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Pediatric brain cancer is the leading cause of disease-related mortality in children, and many aggressive tumors still lack effective treatment strategies. We characterized aberrant alternative splicing across pediatric brain tumors, identifying pediatric high-grade gliomas (HGGs) among the most heterogeneous. Annotating these events with UniProt, we identified 11,940 splice events in 5,368 genes leading to potential protein function changes. We discovered CDC-like kinase 1 ( CLK1 ) is aberrantly spliced to include exon 4, resulting in a gain of two phosphorylation sites and subsequent activation. Inhibition of CLK1 with Cirtuvivint significantly decreased both cell viability and proliferation in the pediatric HGG KNS-42 cell line. Morpholino-mediated depletion of CLK1 exon 4 splicing reduced RNA expression, protein abundance, and cell viability with concurrent differential expression of 78 cancer genes and differential splicing at functional sites in 193 cancer genes. Our findings highlight a dependency of pediatric HGGs on CLK1 and represent a promising therapeutic strategy., Competing Interests: Conflicts of Interest The authors declare no conflicts of interest.

Introduction: Inverted papilloma (IP) is a sinonasal tumor with a well-known potential for malignant transformation. The role of human papillomavirus (HPV) in its pathogenesis has been controversial. The purpose of this study was to determine the virome associated with IP, with progression to carcinoma in situ (CIS), and invasive carcinoma., Methods: To determine the HPV-specific types, a metagenomics assay that contains 62,886 probes targeting viral genomes in a microarray format was used. The platform screens DNA and RNA from fixed tissues from eight controls, 16 IP without dysplasia, five IP with CIS, and 13 IP-associated squamous cell carcinoma (IPSCC). Paired with next-generation sequencing, 48 types of HPV with 857 region-specific probes were interrogated against the tumors., Results: The prevalence of HPV-16 was 14%, 42%, 70%, and 73% in control tissue, IP without dysplasia, IP with CIS, and IPSCC, respectively. The prevalence of HPV-18 had a similar progressive increase in prevalence, with 14%, 27%, 67%, and 74%, respectively. The assay allowed region-specific analysis, which identified the only oncogenic HPV-18 E6 to be statistically significant when compared with control tissue. The prevalence of HPV-18 E6 was 0% in control tissue, 25% in IP without dysplasia, 60% in IP with CIS, and 77% in IPSCC., Conclusions: There are over 200 HPV types that infect human epithelial cells, of which only a few are known to be high-risk. Our study demonstrated a trend of increasing prevalence of HPV-18 E6 that correlated with histologic severity, which is novel and supports a potential role for HPV in the pathogenesis of IP., (© 2023 ARS-AAOA, LLC.)

Increasing evidence suggests that besides mutational and molecular alterations, the immune component of the tumor microenvironment also substantially impacts tumor behavior and complicates treatment response, particularly to immunotherapies. Although the standard method for characterizing tumor immune profile is through performing integrated genomic analysis on tissue biopsies, the dynamic change in the immune composition of the tumor microenvironment makes this approach not feasible, especially for brain tumors. Radiomics is a rapidly growing field that uses advanced imaging techniques and computational algorithms to extract numerous quantitative features from medical images. Recent advances in machine learning methods are facilitating biological validation of radiomic signatures and allowing them to "mine" for a variety of significant correlates, including genetic, immunologic, and histologic data. Radiomics has the potential to be used as a non-invasive approach to predict the presence and density of immune cells within the microenvironment, as well as to assess the expression of immune-related genes and pathways. This information can be essential for patient stratification, informing treatment decisions and predicting patients' response to immunotherapies. This is particularly important for tumors with difficult surgical access such as gliomas. In this review, we provide an overview of the glioma microenvironment, describe novel approaches for clustering patients based on their tumor immune profile, and discuss the latest progress on utilization of radiomics for immune profiling of glioma based on current literature., (© 2023. The Author(s).)

Pediatric solid and central nervous system tumors are the leading cause of cancer-related death among children. Identifying new targeted therapies necessitates the use of pediatric cancer models that faithfully recapitulate the patient's disease. However, the generation and characterization of pediatric cancer models has significantly lagged behind adult cancers, underscoring the urgent need to develop pediatric-focused cell line resources. Herein, we establish a single-site collection of 261 cell lines, including 224 pediatric cell lines representing 18 distinct extracranial and brain childhood tumor types. We subjected 182 cell lines to multi-omics analyses (DNA sequencing, RNA sequencing, DNA methylation), and in parallel performed pharmacological and genetic CRISPR-Cas9 loss-of-function screens to identify pediatric-specific treatment opportunities and biomarkers. Our work provides insight into specific pathway vulnerabilities in molecularly defined pediatric tumor classes and uncovers biomarker-linked therapeutic opportunities of clinical relevance. Cell line data and resources are provided in an open access portal., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Pediatric brain tumors are the leading cause of cancer-related death in children in the United States and contribute a disproportionate number of potential years of life lost compared to adult cancers. Moreover, survivors frequently suffer long-term side effects, including secondary cancers. The Children's Brain Tumor Network (CBTN) is a multi-institutional international clinical research consortium created to advance therapeutic development through the collection and rapid distribution of biospecimens and data via open-science research platforms for real-time access and use by the global research community. The CBTN's 32 member institutions utilize a shared regulatory governance architecture at the Children's Hospital of Philadelphia to accelerate and maximize the use of biospecimens and data. As of August 2022, CBTN has enrolled over 4700 subjects, over 1500 parents, and collected over 65,000 biospecimen aliquots for research. Additionally, over 80 preclinical models have been developed from collected tumors. Multi-omic data for over 1000 tumors and germline material are currently available with data generation for > 5000 samples underway. To our knowledge, CBTN provides the largest open-access pediatric brain tumor multi-omic dataset annotated with longitudinal clinical and outcome data, imaging, associated biospecimens, child-parent genomic pedigrees, and in vivo and in vitro preclinical models. Empowered by NIH-supported platforms such as the Kids First Data Resource and the Childhood Cancer Data Initiative, the CBTN continues to expand the resources needed for scientists to accelerate translational impact for improved outcomes and quality of life for children with brain and spinal cord tumors., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. David S. Ziegler is a consultant, or on the advisory board, of Bayer, AstraZeneca, Accendatech, Novartis, Day One, FivePhusion, Amgen, Alexion, and Norgine. Angela J. Waanders is on the advisory board of Alexion and Day One., (Copyright © 2022. Published by Elsevier Inc.)

Background: Inverted papilloma (IP) is a sinonasal tumor with a well-known potential for malignant transformation. The purpose of this study was to identify the genes and pathways associated with IP, with progression to carcinoma-in-situ and invasive carcinoma., Methods: To determine genes and molecular pathways that may indicate progression and correlate with histologic changes, we analyzed six IP without dysplasia, five IP with carcinoma-in-situ, and 13 squamous cell carcinoma ex-IP by targeted sequencing. The HTG EdgeSeq Oncology Biomarker Panel coupled with next-generation sequencing was used to evaluate 2560 transcripts associated with solid tumors., Results: Progressive upregulation of 11 genes were observed (CALD1, COL1A1, COL3A1, COL4A2, COL5A2, FN1, ITGA5, LGALS1, MMP11, SERPINH1, SPARC) in the order of invasive carcinoma > carcinoma-in-situ > IP without dysplasia. When compared with IP without dysplasia, more genes are differentially expressed in invasive carcinoma than carcinoma-in-situ samples (341 downregulated/333 upregulated vs. 195 downregulated/156 upregulated). Gene set enrichment analysis determined three gene sets in common between the cohorts (epithelial mesenchymal transition, extracellular matrix organization, and coagulation)., Conclusions: Progressive upregulation of genes specific to IP malignant degeneration has significant clinical implications. This panel of 11 genes will improve concordance of histologic classification, which can directly impact treatment and patient outcomes. Additionally, future studies on larger tumor sets may observe upregulation in the gene panel that preceded histologic changes, which may be useful for further risk stratification., (© 2021 ARS-AAOA, LLC.)

We report a comprehensive proteogenomics analysis, including whole-genome sequencing, RNA sequencing, and proteomics and phosphoproteomics profiling, of 218 tumors across 7 histological types of childhood brain cancer: low-grade glioma (n = 93), ependymoma (32), high-grade glioma (25), medulloblastoma (22), ganglioglioma (18), craniopharyngioma (16), and atypical teratoid rhabdoid tumor (12). Proteomics data identify common biological themes that span histological boundaries, suggesting that treatments used for one histological type may be applied effectively to other tumors sharing similar proteomics features. Immune landscape characterization reveals diverse tumor microenvironments across and within diagnoses. Proteomics data further reveal functional effects of somatic mutations and copy number variations (CNVs) not evident in transcriptomics data. Kinase-substrate association and co-expression network analysis identify important biological mechanisms of tumorigenesis. This is the first large-scale proteogenomics analysis across traditional histological boundaries to uncover foundational pediatric brain tumor biology and inform rational treatment selection., Competing Interests: Declaration of Interests E.E.S. serves as chief executive officer for Sema4 and has an equity interest in this company., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Medulloblastoma is a highly heterogeneous pediatric brain tumor with five molecular subtypes, Sonic Hedgehog TP53-mutant, Sonic Hedgehog TP53-wildtype, WNT, Group 3, and Group 4, defined by the World Health Organization. The current mechanism for classification into these molecular subtypes is through the use of immunostaining, methylation, and/or genetics. We surveyed the literature and identified a number of RNA-Seq and microarray datasets in order to develop, train, test, and validate a robust classifier to identify medulloblastoma molecular subtypes through the use of transcriptomic profiling data. We have developed a GPL-3 licensed R package and a Shiny Application to enable users to quickly and robustly classify medulloblastoma samples using transcriptomic data. The classifier utilizes a large composite microarray dataset (15 individual datasets), an individual microarray study, and an RNA-Seq dataset, using gene ratios instead of gene expression measures as features for the model. Discriminating features were identified using the limma R package and samples were classified using an unweighted mean of normalized scores. We utilized two training datasets and applied the classifier in 15 separate datasets. We observed a minimum accuracy of 85.71% in the smallest dataset and a maximum of 100% accuracy in four datasets with an overall median accuracy of 97.8% across the 15 datasets, with the majority of misclassification occurring between the heterogeneous Group 3 and Group 4 subtypes. We anticipate this medulloblastoma transcriptomic subtype classifier will be broadly applicable to the cancer research and clinical communities., Competing Interests: The authors have declared that no competing interests exist.

CT10 regulator of kinase (Crk) and Crk-like (CrkL) are the cellular counterparts of the viral oncogene v-Crk Elevated levels of Crk and CrkL have been observed in many human cancers; inhibition of Crk and CrkL expression reduced the tumor-forming potential of cancer cell lines. Despite a close relationship between the Crk family proteins and tumorigenesis, how Crk and CrkL contribute to cell growth is unclear. We ablated endogenous Crk and CrkL from cultured fibroblasts carrying floxed alleles of Crk and CrkL by transfection with synthetic Cre mRNA (synCre). Loss of Crk and CrkL induced by synCre transfection blocked cell proliferation and caused shrinkage of the cytoplasm and the nucleus, formation of adherens junctions, and reduced cell motility. Ablation of Crk or CrkL alone conferred a much more modest reduction in cell proliferation. Reintroduction of CrkI, CrkII, or CrkL individually rescued cell proliferation in the absence of the endogenous Crk and CrkL, suggesting that Crk and CrkL play overlapping functions in regulating fibroblast growth. Serum and basic FGF induced phosphorylation of Akt, MAP kinases, and S6 kinase and Fos expression in the absence of Crk and CrkL, suggesting that cells lacking Crk and CrkL are capable of initiating major signal transduction pathways in response to extracellular stimuli. Furthermore, cell cycle and cell death analyses demonstrated that fibroblasts lacking Crk and CrkL become arrested at the G 1 -S transition and undergo a modest apoptosis. Taken together, our results suggest that Crk and CrkL play essential overlapping roles in fibroblast growth., (© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.)

Leukemias expressing constitutively activated mutants of ABL1 tyrosine kinase (BCR-ABL1, TEL-ABL1, NUP214-ABL1) usually contain at least 1 normal ABL1 allele. Because oncogenic and normal ABL1 kinases may exert opposite effects on cell behavior, we examined the role of normal ABL1 in leukemias induced by oncogenic ABL1 kinases. BCR-ABL1-Abl1(-/-) cells generated highly aggressive chronic myeloid leukemia (CML)-blast phase-like disease in mice compared with less malignant CML-chronic phase-like disease from BCR-ABL1-Abl1(+/+) cells. Additionally, loss of ABL1 stimulated proliferation and expansion of BCR-ABL1 murine leukemia stem cells, arrested myeloid differentiation, inhibited genotoxic stress-induced apoptosis, and facilitated accumulation of chromosomal aberrations. Conversely, allosteric stimulation of ABL1 kinase activity enhanced the antileukemia effect of ABL1 tyrosine kinase inhibitors (imatinib and ponatinib) in human and murine leukemias expressing BCR-ABL1, TEL-ABL1, and NUP214-ABL1. Therefore, we postulate that normal ABL1 kinase behaves like a tumor suppressor and therapeutic target in leukemias expressing oncogenic forms of the kinase., (© 2016 by The American Society of Hematology.)

Crk and CrkL are SH2- and SH3-containing cytosolic adaptor proteins that can induce anchorage-independent growth of fibroblasts. Crk and CrkL play key roles in maintaining cytoskeletal integrity, cell motility and migration. We investigated the role of these two proteins in oncogenic transformation induced by v-fos and v-ras oncogenes using cell lines and fibroblasts carrying conditional alleles of Crk or CrkL. Transformation was assessed by cell morphology, saturation density and anchorage-independent growth in soft agar. We found that cell lines expressing v-fos or v-ras in the absence of Crk or CrkL displayed no evident morphological alterations and reduced anchorage-independent growth compared to those retaining Crk and CrkL. Similarly, overexpression of v-fos in mouse embryonic fibroblasts conferred a growth advantage and induced morphological changes, both of which were abrogated in the absence of either Crk or CrkL. In contrast, Crk, but not CrkL, contributed to v-ras-induced transformation of embryonic fibroblasts. These results suggest that both Crk and CrkL are required for the acquisition of cellular transformation by v-fos, whereas Crk plays a more prominent role than CrkL in v-ras-induced transformation., (© 2015 Wiley Periodicals, Inc.)

Although poorly understood, androgen receptor (AR) signaling is sustained despite treatment of prostate cancer with antiandrogens and potentially underlies development of incurable castrate-resistant prostate cancer. However, therapies targeting the AR signaling axis eventually fail when prostate cancer progresses to the castrate-resistant stage. Stat5a/b, a candidate therapeutic target protein in prostate cancer, synergizes with AR to reciprocally enhance the signaling of both proteins. In this work, we demonstrate that Stat5a/b sequesters antiandrogen-liganded (MDV3100, bicalutamide, flutamide) AR in prostate cancer cells and protects it against proteasomal degradation in prostate cancer. Active Stat5a/b increased nuclear levels of both unliganded and antiandrogen-liganded AR, as demonstrated in prostate cancer cell lines, xenograft tumors, and clinical patient-derived prostate cancer samples. Physical interaction between Stat5a/b and AR in prostate cancer cells was mediated by the DNA-binding domain of Stat5a/b and the N-terminal domain of AR. Moreover, active Stat5a/b increased AR occupancy of the prostate-specific antigen promoter and AR-regulated gene expression in prostate cancer cells. Mechanistically, both Stat5a/b genetic knockdown and antiandrogen treatment induced proteasomal degradation of AR in prostate cancer cells, with combined inhibition of Stat5a/b and AR leading to maximal loss of AR protein and prostate cancer cell viability. Our results indicate that therapeutic targeting of AR in prostate cancer using antiandrogens may be substantially improved by targeting of Stat5a/b., (©2014 American Association for Cancer Research.)

Genomic instability is a hallmark of chronic myeloid leukemia in chronic phase (CML-CP) resulting in BCR-ABL1 mutations encoding resistance to tyrosine kinase inhibitors (TKIs) and/or additional chromosomal aberrations leading to disease relapse and/or malignant progression. TKI-naive and TKI-treated leukemia stem cells (LSCs) and leukemia progenitor cells (LPCs) accumulate high levels of reactive oxygen species (ROS) and oxidative DNA damage. To determine the role of TKI-refractory LSCs in genomic instability, we used a murine model of CML-CP where ROS-induced oxidative DNA damage was elevated in LSCs, including quiescent LSCs, but not in LPCs. ROS-induced oxidative DNA damage in LSCs caused clinically relevant genomic instability in CML-CP-like mice, such as TKI-resistant BCR-ABL1 mutations (E255K, T315I, H396P), deletions in Ikzf1 and Trp53, and additions in Zfp423 and Idh1. Despite inhibition of BCR-ABL1 kinase, imatinib did not downregulate ROS and oxidative DNA damage in TKI-refractory LSCs to the levels detected in normal cells, and CML-CP-like mice treated with imatinib continued to accumulate clinically relevant genetic aberrations. Inhibition of class I p21-activated protein kinases by IPA3 downregulated ROS in TKI-naive and TKI-treated LSCs. Altogether, we postulate that genomic instability may originate in the most primitive TKI-refractory LSCs in TKI-naive and TKI-treated patients.

Chronic myeloid leukemia in chronic phase (CML-CP) is induced by BCR-ABL1 oncogenic tyrosine kinase. Tyrosine kinase inhibitors eliminate the bulk of CML-CP cells, but fail to eradicate leukemia stem cells (LSCs) and leukemia progenitor cells (LPCs) displaying innate and acquired resistance, respectively. These cells may accumulate genomic instability, leading to disease relapse and/or malignant progression to a fatal blast phase. In the present study, we show that Rac2 GTPase alters mitochondrial membrane potential and electron flow through the mitochondrial respiratory chain complex III (MRC-cIII), thereby generating high levels of reactive oxygen species (ROS) in CML-CP LSCs and primitive LPCs. MRC-cIII-generated ROS promote oxidative DNA damage to trigger genomic instability, resulting in an accumulation of chromosomal aberrations and tyrosine kinase inhibitor-resistant BCR-ABL1 mutants. JAK2(V617F) and FLT3(ITD)-positive polycythemia vera cells and acute myeloid leukemia cells also produce ROS via MRC-cIII. In the present study, inhibition of Rac2 by genetic deletion or a small-molecule inhibitor and down-regulation of mitochondrial ROS by disruption of MRC-cIII, expression of mitochondria-targeted catalase, or addition of ROS-scavenging mitochondria-targeted peptide aptamer reduced genomic instability. We postulate that the Rac2-MRC-cIII pathway triggers ROS-mediated genomic instability in LSCs and primitive LPCs, which could be targeted to prevent the relapse and malignant progression of CML.

The search for new therapeutic strategies for prostate and breast cancer is of significant interest. Signal transducer and activator of transcription 5a/b (Stat5a/b) controls viability and growth of prostate cancer. Nuclear active Stat5a/b expression is clustered to high grade prostate cancers, predicts early disease recurrence and promotes metastatic dissemination of prostate cancer. In breast cancer, the role of Stat5a/b is more complex. In rodent model systems, Stat5a/b may promote malignant transformation and enhance growth of the breast tumors. In contrast, Stat5a/b activation in established human breast cancer positively correlates with tumor differentiation, prevents metastatic dissemination, and predicts favorable clinical outcome of node-negative breast cancer. Here we review the molecular structure and biological functions of Stat5a/b and discuss the potential applications of Stat5a/b for therapy development and as a prognostic marker for prostate and breast cancer., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Chronic myeloid leukemia in chronic phase (CML-CP) cells that harbor oncogenic BCR-ABL1 and normal ABL1 allele often become resistant to the ABL1 kinase inhibitor imatinib. Here, we report that loss of the remaining normal ABL1 allele in these tumors, which results from cryptic interstitial deletion in 9q34 in patients who did not achieve a complete cytogenetic remission (CCyR) during treatment, engenders a novel unexpected mechanism of imatinib resistance. BCR-ABL1-positive Abl1(-/-) leukemia cells were refractory to imatinib as indicated by persistent BCR-ABL1-mediated tyrosine phosphorylation, lack of BCR-ABL1 protein degradation, increased cell survival, and clonogenic activity. Expression of ABL1 kinase, but not a kinase-dead mutant, restored the antileukemic effects of imatinib in ABL1-negative chronic myelogenous leukemia (CML) cells and in BCR-ABL1-positive Abl1(-/-) murine leukemia cells. The intracellular concentration of imatinib and expression of its transporters were not affected, although proteins involved in BCR-ABL1 degradation were downregulated in Abl1(-/-) cells. Furthermore, 12 genes associated with imatinib resistance were favorably deregulated in Abl1(-/-) leukemia. Taken together, our results indicate that loss of the normal ABL1 kinase may serve as a key prognostic factor that exerts major impact on CML treatment outcomes.

Myeloproliferative disorders (MPD) are stem cell-derived clonal diseases arising as a consequence of acquired aberrations in c-ABL, Janus-activated kinase 2 (JAK2), and platelet-derived growth factor receptor (PDGFR) that generate oncogenic fusion tyrosine kinases (FTK), including BCR/ABL, TEL/ABL, TEL/JAK2, and TEL/PDGFbetaR. Here, we show that FTKs stimulate the formation of reactive oxygen species and DNA double-strand breaks (DSB) both in hematopoietic cell lines and in CD34(+) leukemic stem/progenitor cells from patients with chronic myelogenous leukemia (CML). Single-strand annealing (SSA) represents a relatively rare but very unfaithful DSB repair mechanism causing chromosomal aberrations. Using a specific reporter cassette integrated into genomic DNA, we found that BCR/ABL and other FTKs stimulated SSA activity. Imatinib-mediated inhibition of BCR/ABL abrogated this effect, implicating a kinase-dependent mechanism. Y253F, E255K, T315I, and H396P mutants of BCR/ABL that confer imatinib resistance also stimulated SSA. Increased expression of either nonmutated or mutated BCR/ABL kinase, as is typical of blast phase cells and very primitive chronic phase CML cells, was associated with higher SSA activity. BCR/ABL-mediated stimulation of SSA was accompanied by enhanced nuclear colocalization of RAD52 and ERCC1, which play a key role in the repair. Taken together, these findings suggest a role of FTKs in causing disease progression in MPDs by inducing chromosomal instability through the production of DSBs and stimulation of SSA repair.

BCR/ABL kinase-positive chronic myelogenous leukemia (CML) cells display genomic instability leading to point mutations in various genes including bcr/abl and p53, eventually causing resistance to imatinib and malignant progression of the disease. Mismatch repair (MMR) is responsible for detecting misincorporated nucleotides, resulting in excision repair before point mutations occur and/or induction of apoptosis to avoid propagation of cells carrying excessive DNA lesions. To assess MMR activity in CML, we used an in vivo assay using the plasmid substrate containing enhanced green fluorescent protein (EGFP) gene corrupted by T:G mismatch in the start codon; therefore, MMR restores EGFP expression. The efficacy of MMR was reduced approximately 2-fold in BCR/ABL-positive cell lines and CD34(+) CML cells compared with normal counterparts. MMR was also challenged by N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), which generates O(6)-methylguanine and O(4)-methylthymine recognized by MMR system. Impaired MMR activity in leukemia cells was associated with better survival, accumulation of p53 but not of p73, and lack of activation of caspase 3 after MNNG treatment. In contrast, parental cells displayed accumulation of p53, p73, and activation of caspase 3, resulting in cell death. Ouabain-resistance test detecting mutations in the Na(+)/K(+) ATPase was used to investigate the effect of BCR/ABL kinase-mediated inhibition of MMR on mutagenesis. BCR/ABL-positive cells surviving the treatment with MNNG displayed approximately 15-fold higher mutation frequency than parental counterparts and predominantly G:C-->A:T and A:T-->G:C mutator phenotype typical for MNNG-induced unrepaired lesions. In conclusion, these results suggest that BCR/ABL kinase abrogates MMR activity to inhibit apoptosis and induce mutator phenotype.

The large T-antigen from human polyomavirus JC (JCV T-antigen) is suspected to play a role in malignant transformation. Previously, we reported that JCV T-antigen requires the presence of a functional insulin-like growth factor I receptor (IGF-IR) for transformation of fibroblasts and for survival of medulloblastoma cell lines; that IGF-IR is phosphorylated in medulloblastoma biopsies and that JCV T-antigen inhibits homologous recombination-directed DNA repair, causing accumulation of mutations. Here we are evaluating whether JCV T-antigen positive and negative mouse medulloblastoma cell lines, which significantly differ in their tumorigenic properties, are also different in their abilities to repair double strand breaks of DNA (DSBs). Our results show that despite much stronger tumorigenic potential, JCV T-antigen positive medulloblastoma cells are more sensitive to genotoxic agents (cisplatin and gamma-irradiation). Subsequent analysis of DNA repair of DSBs indicated that homologous recombination-directed DNA repair (HRR) was selectively attenuated in JCV T-antigen positive medulloblastoma cells. JCV T-antigen did not affect HRR directly. In the presence of JCV T-antigen, insulin receptor substrate 1 (IRS-1) translocated to the nucleus where it co-localized with Rad51, possibly attenuating HRR., (Copyright (c) 2006 Wiley-Liss, Inc.)

Mutations in the BCR/ABL kinase domain play a major role in resistance to imatinib mesylate (IM). We report here that BCR/ABL kinase stimulates reactive oxygen species (ROS), which causes oxidative DNA damage, resulting in mutations in the kinase domain. The majority of mutations involved A/T-->G/C and G/C-->A/T transitions, a phenotype detected previously in patients, which encoded clinically relevant amino acid substitutions, causing IM resistance. This effect was reduced in cells expressing BCR/ABL(Y177F) mutant, which does not elevate ROS. Inhibition of ROS in leukemia cells by the antioxidants pyrrolidine dithiocarbamate (PDTC), N-acetylcysteine (NAC), and vitamin E (VE) decreased the mutagenesis rate and frequency of IM resistance. Simultaneous administration of IM and an antioxidant exerted better antimutagenic effect than an antioxidant alone. Therefore, inhibition of ROS should diminish mutagenesis and enhance the effectiveness of IM.

BCR/ABL-positive leukemia cells accumulated more replication-dependent DNA double-strand breaks (DSBs) than normal counterparts after treatment with cisplatin and mitomycin C (MMC, as assessed by pulse field gel electrophoresis (PFGE) and neutral comet assay. In addition, leukemia cells could repair these lesions more efficiently than normal cells and eventually survive genotoxic treatment. Elevated levels of drug-induced DSBs in leukemia cells were associated with higher activity of ATR kinase, and enhanced phosphorylation of histone H2AX on serine 139 (gamma-H2AX). gamma-H2AX eventually started to disappear in BCR/ABL cells, while continued to increase in parental cells. In addition, the expression and ATR-mediated phosphorylation of Chk1 kinase on serine 345 were often more abundant in BCR/ABL-positive leukemia cells than normal counterparts after genotoxic treatment. Inhibition of ATR kinase by caffeine but not Chk1 kinase by indolocarbazole inhibitor, SB218078 sensitized BCR/ABL leukemia cells to MMC in a short-term survival assay. Nevertheless, both caffeine and SB218078 enhanced the genotoxic effect of MMC in a long-term clonogenic assay. This effect was associated with the abrogation of transient accumulation of leukemia cells in S and G2/M cell cycle phases after drug treatment. In conclusion, ATR-Chk1 axis was strongly activated in BCR/ABL-positive cells and contributed to the resistance to DNA cross-linking agents causing numerous replication-dependent DSBs.

The oncogenic BCR/ABL tyrosine kinase facilitates the repair of DNA double-strand breaks (DSBs). We find that after gamma-irradiation BCR/ABL-positive leukemia cells accumulate more DSBs in comparison to normal cells. These lesions are efficiently repaired in a time-dependent fashion by BCR/ABL-stimulated non-homologous end-joining (NHEJ) followed by homologous recombination repair (HRR) mechanisms. However, mutations and large deletions were detected in HRR and NHEJ products, respectively, in BCR/ABL-positive leukemia cells. We propose that unfaithful repair of DSBs may contribute to genomic instability in the Philadelphia chromosome-positive leukemias.

Bloom protein (BLM) is a 3'-5' helicase, mutated in Bloom syndrome, which plays an important role in response to DNA double-strand breaks and stalled replication forks. Here, we show that BCR/ABL tyrosine kinase, which also modulates DNA repair capacity, is associated with elevated expression of BLM. Downregulation of BLM by antisense cDNA or dominant-negative mutant inhibits homologous recombination repair (HRR) and increases sensitivity to cisplatin in BCR/ABL-positive cells. Bone marrow cells from mice heterozygous for BLM mutation, BLM(Cin/+), transfected with BCR/ABL display increased sensitivity to cisplatin compared to those obtained from the wild-type littermates. BCR/ABL promotes interactions of BLM with RAD51, while simultaneous overexpression of BLM and RAD51 in normal cells increases drug resistance. These data suggest that BLM collaborates with RAD51 to facilitate HRR and promotes the resistance of BCR/ABL-positive leukemia cells to DNA-damaging agents.

We employ diffuse optical tomography (DOT) to track treatment progress in a female subject presenting with locally advanced invasive carcinoma of the breast during neoadjuvant chemotherapy. Three-dimensional images of total hemoglobin concentration and scattering identified the tumor. Our measurements reveal tumor shrinkage during the course of chemotherapy, in reasonable agreement with magnetic resonance images of the same subject. A decrease in total hemoglobin concentration contrast between tumor and normal tissue was also observed over time. The results demonstrate the potential of DOT for measuring physiological parameters of breast lesions during chemotherapy.

The oncogenic BCR/ABL tyrosine kinase induces constitutive DNA damage in Philadelphia chromosome (Ph)-positive leukemia cells. We find that BCR/ABL-induced reactive oxygen species (ROSs) cause chronic oxidative DNA damage resulting in double-strand breaks (DSBs) in S and G(2)/M cell cycle phases. These lesions are repaired by BCR/ABL-stimulated homologous recombination repair (HRR) and nonhomologous end-joining (NHEJ) mechanisms. A high mutation rate is detected in HRR products in BCR/ABL-positive cells, but not in the normal counterparts. In addition, large deletions are found in NHEJ products exclusively in BCR/ABL cells. We propose that the following series of events may contribute to genomic instability of Ph-positive leukemias: BCR/ABL --> ROSs --> oxidative DNA damage --> DSBs in proliferating cells --> unfaithful HRR and NHEJ repair.

Tumors expressing the ABL oncoproteins (BCR/ABL, TEL/ABL, v-ABL) can avoid apoptosis triggered by DNA damaging agents. The tumor suppressor protein p53 is an important activator of apoptosis in normal cells; conversely its functional loss may cause drug resistance. The ABL oncoprotein-p53 paradigm represents the relationship between an oncogenic tyrosine kinase and a tumor suppressor gene. Here we show that BCR/ABL oncoproteins employ p53 to induce resistance to DNA damage in myeloid leukemia cells. Cells transformed by the ABL oncoproteins displayed accumulation of p53 upon DNA damage. In contrast, only a modest increase of p53 expression followed by activation of caspase-3 were detected in normal cells expressing endogenous c-ABL. Phosphatidylinositol-3 kinase-like protein kinases (ATR and also ATM) -dependent phosphorylation of p53-Ser15 residue was associated with the accumulation of p53, and stimulation of p21(Waf-1) and GADD45, resulting in G(2)/M delay in BCR/ABL cells after genotoxic treatment. Inhibition of p53 by siRNA or by the temperature-sensitive mutation reduced G(2)/M accumulation and drug resistance of BCR/ABL cells. In conclusion, accumulation of the p53 protein contributed to prolonged G(2)/M checkpoint activation and drug resistance in myeloid cells expressing the BCR/ABL oncoproteins.