David C. Dorn

Hematopoiesis Controlled by Distinct TIF1γ and Smad4 Branches of the TGFβ Pathway

Tissue homeostasis in mammals relies on powerful cytostatic and differentiation signals delivered by the cytokine TGFbeta and relayed within the cell via the activation of Smad transcription factors. Formation of transcription regulatory complexes by the association of Smad4 with receptor-phosphorylated Smads 2 and 3 is a central event in the canonical TGFbeta pathway. Here we provide evidence for a branching of this pathway. The ubiquitious nuclear protein Transcriptional Intermediary Factor 1gamma (TIF1gamma) selectively binds receptor-phosphorylated Smad2/3 in competition with Smad4. Rapid and robust binding of TIF1gamma to Smad2/3 occurs in hematopoietic, mesenchymal, and epithelial cell types in response to TGFbeta. In human hematopoietic stem/progenitor cells, where TGFbeta inhibits proliferation and stimulates erythroid differentiation, TIF1gamma mediates the differentiation response while Smad4 mediates the antiproliferative response with Smad2/3 participating in both responses. Thus, Smad2/3-TIF1gamma and Smad2/3-Smad4 function as complementary effector arms in the control of hematopoietic cell fate by the TGFbeta/Smad pathway.

The effect of cantharidins on leukemic stem cells

To identify an agent with specific activity against leukemic stem cells (LSCs), we evaluated compounds that targeted hepatic leukemia factor (HLF), a gene implicated in hematopoietic stem cell (HSCs) regulation, that we found overexpressed in LSCs. Cantharidin, a natural toxin from blister beetles, used as medicinal agent since antiquity, has been described to modulate the HLF competitor NFIL3 and is under clinical evaluation as an antitumor and antimetastatic agent. The molecule is not a substrate for multidrug resistant pumps and does not cause myelosuppression, and therefore it represents a promising compound for selective ablation of LSCs. Cantharidin and norcantharidin, a derivative with reduced toxicity, decreased HLF protein levels and induced apoptosis in the AML cell line MV4‐11 by modulating the expression of several molecules that govern survival pathway, including HLF, SLUG, NFIL3 and c‐myc, thereby inducing p53 and the mitochondrial caspase cascade. In vitro, cantharidin readily targeted primary AML stem and progenitor cells in contrast to conventional chemotherapeutic agents, such as Ara‐C and daunorubicin, that mainly targeted more differentiated leukemic cells. In vitro the compound did not exhibit a therapeutic window, being equally toxic to normal HSCs and LSCs. In vivo cantharidin did not produce myelosuppression. Because of dose‐limiting toxicity in vivo, neither cantharidin nor norcantharidin proved therapeutical benefit in AML xenograft models as a single agent. However, its potent in vitro LSC activity and pathway targeting may still be exploited clinically with a new generation of cantharidin derivatives or formulations and with appropriate drug combinations.

Cellular and humoral immunogenicity of hamster polyomavirus-derived virus-like particles harboring a mucin 1 cytotoxic T-cell epitope

In this study, we examined hamster polyomavirus (HaPyV) major capsid protein VP1-derived virus-like particles (VLPs) as a carrier for a human tumor-associated cytotoxic T lymphocyte (CTL) epitope. The VP1 tolerated the insertion of an HLA-*A2-restricted CTL epitope from human mucin 1 (MUC1) into two sites independently and simultaneously, without interfering with assembly of chimeric VLPs. Chimeric VLPs did not differ in the entry pathway or maturation potential of human dendritic cells (hDCs) compared to unmodified VLPs. Recently we demonstrated that immunization of BALB/c mice with chimeric VLPs harboring two MUC1 insertions resulted in the generation of MUC1-specific monoclonal antibodies. Here we demonstrate that the monoclonal antibodies generated react specifically with human tumor cells. Co-cultivation of chimeric VLP-primed hDCs with autologous peripheral blood leukocytes resulted in the activation of MUC1 epitope-specific CD8(+) T cells. This was evidenced by IFN-gamma secretion of an expanded MUC1-specific CD8(+) T-cell pool. The induction of epitope-specific T cells in a human in vitro model and of murine MUC1-reactive antibodies in vivo indicate the potential of chimeric HaPyV VP1-derived VLPs as a delivery vehicle for immunotherapeutic targets.

CDK4/6 inhibitor PD 0332991 sensitizes acute myeloid leukemia to cytarabine-mediated cytotoxicity

Cyclin-dependent kinase (CDK)4 and CDK6 are frequently overexpressed or hyperactivated in human cancers. Targeting CDK4/CDK6 in combination with cytotoxic killing therefore represents a rational approach to cancer therapy. By selective inhibition of CDK4/CDK6 with PD 0332991, which leads to early G1 arrest and synchronous S-phase entry upon release of the G1 block, we have developed a novel strategy to prime acute myeloid leukemia (AML) cells for cytotoxic killing by cytarabine (Ara-C). This sensitization is achieved in part through enrichment of S-phase cells, which maximizes the AML populations for Ara-C incorporation into replicating DNA to elicit DNA damage. Moreover, PD 0332991 triggered apoptosis of AML cells through inhibition of the homeobox (HOX)A9 oncogene expression, reducing the transcription of its target PIM1. Reduced PIM1 synthesis attenuates PIM1-mediated phosphorylation of the proapoptotic BAD and activates BAD-dependent apoptosis. In vivo, timely inhibition of CDK4/CDK6 by PD 0332991 and release profoundly suppresses tumor growth in response to reduced doses of Ara-C in a xenograft AML model. Collectively, these data suggest selective and reversible inhibition of CDK4/CDK6 as an effective means to enhance Ara-C killing of AML cells at reduced doses, which has implications for the treatment of elderly AML patients who are unable to tolerate high-dose Ara-C therapy.

Quantification of the CD8+ T cell response against a mucin epitope in patients with breast cancer

Introduction:Mucin 1, encoded by the MUC1 gene, is a tumor-associated antigen expressed on the surface of breast cancer cells. It would be of interest to see whether there is a naturally existing T cell immune response against mucin epitopes in cancer patients.Materials and Methods:Using tetramer and interferon-γ assays, the immune response to one MUC1 peptide epitope in the peripheral blood of breast cancer patients was quantified. The data were compared with the clinical course of the patients.Results:CD8+ T cells capable of recognizing the HLA-A*0201-restricted STAPPVHNV epitope were detected in 9 of 19 patients with a frequency ranging 0.01–0.082%. No significant difference was found between the occurrence of epitope-specific CD8+ T cells of patients with progressive disease and disease-free patients. However, all patients with stable disease showed a specific immune response, including both patients with the highest frequency.Conclusions:The results of this study provide further evidence that a natural specific cellular immune response against this mucin epitope exists in breast cancer patients.

Apoptosis of male germ-line stem cells after laser ablation of their niche.

Male germ-line stem cells (GSCs) and their niche-the apical cells or hub cells-display a unique feature at the apices of insect testicular follicles. In the locust, Locusta migratoria, the niche consists of only one large apical cell surrounded by about 60 GSCs. The apical cell can be readily identified in the intact follicle. Using laser ablation it is feasible to destroy the apical cell exclusively without injuring neighboring GSCs or any other cells. The most immediate effect on GSCs is the loss of their structural polarity. Beginning about 3 h after laser treatment chromatin starts to clump and condense in individual GSCs, and some show the first signs of cellular breakdown. These symptoms intensify during the 96-h observation period after laser ablation of the apical cell. TUNEL staining and electron microscopic observations confirm an apoptotic cell death of the GSCs. Laser ablation of individual GSCs had no effect on neighboring GSCs or the apical cell. Destroyed apical cells were not replaced during the observation period. Mitotic divisions of GSCs ceased after about 24 h after apical cell ablation. It is speculated that it might be a general principle in stem cell-niche relationships that stem cells undergo apoptosis when the niche is dysfunctional. This could be a control mechanism to prevent tumor growth of orphaned GSCs.