NFATc1 as a therapeutic target in FLT3-ITD-positive AML.

Internal tandem duplications (ITD) in the Fms-related tyrosine kinase 3 receptor (FLT3) are associated with a dismal prognosis in acute myeloid leukemia (AML). FLT3 inhibitors such as sorafenib may improve outcome, but only few patients display long-term responses, prompting the search for underlying resistance mechanisms and therapeutic strategies to overcome them. Here we identified that the nuclear factor of activated T cells, NFATc1, is frequently overexpressed in FLT3-ITD-positive (FLT3-ITD+) AML. NFATc1 knockdown using inducible short hairpin RNA or pharmacological NFAT inhibition with cyclosporine A (CsA) or VIVIT significantly augmented sorafenib-induced apoptosis of FLT3-ITD+ cells. CsA also potently overcame sorafenib resistance in FLT3-ITD+ cell lines and primary AML. Vice versa, de novo expression of a constitutively nuclear NFATc1-mutant mediated instant and robust sorafenib resistance in vitro. Intriguingly, FLT3-ITD+ AML patients (n=26) who received CsA as part of their rescue chemotherapy displayed a superior outcome when compared with wild-type FLT3 (FLT3-WT) AML patients. Our data unveil NFATc1 as a novel mediator of sorafenib resistance in FLT3-ITD+ AML. CsA counteracts sorafenib resistance and may improve treatment outcome in AML by means of inhibiting NFAT.

Adaptation of cancer cells from different entities to the MDM2 inhibitor nutlin-3 results in the emergence of p53-mutated multi-drug-resistant cancer cells.

Six p53 wild-type cancer cell lines from infrequently p53-mutated entities (neuroblastoma, rhabdomyosarcoma, and melanoma) were continuously exposed to increasing concentrations of the murine double minute 2 inhibitor nutlin-3, resulting in the emergence of nutlin-3-resistant, p53-mutated sublines displaying a multi-drug resistance phenotype. Only 2 out of 28 sublines adapted to various cytotoxic drugs harboured p53 mutations. Nutlin-3-adapted UKF-NB-3 cells (UKF-NB-3(r)Nutlin(10 µM), harbouring a G245C mutation) were also radiation resistant. Analysis of UKF-NB-3 and UKF-NB-3(r)Nutlin(10 µM) cells by RNA interference experiments and lentiviral transduction of wild-type p53 into p53-mutated UKF-NB-3(r)Nutlin(10 µM) cells revealed that the loss of p53 function contributes to the multi-drug resistance of UKF-NB-3(r)Nutlin(10 µM) cells. Bioinformatics PANTHER pathway analysis based on microarray measurements of mRNA abundance indicated a substantial overlap in the signalling pathways differentially regulated between UKF-NB-3(r)Nutlin(10 µM) and UKF-NB-3 and between UKF-NB-3 and its cisplatin-, doxorubicin-, or vincristine-resistant sublines. Repeated nutlin-3 adaptation of neuroblastoma cells resulted in sublines harbouring various p53 mutations with high frequency. A p53 wild-type single cell-derived UKF-NB-3 clone was adapted to nutlin-3 in independent experiments. Eight out of ten resulting sublines were p53-mutated harbouring six different p53 mutations. This indicates that nutlin-3 induces de novo p53 mutations not initially present in the original cell population. Therefore, nutlin-3-treated cancer patients should be carefully monitored for the emergence of p53-mutated, multi-drug-resistant cells.

Regulation of telomerase activity by the p53 family member p73.

The terminal ends of eukaryotic chromosomes, termed telomeres, progressively shorten during each round of cell division eventually leading cells into senescence. Tumor cells typically overcome this barrier to unlimited proliferation by activation of the human telomerase reverse transcriptase (hTERT) gene. In contrast, in most human somatic cells hTERT expression is tightly repressed by multiple tumor suppressors. Here, we studied the regulation of hTERT by the p53 family member p73. We show that forced expression of p73 or activation of endogenous p73 by E2F1 results in the downregulation of telomerase activity. Vice versa, siRNA-mediated knockdown of p73 induces hTERT expression. Responsiveness to p73 is conferred by Sp1 binding sites within the hTERT core promoter. In tumor cells, p73 isoforms lacking the transactivation domain (DeltaNp73) are frequently overexpressed and believed to function as oncogenes. We show that DeltaNp73 antagonizes the repressive effect of the proapoptotic p53 family members on hTERT expression and, in addition, induces hTERT expression in telomerase-negative cells by interfering with E2F-RB-mediated repression of the hTERT core promoter. These data provide evidence that the p73 gene functions as an important regulator of telomerase activity with implications for embryonic development, cellular differentiation and tumorigenesis.

Antitumor capacity of a dominant-negative RET proto-oncogene mutant in a medullary thyroid carcinoma model.

Gain-of-function mutations in the RET proto-oncogene resulting in a constitutively active receptor tyrosine kinase have been identified as responsible for three subtypes of multiple endocrine neoplasia type 2 (MEN-2) and the development of sporadic medullary and papillary thyroid carcinoma. An important strategy in cancer gene therapy is the inhibition of oncogenic signal transduction by interfering with the molecular mechanisms of activation. In the present study, we tested the therapeutic capacity of an adenovirus expressing a dominant-negative (dn) RET mutant, RET(51).flag, under the control of a synthetic C cell-selective calcitonin promoter (TSE2.CP1) against human medullary thyroid cancer (MTC). Infection of human MTC-derived TT cells with Ad-TSE2.CP1-dn-RET(51).flag resulted in the accumulation of immature RET protein in the endoplasmic reticulum and a strong reduction of oncogenic RET receptor on the cell surface, indicating that RET(51).flag exhibits a dominant-negative effect over endogenous oncogenic protein. Analysis of potential downstream mechanisms associated with the inhibition of oncogenic RET signaling by overexpression of mutant RET(51).flag revealed a significant loss of cell viability in TT cells due to the induction of apoptosis. Finally, we examined the antitumor activity of the dominant-negative RET approach in vivo. Inoculation of Ad-TSE2.CP1- dn-RET(51).flag-expressing MTC cells into nude mice led to complete suppression of tumor growth. Moreover, a single intratumoral injection of Ad-TSE2.CP1-dn-RET(51).flag into established thyroid tumors resulted in prolonged survival of treated mice compared with the controls. Our data suggest that adenoviral delivery of dn-RET(51).flag may be a reliable strategy of effective molecular intervention for RET oncogene-related MTC.

Role of p73 in malignancy: tumor suppressor or oncogene?

The recently identified p53 family member, p73, shows substantial structural and functional homology with p53. However, despite the established role of p53 as a proto-type tumor suppressor, a similar function of p73 in malignancy is questionable. Overexpression of p73 can activate typical p53-responsive genes, and activation of p73 has been implicated in apoptotic cell death induced by aberrant cell proliferation and some forms of DNA-damage. These data together with the localization of TP73 on chromosome 1p36, a region frequently deleted in a variety of human tumors, led to the hypothesis that p73 has tumor suppressor activity just like p53. However, unlike p53-/- mice, p73 knockout mice do not develop tumors. Extensive studies on primary tumor tissues have revealed overexpression of wild-type p73 in the absence of p73 mutations instead, suggesting that p73 may augment, rather than inhibit tumor development. In contrast to p53, differential splicing of the TP73 gene locus gives rise to a complex pattern of interacting p73 isoforms with antagonistic functions. In fact, induction of apoptosis by increased levels of p73 can be blocked by both p53 mutants and the N-terminally truncated p73 isoforms, which were recently shown to possess oncogenic potential. In the light of these new findings the contradictory role of p73 in malignancy will be discussed.

p73 in apoptosis.

The TP53 tumour-suppressor gene belongs to a family that includes the two recently identified homologues TP63 and TP73. Overexpression of p73 can activate typical p53-responsive genes and induce apoptosis like p53. In addition, activation of p73 has been implicated in apoptotic cell death induced by aberrant cell proliferation and some forms of DNA-damage. These data together with the localization of TP73 on chromosome 1p36, a region frequently deleted in a variety of human cancers, led to the hypothesis that p73 has tumour suppressor activity just like p53. However, despite its proapoptotic activity in vitro, the lack of tumour-formation in p73 knock-out mice and primary human tumour data demonstrating overexpression of wild-type p73 currently argue against p73 being a classical tumour suppressor. Interestingly, in contrast to TP53, TP73 gives rise to a complex pattern of pro- and antiapoptotic p73 isoforms generated by differential splicing and alternative promoter usage. Therefore further insight into the function and regulation of these structurally and functionally diverse p73 proteins is needed to elucidate the role of TP73 for apoptosis and human tumorigenesis.

Therapeutic efficacy of E2F1 in pancreatic cancer correlates with TP73 induction.

Pancreatic cancer is particularly resistant to apoptosis by antineoplastic agents, which is partly attributable to the lack of functional p53. Here we show that E2F1 in combination with the most clinically efficient drug, gemcitabine, resulted in a strong induction of apoptosis independent of functional p53, whereas the effect of either therapy alone varied between different cell lines. Intratumoral injection of a helper-dependent adenovirus vector expressing E2F1 plus drug treatment resulted in a significant reduction of tumor volume. The therapeutic effect is directly correlated with the induction of the p53 homologue p73, suggesting that the recently discovered E2F1/p73 pathway plays a critical role in cancer therapy.

Identification of the full-length huntingtin- interacting protein p231HBP/HYPB as a DNA-binding factor.

Neurodegeneration in Huntington's disease (HD) is associated with an elongated glutamine tract in the widely expressed huntingtin protein. Although the pathogenic mechanisms are still unknown, the distinct physical properties of mutant huntingtin in the brain suggest that other factors including huntingtin-interacting proteins might play a specific role. We have previously identified a DNA-binding motif in the proximal E1A promoter of adenovirus serotype 12 as responsible for E1A autoregulation. Here, we identified the p231HBP protein as a DNA-binding factor, the C-terminal portion of which has recently been characterized as the huntingtin-interacting protein HYPB of unknown function. We have determined the full-length cDNA sequence, identified several domains supporting its gene regulatory functions, and mapped the HBP231 gene to chromosome 3p21.2-p21.3. Our results provide an interesting molecular link between huntingtin and a DNA-binding factor, implicating that this interaction might result in the alteration of cellular gene expression involved in HD pathogenesis.

Large nontransplanted hepatocellular carcinoma in woodchucks: treatment with adenovirus-mediated delivery of interleukin 12/B7.1 genes.

BACKGROUND: Cytokine-based gene therapy strategies efficiently stimulate immune responses against many established transplanted tumors, leading to rejection of the tumor. In this study, we investigated the therapeutic potential of cancer immunotherapy in a clinically more relevant model, woodchucks with primary hepatocellular carcinomas induced by woodchuck hepatitis virus. METHODS: Large (2-5 cm), established intrahepatic tumors were given an injection once with 1 x 10(9) plaque-forming units of AdIL-12/B7.1, an adenovirus vector carrying genes for murine interleukin 12 and B7.1, or of AdEGFP, the control virus, and regression of the tumors was then monitored. Five animals were used in total. RESULTS: In four tumor-bearing animals, the antitumor response was assessed by autopsy and histologic analysis within 1-2 weeks after treatment. In all animals treated with AdIL-12/B7.1 therapy versus AdEGFP therapy, we observed substantial tumor regression (P =.006; two-sided unpaired Student's t test) accompanied by a massive infiltration of T lymphocytes. These tumors also contained increased levels of CD4(+) and CD8(+) T cells and interferon gamma (IFN gamma). In continuously growing tumor nodules given an injection of the control virus or in nontumoral liver, no such effects (i.e., tumor regression and increased levels of CD4(+) and CD8(+) T cells and IFN gamma) were detected. In the fifth animal, monitored for long-term antitumor efficacy by magnetic resonance imaging (MRI) after intratumoral vector administration by MRI guidance, the tumor was almost completely eliminated (> or = 95%) 7 weeks after treatment. CONCLUSION: Adenovirus vector-based immunotherapy appears to be an effective treatment of large nontransplanted (orthotopic) tumors that acquire malignant characteristics in a stepwise process, reflecting the real-world scenario of hepatocellular carcinoma in humans.

Role of the p53-homologue p73 in E2F1-induced apoptosis.

Most human cancers harbour aberrations of cell-cycle control, which result in deregulated activity of the E2F transcription factors with concomitant enhanced cell-cycle progression. Oncogenic signalling by E2F1 has recently been linked to stabilization and activation of the tumour suppressor p53 (refs 1,3,4). The p73 protein shares substantial sequence homology and functional similarity with p53 (refs 5-7 ). Hence, several previously considered p53-independent cellular activities may be attributable to p73. Here we provide evidence that E2F1 directly activates transcription of TP73, leading to activation of p53-responsive target genes and apoptosis. Disruption of p73 function by a tumour-derived p53 mutant reduced E2F1-mediated apoptosis. Thus, p73 activation by deregulated E2F1 activity might constitute a p53-independent, anti-tumorigenic safeguard mechanism.

Improved safety through tamoxifen-regulated induction of cytotoxic genes delivered by Ad vectors for cancer gene therapy.

The transfer of pro-apoptotic genes to tumors is one of the most promising strategies for anticancer gene therapy. However, the use of potentially toxic genes, such as tumor suppressor genes or apoptotic genes, needs controllable transgene activation. To achieve regulation of the transgene at a desired time, we developed an adenovirus (Ad) vector, in which the apoptotic activity of the target gene has been made 4-OHT-dependent by fusion to the ligand binding-domain of the estrogen receptor (ER). For evaluation of the system in human tumor cells, we used the E2F1 gene which encodes a transcription factor that triggers massive apoptosis in several human cancers. AdER-E2F1 expressed high levels of transgene over at least 1 week. Upon activation of E2F1 by the ligand 4-hydroxy-tamoxifen (4-OHT) the ER-E2F1 fusion protein correctly translocated from the cytosol to the nucleus, transactivated E2F-dependent promoters, and rapidly induced substantial E2F1-related toxicity. Finally, experiments in nude mice showed tightly regulated tumor growth suppression in vivo. Taken together, our system represents a powerful approach for tight regulation and rapid induction of cytotoxicity as the major criteria for safe gene delivery.

E1A overcomes the apoptosis block in BCR-ABL+ leukemia cells and renders cells susceptible to induction of apoptosis by chemotherapeutic agents.

A crucial function of the BCR-ABL chimeric gene in chronic myeloid leukemia is the prolongation of cell survival by inhibition of apoptosis. BCR-ABL expression confers cross-resistance to multiple genotoxic anticancer drugs by inhibition of the apoptotic response to DNA damage in association with cell cycle arrest at the G2-M restriction point. Previous reports indicated that BCR-ABL exerts its antiapoptotic effect against various apoptotic stimuli upstream to the cleavage and activity of caspase-3. Here we show that the adenovirus E1A protein induces substantial apoptosis in BCR-ABL expressing K562 and LAMA-84 leukemia cells. This apoptotic activity of E1A is accompanied by processing of caspase-3 and cleavage of poly(ADP-ribose) polymerase and can be significantly blocked by z-VAD-fmk Z-Val-Ala-Asp(OCH3)-CH2F and the caspase-3-specific inhibitor Z-DEVD-FMK Z-Asp(OCH3)-Glu-Val-Asp(OCH3)-CH2F. Moreover, E1A renders K562 cells, which are particularly resistant to cell death irrespective of the inducing agent, susceptible to induction of apoptosis by the chemotherapeutic agents etoposide and daunorubicin. Counteracting the DNA damage-induced inactivation of cdc2 kinase, E1A reverses the drug-induced G2-M arrest These results indicate that solitary delivery of E1A significantly antagonizes BCR-ABL-induced antiapoptotic functions and circumvents the inherent resistance to DNA damage-induced apoptosis, supporting the use of E1A in combination with chemotherapeutic agents as a promising therapeutic strategy for successful treatment of Philadelphia chromosome-positive leukemia in vivo.

E1A is sufficient by itself to induce apoptosis independent of p53 and other adenoviral gene products.

Induction of apoptosis seems to be a key function in maintaining normal cell growth by exerting negative controls on cell proliferation and suppressing tumorigenesis. The adenovirus E1A oncogene shows both cell cycle progression and apoptotic functions. To understand the mechanism of E1A-induced apoptosis, the apoptotic function of E1A 13S was investigated in p53-null cells. We show here that E1A is sufficient by itself to induce substantial apoptosis independent of p53 and other adenoviral genes. The apoptotic function of E1A is accompanied by processing of caspase-3 and cleavage of poly(ADP-ribose)-polymerase. Cell death is significantly blocked by the caspase inhibitor zVAD-fmk and when coexpressed with E1B19K, Bcl-2 or the retinoblastoma protein (RB). Analyses of E1A mutants indicated that the apoptotic activity of E1A correlates closely with the ability to bind the key regulators of E2F1-induced apoptosis, p300 and RB. Finally, in vivo relevance of down-modulation of p53-independent apoptosis for efficient transformation is demonstrated.