Casein Kinase 1D Encodes a Novel Drug Target in Hedgehog-GLI-Driven Cancers and Tumor-Initiating Cells Resistant to SMO Inhibition.

(1) Background: Aberrant activation of the hedgehog (HH)-GLI pathway in stem-like tumor-initiating cells (TIC) is a frequent oncogenic driver signal in various human malignancies. Remarkable efficacy of anti-HH therapeutics led to the approval of HH inhibitors targeting the key pathway effector smoothened (SMO) in basal cell carcinoma and acute myeloid leukemia. However, frequent development of drug resistance and severe adverse effects of SMO inhibitors pose major challenges that require alternative treatment strategies targeting HH-GLI in TIC downstream of SMO. We therefore investigated members of the casein kinase 1 (CSNK1) family as novel drug targets in HH-GLI-driven malignancies. (2) Methods: We genetically and pharmacologically inhibited CSNK1D in HH-dependent cancer cells displaying either sensitivity or resistance to SMO inhibitors. To address the role of CSNK1D in oncogenic HH signaling and tumor growth and initiation, we quantitatively analyzed HH target gene expression, performed genetic and chemical perturbations of CSNK1D activity, and monitored the oncogenic transformation of TIC in vitro and in vivo using 3D clonogenic tumor spheroid assays and xenograft models. (3) Results: We show that CSNK1D plays a critical role in controlling oncogenic GLI activity downstream of SMO. We provide evidence that inhibition of CSNK1D interferes with oncogenic HH signaling in both SMO inhibitor-sensitive and -resistant tumor settings. Furthermore, genetic and pharmacologic perturbation of CSNK1D decreases the clonogenic growth of GLI-dependent TIC in vitro and in vivo. (4) Conclusions: Pharmacologic targeting of CSNK1D represents a novel therapeutic approach for the treatment of both SMO inhibitor-sensitive and -resistant tumors.

DYRK1B as therapeutic target in Hedgehog/GLI-dependent cancer cells with Smoothened inhibitor resistance.

A wide range of human malignancies displays aberrant activation of Hedgehog (HH)/GLI signaling, including cancers of the skin, brain, gastrointestinal tract and hematopoietic system. Targeting oncogenic HH/GLI signaling with small molecule inhibitors of the essential pathway effector Smoothened (SMO) has shown remarkable therapeutic effects in patients with advanced and metastatic basal cell carcinoma. However, acquired and de novo resistance to SMO inhibitors poses severe limitations to the use of SMO antagonists and urgently calls for the identification of novel targets and compounds.Here we report on the identification of the Dual-Specificity-Tyrosine-Phosphorylation-Regulated Kinase 1B (DYRK1B) as critical positive regulator of HH/GLI signaling downstream of SMO. Genetic and chemical inhibition of DYRK1B in human and mouse cancer cells resulted in marked repression of HH signaling and GLI1 expression, respectively. Importantly, DYRK1B inhibition profoundly impaired GLI1 expression in both SMO-inhibitor sensitive and resistant settings. We further introduce a novel small molecule DYRK1B inhibitor, DYRKi, with suitable pharmacologic properties to impair SMO-dependent and SMO-independent oncogenic GLI activity. The results support the use of DYRK1B antagonists for the treatment of HH/GLI-associated cancers where SMO inhibitors fail to demonstrate therapeutic efficacy.

Cancer immunotherapeutic potential of novel small molecule TLR7 and TLR8 agonists.

Toll-like receptor (TLR)-mediated signaling is proposed as an immunotherapeutic target against tumorigenesis. Natural killer (NK) cells play a critical role in host defense against tumors. Specifically, formation of tumor metastasis in various organs can be suppressed by the local activity of NK cells. In this study, we present a novel TLR7 agonist (termed SC-1) that induces pro-inflammatory cytokines in human blood cells, activates NK cell function, and is highly efficient in preventing lung metastases in a pulmonary metastatic Renca model. Furthermore, a second compound (termed SC-2), acting as dual-specific TLR7 and TLR8 agonist, was evaluated with respect to its immunostimulatory and NK cell-activating capacities. The release of pro-inflammatory cytokines was shown to be even more pronounced with this compound. Additional experiments showed a significant up-regulation of activation marker CD69 on NK cells and increased cytolytic activity of peripheral blood cells compared to the effect of a monospecific TLR7 agonist SC-1. Normally, TLR7 and TLR8 are expressed on different immune cell subpopulations. TLR7 expression on antigen-presenting cells is detected in plasmacytoid dendritic cells, CD34+-derived dendritic cells, and B-cells, whereas TLR8 is mainly expressed on cells of the myeloid lineage, such as monocytes, macrophages, and myeloid dendritic cells. Therefore, a compound that activates both TLR7 and TLR8 would result in a highly efficient immune system activation and may give rise to an enhanced anti-tumor activity in vivo compared to that elicited by a monospecific TLR7 agonist.

Tapping an unexploited repository: Carnoy's fixed cell pellets for proteomic biomarker research in leukemia.

For chromosomal analysis in tumor genetics, cells from blood and bone marrow are prepared and preserved virtually indefinitely in Carnoy's fixative (methanol/acetic acid). Numerous samples are stored unvalued in hospitals and institutes worldwide. We developed a method to analyze proteins from even a small amount of these cells by mass spectrometry using affinity chromatographic surfaces (SELDI), and demonstrated the application of proteomic biomarker research in cases of acute myeloid leukemia.

Cytogenetic characterisation and proteomic profiling of the Imatinib-resistant cell line KCL22-R.

Approximately 30% of chronic myeloid leukemia patients show initially no response to Imatinib, a potent inhibitor of BCR-ABL. This intrinsic resistance may be due to BCR-ABL-independent cell growth. Here we analysed the cytogenetic anomalies and the proteomic profiling in KCL22-S and KCL22-R, two Imatinib-sensitive and -resistant derivative cell lines of KCL22. A tetrasomy 8 and a non-reciprocal translocation +der(6)t(6;13)(p11.1;q12) were found only in KCL22-R as new evolved anomalies. Chromosome der(6)t(6;13) showed four variants differing in the chromatin content of 13q14-13qter including the retinoblastoma gene. Due to these sub-clones, approximately 65-79% of the Imatinib-treated KCL22-R cells showed a disomy and 21-35% a monosomy for 13q14. Imatinib removal reduced the main clone to approximately 20% in the benefit of the monosomic sub-clones. This was accompanied by an increased apoptosis rate and was revertible by Imatinib re-treatment. This effect may be connected with genes located in 13q14-qter. Proteomic profiling of the cell lines performed with ProteinChip technology (SELDI) revealed several differentially expressed proteins (n=45). In summary, we demonstrate here the complex changes on the cytogenetic and proteomic level which could be caused by Imatinib and the resistance resulting from it.

Immune escape for renal cell carcinoma: CD70 mediates apoptosis in lymphocytes.

Tumors can escape immune recognition and destruction through the induction of apoptosis in lymphocytes. Although renal cell carcinoma (RCC) is able to prevent immune recognition, only a few genes (such as FasL) that are relevant for RCC immune escape have been identified so far. We have previously shown that some apoptosis-inducing genes are overexpressed in RCC. We hypothesized that these genes could be part of the immune-escape strategy of these tumors. Here we report that CD70, a cytokine overexpressed in RCC, promotes lymphocyte apoptosis through interaction with its receptor CD27 and with the intracellular receptor-binding protein SIVA. Apoptosis increased after cocultivating lymphocytes with the RCC cell lines A498 and CAKI2. The addition of recombinant soluble CD70 to both native lymphocytes and a T-cell cell line resulted in increased lymphocyte apoptosis as well. Furthermore, induced apoptosis could be partially blocked with anti-CD27 and anti-CD70 antibodies. Our results strongly indicate a role for CD70 and CD27 receptor in lymphocyte apoptosis within the tumor environment. Apoptosis mediated by exposure to the CD70 secreted by tumor cells may contribute to the failure of RCC patients to develop an effective lymphocyte-mediated antitumor response.

Rosai-Dorfman disease with cutaneous manifestation (sinus histiocytosis with massive lymphadenopathy).

We report a 73-year-old woman with the typical findings of "Rosai-Dorfman disease" (RDD) including massive, painless lymphadenopathy, fever and multiple, asymptomatic cutaneous lesions on her face, collum and neck. Histologically, the lesions were marked by characteristic large macrophages exhibiting emperipolesis.

Characteristic genomic imbalances in pediatric pheochromocytoma.

Pheochromocytoma (PCC) in children is rare, genetically not well described, and often related to a poor prognosis. We detected genomic imbalances in all 14 tumors from children analyzed by comparative genomic hybridization. A combinatorial loss of chromatin from 3p and 11p was a common feature in 10 of 14 (72%) patients, which was a result of either a loss of a total chromosome 3 and a total chromosome 11 in 6 of 10 patients, or confined deletions of their p arms in 4 of 10 patients. All patients exhibiting a loss of 3p and 11p carried VHL mutations. The VHL mutations were constitutive in 9 cases and somatic and restricted to tumor DNA in the remaining tumor. On the other hand, VHL mutations were absent in 4 patients, 2 who had other familial syndromes (NF1, SDHD) and 2 with unknown etiology. Our data show that the pattern of imbalances in the tumor DNA of PCC patients strongly correlated with an underlying familial VHL mutation. Furthermore, we show that true sporadic PCC is rare in childhood. Thus, children with PCC should be checked for a related predisposing gene. This would also identify familial syndrome patients requiring long-term monitoring for other syndrome-related malignancies.

Molecular cytogenetic characterization of the mouse cell line WMP2 by spectral karyotyping and multicolor banding applying murine probes.

The Moloney murine leukemia virus-transformed suspension cell line WMP2 is derived from wild mice (Mus musculus) of the WMP/WMP strain. These mice carry nine pairs of metacentric Robertsonian translocation chromosomes. As the chromosomes of the wild-type mouse are all acrocentric, metaphase spreads of the WMP2 cells seam to be highly suited for physical gene mapping. Here we studied the WMP2 line using spectral karyotyping (SKY) combined with new established mouse specific multicolor banding (mcb) probes for the chromosomes X, 3, 4, 6 and 18. SKY revealed that the WMP2 cell line developed further four derivative chromosomes. After application of mcb five previously unrecognizable intrachromosomal rearrangements with 9 breakpoints were detected for the studied chromosomes.

Microdissection-derived murine mcb probes from somatic cell hybrids.

The multicolor-banding (mcb) technique is a fluorescence in situ hybridization (FISH)-banding approach, which is based on region-specific microdissection libraries producing changing fluorescence intensity ratios along the chromosomes. The latter are used to assign different pseudocolors to specific chromosomal regions. Here we present the first three available mcb-probe sets for the Mus musculus chromosomes 3, 6, and 18. In the present work, the creation of the microdissection libraries was done for the first time on mouse/human somatic cell hybrids. During creation of the mcb-probes, the latter enabled an unambiguous identification of the, otherwise in GTG-banding, hardly distinguishable murine chromosomes.

Proteome analysis of maternal serum samples for trisomy 21 pregnancies using ProteinChip arrays and bioinformatics.

A surface-enhanced laser desorption/ionization time of flight (SELDI-TOF)-based ProteinChip System was used as a tool for rapid discovery and identification of protein patterns in serum that discriminate between trisomy 21 and unaffected pregnancies. We analyzed 24 serum samples from women carrying a trisomy 21 pregnancy and 32 with an unaffected pregnancy, ranging from 10.0 to 14.0 weeks of gestation. The resulting protein profiles were submitted to a clustering algorithm, a rule extraction, a rating, and a rule base construction step. For the generated combined rule base, the specificity and sensitivity for the prediction of a trisomy 21 pregnancy reach 97% and 91%, respectively.

Somatic mosaicism in patients with Angelman syndrome and an imprinting defect.

Angelman syndrome is a neurogenetic disorder caused by the loss of function of the imprinted UBE3A gene in 15q11-q13. In a small group of patients, the disease is due to an imprinting defect (ID) that silences the maternal UBE3A allele. The presence of a faint maternal band detected by methylation-specific PCR analysis of the SNURF-SNRPN locus in approximately one-third of patients who have an ID but no imprinting center deletion suggested that these patients are mosaics of ID cells and normal cells. In two patients studied, somatic mosaicism was proven by molecular and cellular cloning, respectively. X inactivation studies of cloned fibroblasts from one patient suggest that ID occurred before the blastocyst stage. To quantify the degree of mosaicism, we developed a novel quantitative methylation assay based on real-time PCR. In 24 patients tested, the percentage of normal cells ranged from <1% to 40%. Regression analysis suggests that patients with a higher percentage of normally methylated cells tend to have milder clinical symptoms than patients with a lower percentage. In conclusion, we suggest that the role of mosaic imprinting defects in mental retardation is underestimated.

Somatic mosaicism for maternal uniparental disomy 15 in a girl with Prader-Willi syndrome: confirmation by cell cloning and identification of candidate downstream genes.

Although uniparental disomy often results from the postzygotic rescue of a meiotic non-disjunction event, mosaicism is usually confined to the placenta. We describe a girl with Prader-Willi syndrome (PWS) who is mosaic for normal cells and cells with maternal uniparental disomy 15 [upd(15)mat] in blood and skin. Somatic mosaicism was confirmed by cloning and genotyping of skin fibroblasts. X inactivation studies indicated that upd occurred prior to X inactivation. RNA samples from the cloned cells were used in DNA microarray experiments to study the effect of upd(15)mat on the gene expression pattern of fibroblasts. Proof of principle was obtained by detecting several chromosome 15 genes known to be imprinted. We did not obtain any evidence for novel 15q genes showing imprinted expression in fibroblasts. Differentially expressed genes on other chromosomes are candidates for downstream genes regulated by an imprinted gene and may play a role in the pathogenesis of PWS. The finding of strongly reduced mRNA levels in upd(15)mat cells of the gene encoding secretogranin II (SCG2), which is a precursor of the dopamine releasing factor secretoneurin, raises the question whether hyperphagia in patients with PWS might be due to a defect in dopamine-modulated food reward circuits.

The DNA-based structure of human chromosome 5 in interphase.

In contrast to those of metaphase chromosomes, the shape, length, and architecture of human interphase chromosomes are not well understood. This is mainly due to technical problems in the visualization of interphase chromosomes in total and of their substructures. We analyzed the structure of chromosomes in interphase nuclei through use of high-resolution multicolor banding (MCB), which paints the total shape of chromosomes and creates a DNA-mediated, chromosome-region-specific, pseudocolored banding pattern at high resolution. A microdissection-derived human chromosome 5-specific MCB probe mixture was hybridized to human lymphocyte interphase nuclei harvested for routine chromosome analysis, as well as to interphase nuclei from HeLa cells arrested at different phases of the cell cycle. The length of the axis of interphase chromosome 5 was determined, and the shape and MCB pattern were compared with those of metaphase chromosomes. We show that, in lymphocytes, the length of the axis of interphase chromosome 5 is comparable to that of a metaphase chromosome at 600-band resolution. Consequently, the concept of chromosome condensation during mitosis has to be reassessed. In addition, chromosome 5 in interphase is not as straight as metaphase chromosomes, being bent and/or folded. The shape and banding pattern of interphase chromosome 5 of lymphocytes and HeLa cells are similar to those of the corresponding metaphase chromosomes at all stages of the cell cycle. The MCB pattern also allows the detection and characterization of chromosome aberrations. This may be of fundamental importance in establishing chromosome analyses in nondividing cells.

Cultivation of fetal erythroid precursors from maternal blood: isolation and characterization by PCR and FISH.

Cultivation of fetal progenitor cells from maternal blood offers the opportunity to produce sufficient fetal cells for prenatal molecular genetic and cytogenetic analysis. For in vitro cultivation, 10 ml of blood was collected from 22 women carrying a male fetus. After triple-density gradient centrifugation, the mononucleated cells were cultivated for 10 to 14 days in special hematopoietic growth medium. Red and white colored cell colonies were individually collected by micromanipulation. A representative sample from each colony was characterized by chromosome Y-specific polymerase chain reaction (PCR) systems. The remaining cells of the Y-positive colonies were used to perform chromosome preparations and fluorescence in situ hybridization (FISH) to detect XY-positive interphase nuclei and metaphases. Y-positive signals could be detected in 15 (68%) of the 22 analyzed blood samples. With SRY PCR 10.5% (40/379) of the collected red colonies were determined to be of fetal origin and 6.1% (32/522) of the colonies analyzed by amelogenin PCR were Y-positive. All collected white cell colonies were Y-negative. FISH analyses of PCR-positive colonies revealed that less than 30% of the cells within a colony are of fetal origin and reflect more precisely the actual situation within a single colony. Moreover the successful preparation of fetal metaphases in non-invasive prenatal diagnosis is presented.