Comprehensive Genomic Profiling Alters Clinical Diagnoses in a Significant Fraction of Tumors Suspicious of Sarcoma.

PURPOSE: Tumor classification is a key component in personalized cancer care. For soft-tissue and bone tumors, this classification is currently based primarily on morphology assessment and IHC staining. However, these standard-of-care methods can pose challenges for pathologists. We therefore assessed how whole-genome and whole-transcriptome sequencing (WGTS) impacted tumor classification and clinical management when interpreted together with histomorphology. EXPERIMENTAL DESIGN: We prospectively evaluated WGTS in routine diagnostics of 200 soft-tissue and bone tumors suspicious for malignancy, including DNA and RNA isolation from the tumor, and DNA isolation from a peripheral blood sample or any non-tumor tissue. RESULTS: On the basis of specific genomic alterations or absence of presumed findings, WGTS resulted in reclassification of 7% (13/197) of the histopathologic diagnoses. Four cases were downgraded from low-grade sarcomas to benign lesions, and two cases were reclassified as metastatic malignant melanomas. Fusion genes associated with specific tumor entities were found in 30 samples. For malignant soft-tissue and bone tumors, we identified treatment relevant variants in 15% of cases. Germline pathogenic variants associated with a hereditary cancer syndrome were found in 22 participants (11%). CONCLUSIONS: WGTS provides an important dimension of data that aids in the classification of soft-tissue and bone tumors, correcting a significant fraction of clinical diagnoses, and identifies molecular targets relevant for precision medicine. However, genetic findings need to be evaluated in their morphopathologic context, just as germline findings need to be evaluated in the context of patient phenotype and family history.

Interdependence of SS18-SSX-driven YAP1 and ß-Catenin Activation in Synovial Sarcoma.

Synovial sarcoma, a rare malignant soft tissue tumor, is characterized by a specific chromosomal translocation t(X;18). The resulting chimeric SS18-SSX fusion protein drives synovial sarcoma pathogenesis by integrating into the BAF complex and dysregulating gene transcription. Because previous functional analyses revealed a connection between SS18-SSX and the activity of the transcriptional coregulators YAP1/TAZ and ß-catenin, respectively, this study examined a potential interdependence between these essential effector proteins in synovial sarcoma. In a large cohort of synovial sarcoma tissue specimens, IHC analyses revealed a substantial subset of synovial sarcoma with concurrent nuclear accumulation of YAP1/TAZ and ß-catenin. In vitro, small-molecule inhibitor treatment, RNAi-mediated knockdown, and vector-based overexpression assays demonstrated that YAP1, TAZ, and ß-catenin transcriptional activity is not only stimulated by the SS18-SSX fusion protein, but that they also mutually enhance each other's activation. These analyses showed the highest cooperative effect with overexpression of YAP1 in combination with ß-catenin. Coimmunoprecipitation experiments detected nuclear interactions between YAP1, ß-catenin, and the SS18-SSX fusion protein, the latter being an integral part of the BAF complex. Disruption of BAF complex assembly affected the coregulation of YAP1 and ß-catenin, indicating that this chromatin remodeling complex plays a crucial role for interdependent YAP1 and ß-catenin activation in synovial sarcoma cells. IMPLICATIONS: This study provides deeper insights into synovial sarcoma tumor biology demonstrating a mutual dependence between YAP1/TAZ and ß-catenin transcriptional activity and a complex interplay with the SS18-SSX fusion protein within the BAF complex.

SS18-SSX drives CREB activation in synovial sarcoma.

PURPOSE: Synovial sarcoma (SySa) is a rare soft tissue tumor characterized by a reciprocal t(X;18) translocation. The chimeric SS18-SSX fusion protein represents the major driver of the disease, acting as aberrant transcriptional dysregulator. Oncogenic mechanisms whereby SS18-SSX mediates sarcomagenesis are incompletely understood, and strategies to selectively target SySa cells remain elusive. Based on results of Phospho-Kinase screening arrays, we here investigate the functional and therapeutic relevance of the transcription factor CREB in SySa tumorigenesis. METHODS: Immunohistochemistry of phosphorylated CREB and its downstream targets (Rb, Cyclin D1, PCNA, Bcl-xL and Bcl-2) was performed in a large cohort of SySa. Functional aspects of CREB activity, including SS18-SSX driven circuits involved in CREB activation, were analyzed in vitro employing five SySa cell lines and a mesenchymal stem cell model. CREB mediated transcriptional activity was modulated by RNAi-mediated knockdown and small molecule inhibitors (666-15, KG-501, NASTRp and Ro 31-8220). Anti-proliferative effects of the CREB inhibitor 666-15 were tested in SySa avian chorioallantoic membrane and murine xenograft models in vivo. RESULTS: We show that CREB is phosphorylated and activated in SySa, accompanied by downstream target expression. Human mesenchymal stem cells engineered to express SS18-SSX promote CREB expression and phosphorylation. Conversely, RNAi-mediated knockdown of SS18-SSX impairs CREB phosphorylation in SySa cells. Inhibition of CREB activity reduces downstream target expression, accompanied by suppression of SySa cell proliferation and induction of apoptosis in vitro and in vivo. CONCLUSION: In conclusion, our data underline an essential role of CREB in SySa tumorigenesis and provides evidence for molecular targeted therapies.

Nuclear IGF1R interacts with NuMA and regulates 53BP1­dependent DNA double­strand break repair in colorectal cancer.

Nuclear insulin­like growth factor 1 receptor (nIGF1R) has been associated with poor overall survival and chemotherapy resistance in various types of cancer; however, the underlying mechanism remains unclear. In the present study, immunoprecipitation­coupled mass spectrometry was performed in an IGF1R­overexpressing SW480­OE colorectal cancer cell line to identify the nIGF1R interactome. Network analysis revealed 197 proteins of interest which were involved in several biological pathways, including RNA processing, DNA double­strand break (DSB) repair and SUMOylation pathways. Nuclear mitotic apparatus protein (NuMA) was identified as one of nIGF1R's colocalizing partners. Proximity ligation assay (PLA) revealed different levels of p53­binding protein 1 (53BP1)­NuMA colocalization between IGF1R­positive (R+) and IGF1R­negative (R­) mouse embryonic fibroblasts following exposure to ionizing radiation (IR). 53BP1 was retained by NuMA in the R­ cells during IR­induced DNA damage. By contrast, the level of NuMA­53BP1 was markedly lower in R+ cells compared with R­ cells. The present data suggested a regulatory role of nIGF1R in 53BP1­dependent DSB repair through its interaction with NuMA. Bright­field PLA analysis on a paraffin­embedded tissue microarray from patients with colorectal cancer revealed a significant association between increased nuclear colocalizing signals of NuMA­53BP1 and a shorter overall survival. These results indicate that nIGF1R plays a role in facilitating 53BP1­dependent DDR by regulating the NuMA­53BP1 interaction, which in turn might affect the clinical outcome of patients with colorectal cancer.

TERT promoter mutation is an objective clinical marker for disease progression in chondrosarcoma.

Chondrosarcomas are the second most common malignant bone tumor. Activating promoter mutations in telomerase reverse transcriptase (TERT) was recently described by us and others as a frequent mutation in high-grade chondrosarcoma. In this study, we investigate the prognostic significance of TERT promoter mutations in 241 chondrosarcomas from 190 patients collected over 24 years (1994-2017). The TERT promoter was sequenced after microdissection of 135 chondrosarcomas from 106 patients in addition to data from our previous cohort. The TERT promoter mutation at -124 C > T was found in 45% of all patients and was significantly associated (p > 0,001) with higher tumor grade, shorter metastasis-free survival, and disease-specific survival. Additionally, TERT promoter-mutated tumors were associated with a more aggressive metastatic pattern. Shorter survival was observed in patients with wild-type primary tumors who developed a mutated metastasis indicative of tumor progression. Primary tumor genetic heterogeneity and altering mutational status between nonsynchronous metastatic lesions suggests that chondrosarcoma is a multiclonal disease progressing through a branching evolution. Conclusion: TERT promoter mutation seems to be a central event in chondrosarcoma progression with association to metastatic disease and disease-related mortality. As an easily analyzed marker, there is future potential to utilize TERT promoter mutation status as a prognostic marker and investigate telomerase-targeted therapy in chondrosarcomas.

Nuclear IGF1R interact with PCNA to preserve DNA replication after DNA-damage in a variety of human cancers.

Nuclear IGF1R has been linked to poor outcome in cancer. We recently showed that nuclear IGF1R phosphorylates PCNA and increases DNA damage tolerance. In this paper we aimed to describe this mechanism in cancer tissue as well as in cancer cell lines. In situ proximity ligation assay identified frequent IGF1R and PCNA colocalization in many cancer types. IGF1R/PCNA colocalization was more frequently increased in tumor cells than in adjacent normal, and more prominent in areas with dysplasia and invasion. However, the interaction was often lost in tumors with poor response to neoadjuvant treatment and most metastatic lesions. In two independent cohorts of serous ovarian carcinomas and oropharyngeal squamous cell carcinomas, stronger IGF1R/PCNA colocalization was significantly associated with a higher overall survival. Ex vivo irradiation of ovarian cancer tissue acutely induced IGF1R/PCNA colocalization together with γH2AX-foci formations. In vitro, RAD18 mediated mono-ubiquitination of PCNA during replication stress was dependent on IGF1R kinase activity. DNA fiber analysis revealed that IGF1R activation could rescue stalled DNA replication forks, but only in cancer cells with baseline IGF1R/PCNA interaction. We believe that the IGF1R/PCNA interaction is a basic cellular mechanism to increase DNA stress tolerance during proliferation, but that this mechanism is lost with tumor progression in conjunction with accumulated DNA damage and aberrant strategies to tolerate genomic instability. To exploit this mechanism in IGF1R targeted therapy, IGF1R inhibitors should be explored in the context of concomitant induction of DNA replication stress as well as in earlier clinical stages than previously tried.

Transcriptome profiling of Ewing sarcomas - treatment resistance pathways and IGF-dependency.

Ewing sarcomas (ESs) are aggressive sarcomas driven by EWS fusion genes. We sought to investigate whether whole-transcriptome sequencing (RNA-seq) could be used to detect patterns associated with chemotherapy response or tumor progression after first-line treatment. Transcriptome sequencing (RNA-seq) of 13 ES cases was performed. Among the differentially expressed pathways, we identified IGF2 expression as a potential driver of chemotherapy response and progression. We investigated the effect of IGF2 on proliferation, radioresistance, apoptosis, and the transcriptome pattern in four ES cell lines and the effect of IGF2 expression in a validation series of 14 patients. Transcriptome analysis identified differentially expressed genes (adj. P < 0.005) and pathways associated with chemotherapy response (285 genes), short overall survival (662 genes), and progression after treatment (447 genes). Imprinting independent promoter P3-mediated IGF2 expression was identified in a subset of cases with aggressive clinical course. In ES cell lines, IGF2 induced proliferation, but promoted radioresistance only in CADO cells. High IGF2 expression was also significantly associated with shorter overall survival in patients with ES. Transcriptome analysis of the clinical samples and the cell lines revealed an IGF-dependent signature, potentially related to a stem cell-like phenotype. Transcriptome analysis is a potentially powerful complementary tool to predict the clinical behavior of ES and may be utilized for clinical trial stratification strategies and personalized oncology. Certain gene signatures, for example, IGF-related pathways, are coupled to biological functions that could be of clinical importance. Finally, our results indicate that IGF inhibition may be successful as a first-line therapy in conjunction with conventional radiochemotherapy for a subset of patients.

Prevalence of the Hippo Effectors YAP1/TAZ in Tumors of Soft Tissue and Bone.

Tumors of soft tissue and bone represent a heterogeneous group of neoplasias characterized by a wide variety of genetic aberrations. Albeit knowledge on tumorigenesis in mesenchymal tumors is continuously increasing, specific insights on altered signaling pathways as a basis for molecularly targeted therapeutic strategies are still sparse. The aim of this study was to determine the involvement of YAP1/TAZ-mediated signals in tumors of soft tissue and bone. Expression levels of YAP1 and TAZ were analyzed by immunohistochemistry in a large cohort of 486 tumor specimens, comprising angiosarcomas (AS), Ewing sarcomas, leiomyosarcomas, malignant peripheral nerve sheath tumors (MPNST), solitary fibrous tumors, synovial sarcomas (SySa), well-differentiated/dedifferentiated/pleomorphic and myxoid liposarcomas (MLS). Moderate to strong nuclear staining of YAP1 and TAZ was detected in 53% and 33%, respectively. YAP1 nuclear expression was most prevalent in MPNST, SySa and MLS, whereas nuclear TAZ was predominately detected in AS, MLS and MPNST. In a set of sarcoma cell lines, immunoblotting confirmed nuclear localization of YAP1 and TAZ, corresponding to their transcriptionally active pool. Suppression of YAP1/TAZ-TEAD mediated transcriptional activity significantly impaired sarcoma cell viability in vitro and in vivo. Our findings identify nuclear YAP1 and TAZ positivity as a common feature in subsets of sarcomas of soft tissue and bone and provide evidence of YAP1/TAZ-TEAD signaling as a specific liability to be considered as a new target for therapeutic intervention. Nuclear YAP1/TAZ expression may represent a biomarker suited to identify patients that could benefit from YAP1/TAZ-TEAD directed therapeutic approaches within future clinical trials.

Dual inhibition of IGF-IR and ALK as an effective strategy to eradicate NPM-ALK+ T-cell lymphoma.

BACKGROUND: Nucleophosmin-anaplastic lymphoma kinase-expressing (NPM-ALK+) T cell lymphoma is an aggressive neoplasm. NPM-ALK, an oncogenic tyrosine kinase, plays a critical role in this lymphoma. Recently, selective ALK inhibitors have emerged as a first-line therapy for this neoplasm. Unfortunately, ALK inhibitors were hindered by emergence of resistance and relapse. We have previously demonstrated that type I insulin-like growth factor receptor (IGF-IR) is commonly expressed and activated in this lymphoma. In addition, IGF-IR and NPM-ALK are physically associated and reciprocally enhance their phosphorylation/activation. Herein, we tested the hypothesis that combined inhibition of IGF-IR and NPM-ALK could significantly improve the effects of inhibiting each kinase alone. METHODS: We used clinically utilized inhibitors of IGF-IR (picropodophyllin; PPP) and ALK (ASP3026) to assess the in vitro cellular effects of combined treatment versus treatment using a single agent. Moreover, we used a systemic NPM-ALK+ T cell lymphoma mouse model to analyze the in vivo effects of PPP and ASP3026 alone or in combination. RESULTS: Our data show that combined treatment with PPP and ASP3026 decreased the viability, proliferation, and anchorage-independent colony formation, and increased apoptosis of NPM-ALK+ T cell lymphoma cells in vitro. The in vitro effects of combined treatment were synergistic and significantly more pronounced than the effects of PPP or ASP3026 alone. Biochemically, simultaneous antagonism of IGF-IR and ALK induced more pronounced decrease in pIGF-IRY1135/1136, pNPM-ALKY646, and pSTAT3Y705 levels than antagonizing IGF-IR or ALK alone. Moreover, combined targeting of IGF-IR and NPM-ALK decreased significantly systemic lymphoma tumor growth and improved mice survival in vivo. Consistent with the in vitro results, the in vivo effects of the combined therapy were more pronounced than the effects of targeting IGF-IR or ALK alone. CONCLUSIONS: Combined targeting of IGF-IR and ALK is more effective than targeting IGF-IR or ALK alone in NPM-ALK+ T cell lymphoma. This strategy might also limit emergence of resistance to high doses of ALK inhibitors. Therefore, it could represent a successful therapeutic approach to eradicate this aggressive lymphoma. Importantly, combined inhibition is feasible because of the clinical availability of IGF-IR and ALK inhibitors. Our findings are applicable to other types of cancer where IGF-IR and ALK are simultaneously expressed.

SS18-SSX-Dependent YAP/TAZ Signaling in Synovial Sarcoma.

PURPOSE: Synovial sarcoma is a soft tissue malignancy characterized by a reciprocal t(X;18) translocation. The chimeric SS18-SSX fusion protein acts as a transcriptional dysregulator representing the major driver of the disease; however, the signaling pathways activated by SS18-SSX remain to be elucidated to define innovative therapeutic strategies. EXPERIMENTAL DESIGN: Immunohistochemical evaluation of the Hippo signaling pathway effectors YAP/TAZ was performed in a large cohort of synovial sarcoma tissue specimens. SS18-SSX dependency and biological function of the YAP/TAZ Hippo signaling cascade were analyzed in five synovial sarcoma cell lines and a mesenchymal stem cell model in vitro. YAP/TAZ-TEAD-mediated transcriptional activity was modulated by RNAi-mediated knockdown and the small-molecule inhibitor verteporfin. The effects of verteporfin were finally tested in vivo in synovial sarcoma cell line-based avian chorioallantoic membrane and murine xenograft models as well as a patient-derived xenograft. RESULTS: A significant subset of synovial sarcoma showed nuclear positivity for YAP/TAZ and their transcriptional targets FOXM1 and PLK1. In synovial sarcoma cells, RNAi-mediated knockdown of SS18-SSX led to significant reduction of YAP/TAZ-TEAD transcriptional activity. Conversely, SS18-SSX overexpression in SCP-1 cells induced aberrant YAP/TAZ-dependent signals, mechanistically mediated by an IGF-II/IGF-IR signaling loop leading to dysregulation of the Hippo effectors LATS1 and MOB1. Modulation of YAP/TAZ-TEAD-mediated transcriptional activity by RNAi or verteporfin treatment resulted in significant growth inhibitory effects in vitro and in vivo. CONCLUSIONS: Our preclinical study identifies an elementary role of SS18-SSX-driven YAP/TAZ signals, highlights the complex network of oncogenic signaling pathways in synovial sarcoma pathogenesis, and provides evidence for innovative therapeutic approaches.

hTERT promoter mutations in chondrosarcomas associate with progression and disease-related mortality.

Chondrosarcomas are malignant skeletal tumors with chondroid differentiation. Prognosis is largely dependent on histological grading, which suffer from significant interobserver variability. Telomerase activity and abundant telomerase reverse transcriptase (hTERT) expression has previously been associated with chondrosarcoma grade and metastasis. We therefore analyzed the hTERT promoter in clinicopathologically well-characterized chondrosarcomas (grade 1-3) from 87 patients. Using Sanger sequencing we identified an activating -124 C > T mutation in 23 cases (26%). Promoter mutations were significantly associated with increased histological grade (8% of grade 1, 32% of grade 2 and 46% of grade 3, P = 0.002), suggesting a role in tumor progression. In four chondrosarcomas where the histopathological grade was heterogenous, the hTERT mutation was only identified in the higher-grade areas. Additionally, hTERT promoter mutations were significantly associated with worse metastasis-free survival (P = 0.018), chondrosarcoma-specific survival (P = 0.022) and older patient age (P = 0.003). These data suggest that hTERT promoter mutations are common in high grade conventional chondrosarcomas. Granted that additional studies can confirm these findings; hTERT promoter analysis could potentially serve as an adjuvant prognostic marker in routine chondrosarcoma grading. This study reinforces the rationale of telomerase targeted therapy in a subset of chondrosarcomas.

Characterization of an activating R1353H insulin-like growth factor 1 receptor variant in a male with extreme tall height.

OBJECTIVE: The insulin-like growth factor1 receptor (IGF1R) is important in growth and development, and inactivating IGF1R mutations cause short stature and relatively high levels of serum IGF-I. We identified an unclassified IGF1RR1353H variant in a male with extreme tall height, very low levels of serum IGF-I and delayed and prolonged growth spurt. The index case's mother and three sons all carried the variant, but so far only the eldest son (age 18 years) presented with tall height. We hypothesized that the variant could constitute an activating mutation. DESIGN: The IGF1RR1353H variant was investigated in Igf1r-/- mouse embryonic fibroblasts (R-cells) by cell cycle, colony formation and transcriptome analyses. RESULTS: The IGF1RR1353H (R-1353) exhibited significantly increased cell proliferation, G1-S progression and colony formation in soft agar. RNA sequencing identified 195 differentially expressed genes between R-WT and R-1353 (adjusted P < 1E-100). Most genes were upregulated in R-1353, including the gene encoding the androgen receptor (AR). Gene expression profiling showed the most significant enrichment in extracellular matrix organization (P = 2.76E-7), collagen biosynthesis (P = 1.21E-5) and cell adhesion (P = 7.38E-5). Retrospective biochemical analysis of the index case revealed decreased testosterone and sex hormone-binding globulin levels, whereas LH and FSH were within normal ranges. This profile suggests an increased sensitivity to androgen, which is compatible with the enhanced expression of Ar in R-1353 cells. CONCLUSIONS: Our findings suggest that R1353H constitutes an activating IGF1R variant. The possible deregulation of collagen turnover and increased androgen sensitivity implicates an association to tall phenotype in male carriers.

Telomerase promoter mutations and copy number alterations in solitary fibrous tumours.

AIMS: Solitary fibrous tumour (SFT) is an infrequently metastasising mesenchymal tumour defined by the NAB2-STAT6 fusion gene. Activating mutations in the telomerase reverse transcriptase (hTERT) gene promoter has been reported to associate with adverse patient outcome in SFTs. METHODS: We analysed the hTERT gene for promoter mutations and copy number alterations in 43 primary extrameningeal SFTs (9 malignant and 34 benign tumours according to WHO 2013 criteria), six local recurrences and three metastatic lesions. RESULTS: Activating -124 C>T (n=12) or -148 C>T (n=2) mutations were found in 33% of the tumours and associated with older age (P=0.006), necrosis (P=0.009), higher mitotic rate (P=0.003), nuclear atypia (P=0.002), malignant histological diagnosis (P=0.04) and worse progression-free survival (P=0.023). We also observed frequent (24%) hTERT promoter mutations in histologically benign tumours without metastasis (mean follow-up >9 years), and in 14%-18% of low-risk SFTs as determined by three risk-stratification models. Mutations were seen in 2/6 metastatic tumours and metastatic lesions. hTERT copy number gain was seen in 11/28 hTERT promoter wild-type cases. CONCLUSIONS: Activating hTERT promoter mutations associate with aggressive histopathological features, indicating a role in tumour progression. Given the comparatively high prevalence of hTERT promoter mutations in low-risk and non-metastasising lesions, further studies are required to clarify the prognostic value of hTERT promoter analysis before implementing the analysis in clinical diagnostics.

FNA cytology of solitary fibrous tumors and the diagnostic value of STAT6 immunocytochemistry.

BACKGROUND: Solitary fibrous tumors (SFTs) are rare mesenchymal tumors commonly located in the pleura, soft tissues, or meninges and are characterized by the NGFI-A-binding protein 2 (NAB2)-signal transducer and activator of transcription 6 (STAT6) fusion gene. Recent studies have indicated that nuclear STAT6 immunohistochemistry is a specific marker for SFTs. METHODS: The authors reviewed fine-needle aspiration (FNA) specimens from extracranial SFTs diagnosed at their institution between 1993 and 2017. Histologic blocks and available formalin-fixed smears of FNA specimens from SFTs were investigated for STAT6 immunoreactivity using a monoclonal antibody. STAT6 immunocytochemistry was also investigated in schwannomas and spindle cell lipomas. Cytopathologic and clinical characteristics were described. RESULTS: Nineteen benign and 9 malignant SFTs were identified. Both benign and malignant SFTs had a female predominance (female-to-male ratio, 2.8:1 and 1.25, respectively). Localization varied, and approximately one-half of the extrapleural tumors were located in the extremities and frequently were intramuscular. Benign and malignant primary tumors had limited differences in cytologic presentation, the most notable feature being nuclear pleomorphism. Cytomorphologic features included low-to-moderate cellularity of mixed oval, elongated, round, and stellate cells with pink collagenous stroma and hypercellular clusters with infrequent atypia. In metastatic SFTs, the cytopathology was suggestive of sarcoma. Immunohistochemistry revealed nuclear STAT6 immunoreactivity in SFTs (n = 5) with cytoplasmic reactivity in cytologic mimickers. CONCLUSIONS: Benign and malignant SFTs have common cytopathologic features, and the ability to distinguish between them is limited. Nuclear STAT6 immunoreactivity is a valuable cytologic marker for SFTs. Cancer Cytopathol 2018;126:36-43. © 2017 American Cancer Society.

Phenotypic Screen Identifies a Small Molecule Modulating ERK2 and Promoting Stem Cell Proliferation.

Stem cells display a fundamentally different mechanism of proliferation control when compared to somatic cells. Uncovering these mechanisms would maximize the impact in drug discovery with a higher translational applicability. The unbiased approach used in phenotype-based drug discovery (PDD) programs can offer a unique opportunity to identify such novel biological phenomenon. Here, we describe an integrated phenotypic screening approach, employing a combination of in vitro and in vivo PDD models to identify a small molecule increasing stem cell proliferation. We demonstrate that a combination of both in vitro and in vivo screening models improves hit identification and reproducibility of effects across various PDD models. Using cell viability and colony size phenotype measurement we characterize the structure activity relationship of the lead molecule, and identify that the small molecule inhibits phosphorylation of ERK2 and promotes stem cell proliferation. This study demonstrates a PDD approach that employs combinatorial models to identify compounds promoting stem cell proliferation.

Phase I clinical trial of AXL1717 for treatment of relapsed malignant astrocytomas: analysis of dose and response.

PURPOSE: Early phase I study of safety of AXL1717 in patients with recurrent or progressive malignant astrocytomas and evaluation of preliminary anti-tumor efficacy. PATIENTS AND METHODS: Nine patients fulfilling the set criteria were enrolled. Eight had recurrent glioblastoma and one gliosarcoma. Patients were treated with an oral suspension of AXL1717 (215-400 mg bid) cycle-by-cycle in 35-day cycles (28 days bid and 7 days off). Patients with progressive disease and/or toxicity-related dose delay of more than 14 days were withdrawn. RESULTS: Four patients had tumor responses (44%) to AXL1717 treatment. Two of these had stable disease for 12 months (10 cycles at 215-300 mg bid). Due to MRI-detected progression they were then taken off the study. They died 8 and 12 months later, respectively. One patient was treated 8 months (6 cycles with 215 mg bid). He was withdrawn because of disease progression but died after another 25 months. The fourth patient having stable disease died of sepsis due to pancytopenia in the end of cycle 2 on 400 mg bid. A fifth patient underwent surgery after two cycles with 300 mg bid. Pathological analysis demonstrated abundant necrosis and small areas of viable tumor. After one more cycle with 300 mg bid he was withdrawn due to clinical and radiographic worsening and died 11 months later. The other 4 patients did not have any detectable responses and died within 3-13 months after trial entry. Neutropenia was the main adverse effect, which was easily detected and reversible in all but one patient. CONCLUSION: This clinical phase I study indicates that AXL1717 as a single agent is capable of producing prolonged stable disease and survival of patients with relapsed malignant astrocytomas. The drug was well tolerated. A new formulation of the drug will be used in further investigations in order to better define the optimal dose.

Nuclear insulin-like growth factor 1 receptor phosphorylates proliferating cell nuclear antigen and rescues stalled replication forks after DNA damage.

We have previously shown that the insulin-like growth factor 1 receptor (IGF-1R) translocates to the cell nucleus, where it binds to enhancer-like regions and increases gene transcription. Further studies have demonstrated that nuclear IGF-1R (nIGF-1R) physically and functionally interacts with some nuclear proteins, i.e. the lymphoid enhancer-binding factor 1 (Lef1), histone H3, and Brahma-related gene-1 proteins. In this study, we identified the proliferating cell nuclear antigen (PCNA) as a nIGF-1R-binding partner. PCNA is a pivotal component of the replication fork machinery and a main regulator of the DNA damage tolerance (DDT) pathway. We found that IGF-1R interacts with and phosphorylates PCNA in human embryonic stem cells and other cell lines. In vitro MS analysis of PCNA co-incubated with the IGF-1R kinase indicated tyrosine residues 60, 133, and 250 in PCNA as IGF-1R targets, and PCNA phosphorylation was followed by mono- and polyubiquitination. Co-immunoprecipitation experiments suggested that these ubiquitination events may be mediated by DDT-dependent E2/E3 ligases (e.g. RAD18 and SHPRH/HLTF). Absence of IGF-1R or mutation of Tyr-60, Tyr-133, or Tyr-250 in PCNA abrogated its ubiquitination. Unlike in cells expressing IGF-1R, externally induced DNA damage in IGF-1R-negative cells caused G1 cell cycle arrest and S phase fork stalling. Taken together, our results suggest a role of IGF-1R in DDT.