicts of interest to declare.

Abstract 3969 Poster Board III-905 Because T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive lymphoid leukemia with a high rate of relapse in both children and adults, research is urgently needed to discover molecular pathways that could be targeted in therapy. Recent studies have identified NOTCH1 activating mutations, genomic classifiers, and other molecular pathways that are disrupted in T-ALL, although these findings have not yet translated into better therapy or improved long-term survival. Thus, we are investigating molecular pathways that play a causative role in T-ALL and could be modulated by therapy. We previously showed that HMGA1a is overexpressed in human T-ALL. Further, HMGA1a transgenic mice develop aggressive T-ALL with complete penetrance, indicating that HMGA1a induces leukemogenic transformation in vivo. Disruption of the CDKN2A (INK4A/ARF) tumor suppressor locus has been reported in up to 90% of T-ALL, suggesting that this contributes to leukemic transformation in T-cells. To determine if loss of function of the INK4A/ARF tumor suppressor locus could cooperate with HMGA1a in T-ALL, we crossed our HMGA1a transgenics with mice that are null for the INK4A/ARF locus. We found that T-ALL was markedly accelerated in the HMGA1a transgenics that are also null at this tumor suppressor locus. These mice died significantly earlier than the HMGA1 transgenics that are wildtype at the INK4A/ARF locus (4.8 months+/- 1.0 month versus 10.5 +/- 2.5 months, p < 0.0001 by the student's t-test). The HMGA1a transgenic, INK4A/ARF null mice recapitulate salient clinical and pathologic features of human T-ALL. All mice appeared ill and exhibit marked organomegaly with extensive leukemic infiltration. Immunophenotyping by fluorescence activated cell sorting showed T-ALL in all cases with identical T-cell markers (CD3, CD8, and αβ-TCR) to those previously reported for the HMGA1a transgenics at 8-10 months. Interestingly, the HMGA1a-INK4A/ARF+/- mice also developed early lymphoid malignancy in 6/17 cases. The accelerated lymphoid malignancy in these mice suggests that there is loss of heterozygosity at the INK4A/ARF locus. Alternatively, haploinsufficiency of INK4A and/or ARF may be sufficient to promote lymphoid tumorigenesis in the setting of HMGA1a overexpression. To determine if HMGA1 overexpression and loss of INK4A/ARF expression occur in human T-ALL, we analyzed gene expression profile analysis in leukemic blasts from children and young adults with T-ALL. We found high levels of HMGA1 expression and low levels of INK4A/ARF expression in most patients. Taken together, our results indicate that overexpression of HMGA1 and loss of function at the INK4A/ARF (CDKN2A) tumor suppressor locus cooperate in T-cell leukemogenesis and these pathways could represent rational therapeutic targets. Further, our mice provide a novel preclinical model for T-ALL. Disclosures: No relevant conflicts of interest to declare.

G A A b st ra ct s the increase in DNMT1 levels following IL-6 was not due to do novo gene expression but was due to stabilization of the DNMT1 protein and was blocked by AKT inhibitor triciribine. Increased levels of DNMT1 were accompanied by increased promoter methylation of a panel of genes associated with tumour suppression, cell adhesion and apoptosis resistance. Expression of 4 of these transcripts (IRF-7, Maspin, PAI-1 and IL-4) was confirmed to be down-regulated between 2 and 5-fold by IL-6. IL-6-stimulated down-regulation of these transcripts was prevented by DNMT inhibitor 5-aza-CdR, indicating that the effect of IL-6 on these genes is DNA methylation-dependent. IL-6 increased HCT116 cell migration and anchorage-independent growth by approximately 4-fold, effects that were completely abrogated by 5-aza-CdR. Conclusions: Our results indicate that IL-6, acting through the AKT pathway, may promote tumorigenesis through DNMT1-mediated gene silencing. Further elucidation of the signaling pathways involved in IL-6 regulation of DNMT1 expression may provide novel opportunity for therapeutic intervention.

The class III receptor tyrosine kinase, FLT3, is expressed by over 90% of B-lineage acute lymphoblastic leukemias (ALL) blasts. In addition, it is expressed at extremely high levels in ALL patients with MLL-rearrangements or hyperdiploidy and sometimes mutated in these same patients. In this report, we investigated the effects of EB10, an anti-human FLT3 monoclonal antibody capable of preventing binding of FLT3 ligand (FL), on ALL cell lines and primary cells. In vitro studies, examining the ability of EB10 to inhibit FLT3 activation and downstream signaling in ALL cell lines and primary blasts, yielded variable results. In some cell lines FLT3 phosphorylation was inhibited and with it, downstream activation of pathways involving MAPK, AKT, and STAT5 phosphorylation. However, several cell lines actually exhibited FLT3 activation upon antibody treatment, possibly because of antibody-mediated receptor dimerization, and subsequent activation of downstream pathways. Nevertheless, through antibody-mediated cellular cytotoxicity (ADCC) such an antibody could still prove efficacious against leukemia cells in vivo. In fact, EB10 treatment significantly prolongs survival and/or reduces engraftment of several ALL cell lines and some primary ALL samples in NOD/SCID mice, even when EB10 treatment results in FLT3 activation of those cell lines in vitro. Moreover, FACS and PCR analysis of EB10 treated NOD/SCID mice surviving 150 days post leukemic cell injection revealed that FLT3 immunotherapy eliminated leukemic engraftment. The leukemic cells surviving EB10 treatment in the mice were characterized by FACS analysis and found to express lower levels of FLT3. To assess for resistance, cells surviving EB10 treatment were injected into NOD/SCID mice and treated with a single dose of EB10. FACS analysis revealed that these cells remain sensitive to EB10 treatment. Taken together, these data demonstrate that EB10 is cytotoxic to ALL blasts in vivo and EB10 treatment did not select for resistant clones. Such an antibody, either naked or conjugated to radioactive isotopes or cytotoxic agents, may prove useful in the therapy of infant ALL as well as childhood and adult ALL patients whose blasts typically express FLT3.

The class III receptor tyrosine kinase, FLT3, is expressed by >90% of B-lineage acute lymphoblastic leukemias (ALL) blasts. In addition, it is expressed at extremely high levels in ALL patients with MLL-rearrangements or hyperdiploidy and sometimes mutated in these same patients. In this report, we investigated the effects of EB10, an anti-human FLT3 monoclonal antibody capable of preventing binding of FLT3 ligand (FL), on ALL cell lines and primary cells. In vitro studies, examining the ability of EB10 to inhibit FLT3 activation and downstream signaling in ALL cell lines and primary blasts, yielded variable results. In some cell lines FLT3 phosphorylation was inhibited and with it, downstream activation of pathways involving MAPK, AKT, and STAT5 phosphorylation. However, several cell lines actually exhibited FLT3 activation upon antibody treatment, possibly because of antibody-mediated receptor dimerization, and subsequent activation of downstream pathways. Nevertheless, through antibody-mediated cellular cytotoxicity (ADCC) such an antibody could still prove efficacious against leukemia cells in vivo. In fact, EB10 treatment significantly prolongs survival and/or reduces engraftment of ALL cell lines and primary ALL blasts in NOD/SCID mice. This effect might be even more pronounced in a host that was less immune compromised than are NOD/SCID mice. The leukemic cells surviving EB10 treatment in the mice were characterized by FACS analysis and found to express low levels or no FLT3. In contrast to the reduction in engraftment of human ALL primary blasts, EB10 treatment of NOD/SCID mice did not reduce engraftment of human hematopoietic CD34+ cells. Taken together, these data demonstrate that EB10 is selectively cytotoxic to ALL blasts while having little effect on normal hematopoiesis. Such an antibody, either naked or conjugated to radioactive isotopes or cytotoxic agents, may prove useful in the therapy of infant ALL as well as childhood and adult ALL patients whose blasts typically express FLT3.

Suitable animal models are needed to study monkeypox virus (MPXV) as human monkeypox clinically resembles smallpox and MPXV is a zoonotic and potential bioterroristic agent. We have demonstrated that a species of African dormice, Graphiurus kelleni, is susceptible to a lethal infection of MPXV and that MPXV replicated in multiple organs of this species. Following intranasal administration, MPXV replicated locally in the nasal mucosa causing necrosis and hemorrhage with subsequent systemic spread to lymph nodes, spleen, liver, and other tissues where it caused severe necrosis and/or hemorrhage leading to death. The dormouse model was validated for testing prophylactic (Dryvax vaccine) and therapeutic (cidofovir) test articles against intranasal challenges with MPXV.

Alternative splicing of the gene for Stat3, a transcription factor activated by the IL-6 family of cytokines, produces two isoforms: Stat3alpha and a dominant-negative variant, Stat3beta. Stat3beta-deficient mice were generated by gene targeting. Despite intact expression and phosphorylation of Stat3alpha, overall Stat3 activity was impaired in Stat3beta(-/-) cells. Global comparison of transcription in Stat3beta(+/+) and Stat3beta(-/-) cells revealed stable differences. Stat3beta-deficient mice exhibit diminished recovery from endotoxic shock and hyperresponsiveness of a subset of endotoxin-inducible genes in liver. The hepatic response to endotoxin in wild-type mice is accompanied by a transient increase in the ratio of Stat3beta to Stat3alpha. These findings indicate a critical role for Stat3beta in the control of systemic inflammation.

SummaryPIK3CA is mutated in diverse human cancers, but the functional effects of these mutations have not been defined. To evaluate the consequences of PIK3CA alterations, the two most common mutations were inactivated by gene targeting in colorectal cancer (CRC) cells. Biochemical analyses of these cells showed that mutant PIK3CA selectively regulated the phosphorylation of AKT and the forkhead transcription factors FKHR and FKHRL1. PIK3CA mutations had little effect on growth under standard conditions, but reduced cellular dependence on growth factors. PIK3CA mutations resulted in attenuation of apoptosis and facilitated tumor invasion. Treatment with the PI3K inhibitor LY294002 abrogated PIK3CA signaling and preferentially inhibited growth of PIK3CA mutant cells. These data have important implications for therapy of cancers harboring PIK3CA alterations.

Internal tandem duplication (ITD) mutations within the FMS-Like Tyrosine Kinase gene (FLT3) render the receptor constitutively active, driving proliferation and survival in leukemic blasts. Expression of Flt3-ITD from the endogenous promoter in a murine knock-in model results in progenitor expansion and a myeloproliferative neoplasm. In this study, we show that this expansion begins with over-proliferation within a compartment of normally quiescent long-term hematopoietic stem cells (LT-HSCs), which become rapidly depleted. This depletion is reversible upon treatment with the small molecule inhibitor Sorafenib, which also ablates the disease. Although the normal LT-HSC has been defined as Flt3-negative by flow cytometric detection, we demonstrate that Flt3 is capable of playing a role within this compartment by examining the effects of constitutively activated Flt3-ITD. This indicates an important link between stem cell quiescence/homeostasis and myeloproliferative disease while also giving novel insight into the emergence of FLT3-ITD mutations in the evolution of leukemic transformation.

The lentivirus caprine arthritis-encephalitis virus (CAEV) is a pathogen of goats. It is transmitted in milk and causes a persistent infection in goats, which often fail to produce neutralizing antibodies to the virus. Native CAEV particles are remarkably resistant to digestion with proteinase K and are neutralized extremely slowly by immune sera. Our studies showed that the virus particles are heavily sialylated. Studies with highly specific sialyltransferase enzymes identified penultimate carbohydrate linkages typical of O- and N-linked oligosaccharides on the virus and suggested that the virus may be more heavily sialylated on O-linked than on N-linked oligosaccharides. Removal of sialic acids from the virus by neuraminidase treatment did not reduce infectivity of the particles. However, desialylation rendered the virus more susceptible to proteolysis by proteinase K. Desialylation also enhanced the kinetics of neutralization of the virus by goat antibodies. These results suggest that the carbohydrates on the viral surface are important both in protecting viral proteins from digestion by proteases and in protecting the virus from rapid neutralization by antibodies.

Lentiviruses are nononcogenic, replication-competent retroviruses. They are genus specific in animal host range and are transmitted exogenously in nature by blood, milk, or inflammatory exudates. The viruses have been known for several years as specific pathogens of domestic animals, causing infectious anemia in horses, visna-maedi in sheep, arthritis-encephalitis in caprine species, and more recently, acquired immunodeficiency syndrome (AIDS) and AIDS-related complex (ARC) in humans [1–5]. Newer members of the lentivirus family include feline and bovine immunodeficiency viruses and lentiviruses of macaques (SIVmac) and African green monkeys (SlVagm). The viruses persist indefinitely in infected hosts and cause disease after variable and often prolonged incubation periods of months to years. Disease is gradual in onset and becomes progressively worse with time. It involves many organ systems in inflammatory and degenerative processes and leads eventually to cachexia and death [6–10].

Antibodies to visna virus neutralized the virus in fibroblasts and macrophages but specifically enhanced the binding, penetration, and uncoating of the virus in the latter cells. F(ab')2 fragments of the immune antibody neutralized the virus in fibroblasts but did not enhance the early stages of the virus life cycle in macrophages. Furthermore, these fragments did not neutralize infectivity in macrophages but delayed the appearance of infectious virus in cells after the inoculation of preincubated virus-F(ab')2 complexes.