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Thyroid receptor-interacting protein 13 (TRIP13), a protein of the AAA-ATPase family, is upregulated in various human cancers, including colorectal cancer (CRC). This study focused on the inhibition of TRIP13-induced CRC progression and signalling by DCZ0415, a small molecule targeting TRIP13. It demonstrated potent antitumour activity in TRIP13-deregulated cancer cell lines, regardless of their p53, KRAS, BRAF, epidermal growth factor receptor or microsatellite instability status. The treatment of CRC cells with DCZ0415 resulted in decreased cell proliferation, induced cell cycle arrest in the G2-M phase and increased apoptosis. DCZ0415 diminished xenograft tumour growth and metastasis of CRC in immunocompromised mice. DCZ0415 reduced expression of fibroblast growth factor receptor 4 (FGFR4), signal transducer and activator of transcription 3 (STAT3), and proteins associated with the epithelial-mesenchymal transition and nuclear factor kappa B (NF-κB) pathways in cells and xenografts exhibiting high expression of TRIP13. Additionally, DCZ0415 decreased cyclin D1, β-catenin and T-cell factor 1, leading to the inactivation of the Wnt/β-catenin pathway. In a syngeneic CRC model, DCZ0415 treatment induced an immune response by decreasing PD1 and CTLA4 levels and increasing granzyme B, perforin and interferon gamma. In sum, DCZ04145 inhibits the TRIP13-FGFR4-STAT3 axis, inactivates NF-κB and Wnt/β-catenin signalling, activates antitumour immune response and reduces the progression and metastasis of CRC. This study provides a rationale to evaluate DCZ0415 clinically for the treatment of a subset of CRCs that exhibit dysregulated TRIP13 and FGFR4.

Reliable preclinical models are needed for screening new cancer drugs. Thus, we developed an improved 3D tumor organoid model termed “organoid raft cultures” (ORCs). Development of ORCs involved culturing tumors ex vivo on collagen beds (boats) with grid supports to maintain their morphological structure. The ORCs were developed from patient-derived xenografts (PDXs) of colon cancers excised from immune-deficient mice (NOD/SCID/IL2Rgammanull). We utilized these new models to evaluate the efficacy of an investigational drug, Navitoclax (ABT-263). We tested the efficacy of ABT-263, an inhibitor of BCL-2 family proteins, in these ORCs derived from a PDX that showed high expression of antiapoptotic BCL2 family proteins (BCL-2, BCL-XL, and BCL-W). Hematoxylin and eosin staining evaluation of PDXs and corresponding ORCs indicated the retention of morphological and other histological integrity of ORCs. ORCs treated with ABT-263 showed decreased expression of antiapoptotic proteins (BCL2, BCL-XL and BCL-W) and increased proapoptotic proteins (BAX and PUMA), with concomitant activation of caspase 3. These studies support the usefulness of the ORCs, developed from PDXs, as an alternative to PDXs and as faster screening models.

The t(8;21) RUNX1-ETO translocation is one of the most frequent cytogenetic abnormalities in acute myeloid leukemia (AML). In RUNX1-ETO+ patient samples, differing classes of activating c-KIT receptor tyrosine kinase mutations have been observed. The most common (12%-48%) involves mutations, such as D816V, which occur in the tyrosine kinase domain, whereas another involves mutations within exon 8 in a region mediating receptor dimerization (2%-13% of cases). To test whether distinct subtypes of activating c-KIT mutations differ in their leukemogenic potential in association with RUNX1-ETO, we used a retroviral transduction/transplantation model to coexpress RUNX1-ETO with either c-KitD814V or c-KitT417IΔ418-419 in murine hematopoietic stem/progenitor cells used to reconstitute lethally irradiated mice. Analysis of reconstituted animals showed that RUNX1-ETO;c-KitD814V coexpression resulted in 3 nonoverlapping phenotypes. In 45% of animals, a transplantable AML of relatively short latency and frequent granulocytic sarcoma was noted. Other mice exhibited a rapidly fatal myeloproliferative phenotype (35%) or a lethal, short-latency pre-B-cell leukemia (20%). In contrast, RUNX1-ETO;c-KitT417IΔ418-419 coexpression promoted exclusively AML in a fraction (51%) of reconstituted mice. These observations indicate that c-KitD814V promotes a more varied and aggressive leukemic phenotype than c-KitT417IΔ418-419, which may be the result of differing potencies of the activating c-Kit alleles.

In contrast to antigen-specific alphabeta-T cells (adaptive immune system), gammadelta-T cells can recognize and lyse malignantly transformed cells almost immediately upon encounter in a manner that does not require the recognition of tumor-specific antigens (innate immune system). Given the well-documented capacity of gammadelta-T cells to innately kill a variety of malignant cells, efforts are now actively underway to exploit the antitumor properties of gammadelta-T cells for clinical purposes. Here, we present for the first time preclinical in vivo mouse models of gammadelta-T cell-based immunotherapy directed against breast cancer. These studies were explicitly designed to approximate clinical situations in which adoptively transferred gammadelta-T cells would be employed therapeutically against breast cancer. Using radioisotope-labeled gammadelta-T cells, we first show that adoptively transferred gammadelta-T cells localize to breast tumors in a mouse model (4T1 mammary adenocarcinoma) of human breast cancer. Moreover, by using an antibody directed against the gammadelta-T cell receptor (TCR), we determined that localization of adoptively transferred gammadelta-T cells to tumor is a TCR-dependant process. Additionally, biodistribution studies revealed that adoptively transferred gammadelta-T cells traffic differently in tumor-bearing mice compared to healthy mice with fewer gammadelta-T cells localizing into the spleens of tumor-bearing mice. Finally, in both syngeneic (4T1) and xenogeneic (2Lmp) models of breast cancer, we demonstrate that adoptively transferred gammadelta-T cells are both effective against breast cancer and are otherwise well-tolerated by treated animals. These findings provide a strong preclinical rationale for using ex vivo expanded adoptively transferred gammadelta-T cells as a form of cell-based immunotherapy for the treatment of breast cancer. Additionally, these studies establish that clinically applicable methods for radiolabeling gammadelta-T cells allows for the tracking of adoptively transferred gammadelta-T cells in tumor-bearing hosts.

Phospholipase C-gamma (PLCgamma) has been implicated in tumor cell motility required for invasiveness and metastasis. Diminished tumor dissemination has been demonstrated in xenograft models, but studies in naturally-occurring tumors are lacking, having been limited by the timing of the interventions. Therefore, we generated mice that express a doxycycline (DOX)-inducible dominant-negative fragment of PLCgamma, PLCz; this approach avoids the in utero lethality caused by the absence of PLCgamma. As we targeted two de novo-occurring carcinomas of the mammary (MMTV-driven polyoma middle T antigen model, PyVmT) and prostate (TRAMP model) glands, we limited expression to these epithelial cells by driving DOX transactivator from the prostatein C3 promoter. This avoids the confounding variable of potentially abrogating motility in stromal and endothelial cells. These mice developed normally in the presence of DOX, except for limited mammary development if treated before 6 weeks and immaturity of the prostate gland if treated before 2 weeks of age. DOX-mediated induction of PLCz from age 8 to 16 weeks in PyVmT mice decreased the number of lung metastases by >10-fold (P

Although DNA methylation and transcriptional repression are generally associated, a causal role for DNA methylation in silencing of retroviral vectors has not been established. The newer generation murine stem cell virus retroviral vector (MSCV) lacks many of the repressive cis-acting DNA sequences identified in Moloney murine leukemia virus but remains sensitive to transcriptional silencing in various cell types. To determine the contribution of cytosine methylation to MSCV silencing, we mutated CpG dinucleotides located in the MSCV long terminal repeat (LTR) that are clustered in the U3 region and directly spanning the transcription start site in the R region. Effects of the CpG mutations on MSCV silencing were assessed in murine embryonic stem cells. An analysis of numerous clonal proviral integrants showed that mutation of CpGs in both clusters eliminated proviral integrants that were completely silenced. Variegated expression was shown to represent a substantial component of intraclonal silencing and was independent of the presence of CpGs in the LTR. Treatment of transduced cells with 5-azadeoxycytidine delayed establishment of the silenced state but had only a modest effect on expression of some proviral integrants at late times post-transduction. These results are direct evidence for a causal contribution of DNA methylation in the LTR to MSCV silencing and define the promoter region CpGs as a repressive element in embryonic stem cells. Furthermore, distinct mechanisms are suggested for establishment and maintenance of the silenced proviral state.

Monoclonal antibodies (mAb) to epidermal growth factor receptor (EGFr) inhibit tumor cell proliferation and enhance cytotoxicity of chemotherapeutic agents. The purpose of this study was to investigate the interaction of the anti-EGFr antibody C225 combined with radiotherapy (RT) on EGFr expressing A431 human epidermoid cancer cells.Cell proliferation, apoptosis, EGFr expression and phosphorylation, and clonogenic survival were assayed in vitro. A431 tumor growth inhibition and immunohistochemistry analysis of EGFr expression and apoptosis were assessed in vivo.C225 plus RT produced greater inhibition of A431 cell proliferation than C225 or RT alone which was corroborated by enhanced apoptosis. Similar clonogenic survival occurred following the addition of C225 to RT, although colonies were smaller in the presence of C225. C225 produced inhibition of EGF-induced phosphorylation of EGFr without concurrent down-regulation of surface receptor, which was not altered by RT. Combined treatment of mice bearing tumors demonstrated enhancement of complete regressions, reduction in time to tumor size doubling, and prolongation of survival. Significant apoptosis occurred in xenograft tumors treated with C225 with or without RT.These data demonstrate an interaction between C225 and RT. C225-mediated apoptosis and inhibition of EGFr phosphorylation may be critical in the interaction. Studies to define the precise influence of combined modality treatment on the EGFr signal transduction cascade need to be pursued. The combination of growth factor receptor antibodies and RT has potential application in clinical oncology.

Gene knockout experiments in mice have suggested a hierarchical model of early B cell commitment wherein E2A proteins (E47 and E12) activate early B cell factor (Ebf1), which in turn activates expression of the B cell commitment factor, Pax5. In IL-7 receptor alpha (IL-7Rα) knockout mice, B cell development is blocked before B-lineage commitment at the prepro-B cell stage in adult animals. In IL-7R α −/− prepro-B cells, E47 is expressed and yet is insufficient to transcriptionally activate the putative downstream target gene, Ebf1 . In this study, we show that further increases of E47 expression in IL-7R α −/− prepro-B cells fails to activate Ebf1 , but rather leads to a dramatic induction of the E2A inhibitory factors, Id2 and Id3 . In contrast, enforced expression of Ebf1 in IL-7R α −/− bone marrow potently down-regulates Id2 and Id3 mRNA expression and restores B cell differentiation in vivo. Down-regulation of both Id2 and Id3 during B cell specification is essential in that overexpression of either Id2 or Id3 in wild-type bone marrow blocks B cell specification at the prepro-B cell stage. Collectively, these studies suggest a model where Ebf1 induction specifies the B cell fate by dramatically increasing activity of E47 at the posttranslational level.

The inversion of chromosome 16 in the inv(16)(p13q22) is one of the most frequent cytogenetic abnormalities observed in acute myeloid leukemia (AML). The inv(16) fuses the core binding factor (CBF) beta subunit with the coiled-coil rod domain of smooth muscle myosin heavy chain (SMMHC). Expression of CBFβ-SMMHC in mice does not promote AML in the absence of secondary mutations. Patient samples with the inv(16) also possess mutually exclusive activating mutations in either N-RAS, K-RAS, or the receptor tyrosine kinases, c-KIT and FLT3, in almost 70% of cases. To test whether an activating mutation of FLT3 (FLT3-ITD) would cooperate with CBFβ-SMMHC to promote AML, we coexpressed both mutations in hematopoietic progenitor cells used to reconstitute lethally irradiated mice. Analysis of transplanted animals showed strong selection for CBFβ-SMMHC/FLT3-ITD–expressing cells in bone marrow and peripheral blood. Compared with animals transplanted with only CBFβ-SMMHC–expressing cells, FLT3-ITD further restricted early myeloid differentiation and promoted peripheralization of primitive myeloblasts as early as 2.5 weeks after transplantation. FLT3-ITD also accelerated disease progression in all CBFβ-SMMHC/FLT3-ITD–reconstituted animals, which died of a highly aggressive and transplantable AML within 3 to 5 months. These results indicate that FLT3-activating mutations can cooperate with CBFβ-SMMHC in an animal model of inv(16)-associated AML.

Pancreatic ductal adenocarcinoma (PDAC) is one of the most fatal human malignancies, with an overall 5-year survival rate of

Inactivation of p15(Ink4b) expression by promoter hypermethylation occurs in up to 80% of acute myeloid leukemia (AML) cases and is particularly common in the FAB-M2 subtype of AML, which is characterized by the presence of the RUNX1-ETO translocation in 40% of cases. To establish whether the loss of p15(Ink4b) contributes to AML progression in association with RUNX1-ETO, we have expressed the RUNX1-ETO fusion protein from a retroviral vector in hematopoietic progenitor cells isolated from wild-type, p15(Ink4b) or p16(Ink4a) knockout bone marrow. Analysis of lethally irradiated recipient mice reconstituted with RUNX1-ETO-expressing cells showed that neither p15(Ink4b) or p16(Ink4a) loss significantly accelerated disease progression over the time period of one year post-transplantation. Loss of p15(Ink4b) alone resulted in increased myeloid progenitor cell frequencies in bone marrow by 10-month post-transplant and a 19-fold increase in the frequency of Lin(-)c-Kit(+)Sca-1(+) (LKS) cells that was not associated with expansion of long-term reconstituting HSC. These results strongly suggest that p15(Ink4b) loss must be accompanied by additional oncogenic changes for RUNX1-ETO-associated AML to develop.

The inv(16) is one of the most frequent chromosomal translocations associated with acute myeloid leukemia (AML) and creates a chimeric fusion protein consisting of most of the runt-related X1 co-factor, core binding factor beta fused to the smooth muscle myosin heavy chain MYH11. Expression of the ARF tumor suppressor is regulated by runt-related X1, suggesting that the inv(16) fusion protein (IFP) may repress ARF expression. We established a murine bone marrow transplant model of the inv(16) in which wild type, Arf+/-, and Arf-/- bone marrow were engineered to express the IFP. IFP expression was sufficient to induce a myelomonocytic AML even when expressed in wild type bone marrow, yet removal of only a single allele of Arf greatly accelerated the disease, indicating that Arf is haploinsufficient for the induction of AML in the presence of the inv(16).

PU.1 is a member of the ETS family of transcription factors and is required for the development of multiple hematopoietic lineages. PU.1-/- mice die from hematopoietic failure at about embryonic day 18.5 (e18.5) and show a complete absence of B cells, mature T cells, and macrophages. This phenotype suggests that PU.1 may function at the level of the hematopoietic stem cell (HSC) or a multilineage progenitor. To investigate the role of PU.1 in the regulation of HSCs, PU.1-/- embryos were analyzed at various stages of embryonic development. The absolute number and frequency of HSCs were determined by flow cytometric analysis of c-Kit+Thy-1.1loLin-Sca-1+ (KTLS) cells. We found that KTLS cells were absent or severely reduced in PU.1-/- fetal liver from e12.5 to e15.5. Progenitor cells with a c-Kit+Lin-AA4.1+ and c-Kit+Lin-CD34+ phenotype were also severely reduced. In addition, PU.1-/- fetal liver at e14.5 lacked common myeloid progenitors (CMPs) and granulocyte-macrophage progenitors (GMPs) but retained megakaryocyteerythroid progenitors (MEPs). Consistent with the loss of HSC activity, a 10-fold reduction in erythroid progenitors (mature erythroid burst-forming units [BFUEs]) was observed between e14.5 and e16.5. These data suggest that PU.1 plays an important role in the maintenance or expansion of HSC number in murine fetal liver. (Blood. 2004;104:3894-3900)

Signaling through the IL-7 receptor (IL-7R) is necessary for the development of the earliest B- and T-lineage cells. IL-7R is first expressed on common lymphoid progenitor cells and is not detected on primitive common myeloid progenitors. In this study, we show that enforced expression of IL-7R on multipotential stem cells does not influence lymphoid versus myeloid cell fate. T cell development was compatible with sustained IL-7R expression; however, we observed a near complete block in B cell development at the onset of B-lineage commitment. Unlike pre-proB cells from control animals, developmentally-arrested IL-7R(+)B220(+)CD19(-)NK1.1(-)Ly-6C(-) cells failed to express EBF and Pax5. These results suggest that transient downregulation of IL-7R signaling is a necessary event for induction of EBF and Pax5 expression and B-lymphocyte commitment.

Temporally controlled expression of genes in transgenic mice has advanced our understanding of many physiological processes. One of the more common modes of acutely altering gene levels involves the doxycycline-regulated "tet-on/tet-off" systems. There has been concern that the administration of doxycycline in itself might compromise many aspects of wound repair. Here we report that oral ad libitum administration of doxycycline (2 mg/ml in drinking water) to mice does not alter dermal or epidermal wound healing. The healing of both full- and partial-thickness skin wounds proceeded similarly regardless of doxycycline administration; in fact, collagen organization and tensile strength development appeared to accelerate in the presence of doxycycline. These data suggest that wound healing studies incorporating transgene expression can utilize tet-on/tet-off regulation of gene expression without interference from doxycycline.

Progenitor B cells deficient in Pax5 are developmentally multipotent, suggesting that Pax5 is necessary to maintain commitment to the B-cell lineage. Commitment may be mediated, in part, by Pax5 repression of myeloid-specific genes. To determine whether Pax5 expression in multipotential cells is sufficient to restrict development to the B-cell lineage in vivo, we enforced expression of Pax5 in hematopoietic stem cells using a retroviral vector. Peripheral blood analysis of all animals reconstituted with Pax5-expressing cells indicated that more than 90% of Pax5-expressing cells were B220+ mature B cells that were not malignant. Further analysis showed that Pax5 completely blocked T-lineage development in the thymus but did not inhibit myelopoiesis or natural killer (NK) cell development in bone marrow. These results implicate Pax5 as a critical regulator of B- versus T-cell developmental fate and suggest that Pax5 may promote commitment to the B-cell lineage by mechanisms that are independent of myeloid gene repression.