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Purpose: Tumor relapse after radiotherapy is a major hurdle in treating pediatric H3K27M-mutant diffuse midline gliomas (DMG). Radiotherapy-induced stress increases association of BCL2 family of proteins with BH3 pro-apoptotic activators preventing apoptosis. We hypothesized that inhibition of radiotherapy-induced BCL2 with a clinically relevant inhibitor, venetoclax, will block BCL2 activity leading to increased apoptosis. BCL2 has never been implicated in DMG as a radiotherapy-induced resistant mechanism. Experimental Design: We performed an integrated genomic analysis to determine genes responsible for radioresistance and a targeted drug screen to identify drugs that synergize with radiation in DMG. Effect of venetoclax on radiation-naïve and 6 Gy radiation on cells was evaluated by studying cell death, changes in BCL2 phosphorylation, reactive oxygen species (ROS), and apoptosis, as well as BCL2 association with BH3 apoptosis initiators. The efficacy of combining venetoclax with radiation was evaluated in vivo using orthotopic xenograft models. Results: BCL2 was identified as a key regulator of tumor growth after radiation in DMGs. Radiation sensitizes DMGs to venetoclax treatment independent of p53 status. Venetoclax as a monotherapy was not cytotoxic to DMG cells. Postradiation venetoclax treatment significantly increased cell death, reduced BCL2–BIM association, and augmented mitochondrial ROS leading to increased apoptosis. Combining venetoclax with radiotherapy significantly enhanced the survival of mice with DMG tumors. Conclusions: This study shows that venetoclax impedes the antiapoptotic function of radiation-induced BCL2 in DMG, leading to increased apoptosis. Results from these preclinical studies demonstrate the potential use of the BCL2 inhibitor venetoclax combined with radiotherapy for pediatric DMG.

Supplemental Figure 1: TRAIL-resistant BJAB-LexR cells are not resistant to other drugs. Supplemental Figure 2: TRAIL-resistant MDA-MB-231 cells are not resistant to other drugs. Supplemental Figure 3: Knock-down of SLC26A2 does not affect sensitivity tovdoxorubicin and etoposide in TRAIL resistant MDA-MB-231. Supplemental Figure 4: BJAB-LexR cells have increased SLC26A2 and decreased DR4 and DR5. Supplemental Figure 5: SLC26A2 expression correlates with Metastasis prognosis.

Supplemental Table 1: Significantly enriched pathways from GIANT analysis

Supplemental Legends 1-5

TRAIL is a potent death-inducing ligand that mediates apoptosis through the extrinsic pathway and serves as an important endogenous tumor suppressor mechanism. Because tumor cells are often killed by TRAIL and normal cells are not, drugs that activate the TRAIL pathway have been thought to have potential clinical value. However, to date, most TRAIL-related clinical trials have largely failed due to the tumor cells having intrinsic or acquired resistance to TRAIL-induced apoptosis. Previous studies to identify resistance mechanisms have focused on targeted analysis of the canonical apoptosis pathway and other known regulators of TRAIL receptor signaling. To identify novel mechanisms of TRAIL resistance in an unbiased way, we performed a genome-wide shRNA screen for genes that regulate TRAIL sensitivity in sublines that had been selected for acquired TRAIL resistance. This screen identified previously unknown mediators of TRAIL resistance including angiotensin II receptor 2, Crk-like protein, T-Box Transcription Factor 2, and solute carrier family 26 member 2 (SLC26A2). SLC26A2 downregulates the TRAIL receptors, DR4 and DR5, and this downregulation is associated with resistance to TRAIL. Its expression is high in numerous tumor types compared with normal cells, and in breast cancer, SLC26A2 is associated with a significant decrease in relapse-free survival.Implication: Our results shed light on novel resistance mechanisms that could affect the efficacy of TRAIL agonist therapies and highlight the possibility of using these proteins as biomarkers to identify TRAIL-resistant tumors, or as potential therapeutic targets in combination with TRAIL. Mol Cancer Res; 15(4); 382–94. ©2017 AACR.

Supplementary Table 3: GSEA analysis for upregulated (enriched) pathways in TRAIL treated MDA-231 TRAILR vs. SENS

Supplemental Table 2: GSEA analysis for upregualted (enriched) pathways in TRAIL treated BJAB-LEXR vs. BJAB WT

Purpose:Tumor relapse after radiotherapy is a major hurdle in treating pediatric H3K27M-mutant diffuse midline gliomas (DMG). Radiotherapy-induced stress increases association of BCL2 family of proteins with BH3 pro-apoptotic activators preventing apoptosis. We hypothesized that inhibition of radiotherapy-induced BCL2 with a clinically relevant inhibitor, venetoclax, will block BCL2 activity leading to increased apoptosis. BCL2 has never been implicated in DMG as a radiotherapy-induced resistant mechanism.Experimental Design:We performed an integrated genomic analysis to determine genes responsible for radioresistance and a targeted drug screen to identify drugs that synergize with radiation in DMG. Effect of venetoclax on radiation-naïve and 6 Gy radiation on cells was evaluated by studying cell death, changes in BCL2 phosphorylation, reactive oxygen species (ROS), and apoptosis, as well as BCL2 association with BH3 apoptosis initiators. The efficacy of combining venetoclax with radiation was evaluated in vivo using orthotopic xenograft models.Results:BCL2 was identified as a key regulator of tumor growth after radiation in DMGs. Radiation sensitizes DMGs to venetoclax treatment independent of p53 status. Venetoclax as a monotherapy was not cytotoxic to DMG cells. Postradiation venetoclax treatment significantly increased cell death, reduced BCL2–BIM association, and augmented mitochondrial ROS leading to increased apoptosis. Combining venetoclax with radiotherapy significantly enhanced the survival of mice with DMG tumors.Conclusions:This study shows that venetoclax impedes the antiapoptotic function of radiation-induced BCL2 in DMG, leading to increased apoptosis. Results from these preclinical studies demonstrate the potential use of the BCL2 inhibitor venetoclax combined with radiotherapy for pediatric DMG.

Supplementary Figure from Venetoclax Cooperates with Ionizing Radiation to Attenuate Diffuse Midline Glioma Tumor Growth

Supplementary Table from Venetoclax Cooperates with Ionizing Radiation to Attenuate Diffuse Midline Glioma Tumor Growth

BACKGROUND: H3K27M diffuse midline gliomas (DMGs) are highly aggressive pediatric tumors of pons, thalamus or spinal cord. The only standard-of-care for DMGs is radiation therapy (RT) since the anatomical location of such tumors does not allow surgical resection. Tumor response to RT is at best transient as tumor becomes refractory due to radioresistance. Tumor relapse after RT is a major hurdle in treating DMGs. The mechanism of development of radioresistance due to RT-induced stress has not been studied in DMGs yet. METHODS: We performed an integrated genomic analysis to determine genes responsible for radioresistance and a targeted drug screen to identify drugs synergizing with radiation in DMG. Effect of venetoclax on radiation-naïve and 6Gy radiated DMG cells was evaluated by studying cell death and apoptosis. The efficacy of combining venetoclax with radiation was evaluated in vivo using orthotopic xenograft models. RESULTS: We identified that BCL2 as a key regulator of tumor growth in DMGs after radiation. Radiation sensitizes DMGs to venetoclax treatment. While venetoclax as a monotherapy was not cytotoxic to DMG cells, post-radiation venetoclax significantly increased cell death and apoptosis. Combining venetoclax with RT significantly enhanced the survival of mice with DMG tumors in vivo. Further, we found that the mechanism of radiation-induced cytotoxic effect of venetoclax is p53-independent in DMGs. CONCLUSIONS: This study shows that venetoclax impedes the anti-apoptotic function of radiation-induced BCL2 in DMG leading to apoptosis. Our results are encouraging because, in clinical settings, majority of the DMG patients, irrespective of the tumor p53 status, will benefit from combining RT with venetoclax treatment. Since venetoclax either alone or in combination with chemotherapy drugs are currently in clinical trials for other pediatric cancers, a phase 1b trial is imminent for treating DMGs with venetoclax in combination with radiation therapy.

BACKGROUND: We identified high expression of CD99 in DIPG tumors and developed a CAR using our newly identified single chain variable fragment (scFv) targeting CD99 incorporating a 4-1BB co-stimulatory domain. This CD99 CAR demonstrated the ability to dramatically shrink the established orthotopic DIPG tumor, however tumor recurrence remains an obstacle to cure, due to a loss of the CAR-T cells as they also express the target antigen, CD99 (fratricide). To overcome this obstacle, we modified these CAR-T by editing out CD99. METHODS: CD99 was knocked-out from the human T cells using CRISPR-cas9 gene-editing and subsequently transduced with our CD99 CAR-encoding virus, and isolated the pure population of CD99KO T-cells. These novel, gene-edited T-cells expressing CD99 CAR (“CD99KO CARs”) and the un-edited ones (“CD99 CAR”) were tested for tumor-lysis function when co-cultured with DIPG cells. DIPG tumor-bearing mice infused with a one-time dose of CD99KO CAR-T cells or CD99 CAR- or CD19 control CAR-T cells and were monitored for changes in the tumor burden. At the endpoint spleen and bone marrow were isolated to test for CAR+ cell persistence. RESULTS: The CD99KO CAR-T cells demonstrated effective tumor-lysis when co-cultured with DIPG cells. CD99KO CAR-T cells targeting CD99 showed complete clearance of DIPG tumor in orthotopic DIPG mouse models, and no tumor recurrence was seen well-beyond the time frame of expected tumor recurrence after treatment with un-edited CD99 CAR-T cells. There was an un-precedented increase in the xenograft survival, > 200 days, in mice treated with CD99KO CARs and at which time point sustained persistence of CAR+ cells were evident in the animal spleen and bone marrow. CONCLUSIONS: We have generated a new and promising CAR-T cell therapy that is effective against DIPG with enhanced persistence in animal models which is critical for clinical translation.

Background Group 3 medulloblastoma (MB) is often accompanied by MYC amplification. PLK1 is an oncogenic kinase that controls cell cycle and proliferation and has been preclinically validated as a cancer therapeutic target. Onvansertib (PCM-075) is a novel, orally available PLK1 inhibitor, which shows tumor growth inhibition in various types of cancer. We aim to explore the effect of onvansertib on MYC-driven medulloblastoma as a monotherapy or in combination with radiation. Methods Crisper-Cas9 screen was used to discover essential genes for MB tumor growth. Microarray and immunohistochemistry on pediatric patient samples were performed to examine the expression of PLK1. The effect of onvansertib in vitro was measure by cell viability, colony-forming assays, extreme limiting dilution assay, and RNA-Seq. ALDH activity, cell-cycle distribution, and apoptosis were analyzed by flow cytometry. DNA damage was assessed by immunofluorescence staining. Medulloblastoma xenografts were generated to explore the monotherapy or radio-sensitizing effect. Results PLK1 is overexpressed in Group 3 MB. The IC50 concentrations of onvansertib in Group 3 MB cell lines were in a low nanomolar range. Onvansertib reduced colony formation, cell proliferation, stem cell renewal and induced G2/M arrest in vitro. Moreover, onvansertib in combination with radiation increased DNA damage and apoptosis compared with radiation treatment alone. The combination radiotherapy resulted in marked tumor regression in xenografts. Conclusions These findings demonstrate the efficacy of a novel PLK1 inhibitor onvansertib in vitro and in xenografts of Group 3 MB, which suggests onvansertib is an effective strategy as monotherapy or in combination with radiotherapy in MB.

BACKGROUND: There is an unmet need to identify novel targeted therapies for Diffuse Midline glioma (DMG) which is currently a refractory disease. Recently, we identified high expression of a cell surface antigen, CD99 in H3K27M-mutant expressing DMGs compared to other normal brain counterparts. We developed a novel chimeric CD99 antibody and tested the anti-tumor efficacy of this antibody in vitro and in vivo. METHOD: Bio-legend cell-surface screening was performed in H3K27M-mutant and WT DMG cells. Functional role of CD99 was studied using CD99 proficient and depleted tumors. Designed and synthesized CD99 antibody with a new binding sequence on a human IgG scaffold and performed cell toxicity and growth-inhibitory studies using DMG tumor and normal cells. We also performed these studies in combination with radiation. Multiple patient-derived orthotopic DMG xenograft models was used to test the antibody efficacy. Different antibody delivery routes, that are clinically relevant were investigated. RESULTS: CD99 expression is transcriptionally regulated by H3K27M and is enriched on the cell surface of K27M tumors compared to WT DMG tumors. Our new CD99 antibody (10D1 clone) significantly reduced DIPG tumor cell proliferation in vitro. Intravenous infusion of this antibody in DIPG tumor bearing mice showed complete clearance of tumor that prolonged animal survival suggesting the enhanced anti-tumor efficacy of 10D1-CD99 and importantly, its ability in crossing the blood-brain-barrier and reaching the pons target site. Loco-regional administration of 10D1 showed similar anti-tumor effects even at much reduced antibody concentrations while toxicity to CD99-expressing T cells was minimum. Radiation increased CD99 expression and enhanced the cytotoxic effect of 10D1-CD99. CONCLUSION: We have developed a novel CNS penetrant CD99 antibody that is an attractive therapeutic strategy in treating DMG. 10D1 is currently in development as a therapeutic.

MYC-driven medulloblastoma is a major therapeutic challenge due to frequent metastasis and a poor 5-year survival rate. MYC gene amplification results in transcriptional dysregulation, proliferation, and survival of malignant cells. To identify therapeutic targets in MYC-amplified medulloblastoma, we employ a CRISPR-Cas9 essentiality screen targeting 1,140 genes. We identify CDK7 as a mediator of medulloblastoma tumorigenesis. Using chemical inhibitors and genetic depletion, we observe cessation of tumor growth in xenograft mouse models and increases in apoptosis. The results are attributed to repression of a core set of MYC-driven transcriptional programs mediating DNA repair. CDK7 inhibition alters RNA polymerase II (RNA Pol II) and MYC association at DNA repair genes. Blocking CDK7 activity sensitizes cells to ionizing radiation leading to accrual of DNA damage, extending survival and tumor latency in xenograft mouse models. Our studies establish the selective inhibition of MYC-driven medulloblastoma by CDK7 inhibition combined with radiation as a viable therapeutic strategy.

Myc-driven Medulloblastoma remains a major therapeutic challenge due to frequent metastasis and a poor 5-year survival rate. Myc gene amplification results in transcriptional dysregulation, proliferation, and survival of malignant cells. To identify therapeutic targets in Myc-amplified medulloblastoma we performed a CRISPR-Cas9 essentiality screen targeting 1140 genes annotated as the druggable genome. CDK7 was identified as a mediator of medulloblastoma tumorigenesis. Using covalent inhibitors and genetic depletion of CDK7 we observe the cessation of tumor growth in xenograft mouse models and increase in apoptotic mechanisms. The results are attributed to repression of a core set of Myc-driven transcriptional programs mediating DNA repair. We further establish that blocking CDK7 activity sensitizes cells to ionizing radiation leading to accrual of DNA damage and extended survival and tumor latency in medulloblastoma xenograft mouse models. Our studies establish a mechanism for selective inhibition of Myc-driven MB by CDK7 inhibition combined with radiation as a viable therapeutic strategy for Myc-amplified medulloblastoma.

Atypical teratoid/rhabdoid tumors (AT/RT) are characterized by loss-of-function mutations in the SMARCB1 component (and less commonly SMARCA4) of the SWI/SNF chromatin-remodeling complex. AT/RT demonstrate an overall silent genomic landscape with epigenetic dysregulation of the genome. CDK7 is a key transcriptional regulator that preferentially phosphorylates the Ser5 and Ser7 positions on RNA Polymerase C terminal domain and is involved early in transcription. In tumor cells, CDK7 is enriched at super enhancers which preferentially regulate genes involved in cell transformation, and expressed at significantly higher levels in transformed tissues than the surrounding normal brain. Our preliminary data shows that CDK7 is expressed in a number of AT/RT tumor cell lines and patient-derived tumor cultures, and that loss of CDK7 function though exposure to the novel CDK7 inhibitor THZ2 results in lack of proliferation at lower doses, and caspase-mediated apoptosis at higher concentrations. shRNA-based inhibition confirms that this effect is due specifically to loss of CDK7. RNA sequencing of cells treated with lower doses of THZ2 show significant alterations in transcript expression consistent with altered balance between antagonistic SWI/SNF and PRC2 chromatin-modeling complex activities, as well as alterations in DNA damage response pathways, cell cycle checkpoints, miRNA transcription, and numerous proliferative factors. THZ2 penetrates the blood brain barrier (BBB), is well tolerated, and results in prolonged survival in murine xenograft models of AT/RT. CDK7 inhibition also synergizes with a number of currently-approved oncology drugs, as well as with ionizing radiation, in order to inhibit AT/RT growth and viability.

Diffuse Intrinsic pontine glioma is an incurable brain tumor of childhood characterized by histone mutations at lysine 27 which results in epigenomic dysregulation. There has been a failure to develop effective treatment for this tumor. We performed a combined RNAi and chemical screen targeting epigenetic regulatory genes and identified the polycomb (PcG) repressive complex 1, component BMI1 as a critical factor for DIPG tumor maintenance in vivo. BMI1 chromatin occupancy was enriched at genes associated with differentiation and tumor suppressors in DIPG cells. Inhibition of BMI1 results in decreased cell self-renewal and attenuation of tumor growth due to induction of senescence. Prolong BMI1 inhibition induces a senescence associated secretory phenotype which promotes tumor recurrence. Clearance of senescent cells using BH3 protein mimetics co-operates with BMI1 inhibition to synergistically attenuate tumor growth in vivo.

Our previous studies have shown that the epigenetic developmental regulator EZH2 is overexpressed in medulloblastoma (MB). EZH2 is known to repress developmental genes in embryonic stem cells to maintain their stem cell characteristics. To identify the role of EZH2 in medulloblastoma, we overexpressed EZH2 (EZH2(Ox)) in the C17.2 Myc immortalized cerebellar neural stem cell line. EZH2 overexpression in combination with Myc in the neural progenitor C17.2 cells generated orthotopic tumors that resembled clinical features of MYC-amplified group3 MB. To examine the mechanism of EZH2 mediated tumorigenesis we performed RNA-seq followed by Gene Set Enrichment Analysis (GSEA) of these EZH2 overexpressing neural stem cells. Transcriptomic analysis reveals that the gene sets associated with apoptosis were suppressed by EZH2 overexpression. Specifically, the p53 regulator and pro-apoptotic gene, HIPK2 was significantly suppressed by EZH2 overexpression. To examine the impact of EZH2 on apoptosis in Myc driven medulloblastoma cells we performed a CRISPR knock out of EZH2 in the D458 group 3 MB cell line. EZH2 deletion resulted in increased apoptosis leading to sensitization of cells to chemotherapy and radiation treatments. Further analysis of these CRISPR-edited EZH2 knockout cells showed a predominant upregulation in the HIPK2 expression compared to control D458 cells. While, overexpressing HIPK2 in D458 cells resulted in increased apoptosis after cisplatin or ionizing radiation, depleting HIPK2 in EZH2 CRISPR-KO cells rescued the proapoptotic phenotype. Treating mice with cisplatin resulted in significantly smaller orthotopic xenograft tumors of EZH2 knockout medulloblastoma cells compared to EZH2 sufficient medulloblastoma cells. Together, our results suggest that EZH2 contributes to treatment-resistance by inhibiting apoptosis via transcriptionally modulating HIPK2 expression in group 3 medulloblastoma. Therefore, targeting EZH2 will effectively sensitize tumors to standard therapies.

Myc-driven Medulloblastoma remains a major therapeutic challenge due to frequent metastasis and a poor 5-year survival rate. Myc overexpression results in transcriptional dysregulation, proliferation, and survival of malignant cells. However direct inhibition of Myc remains an obstacle to therapeutic interventions. To identify therapeutic targets in Myc amplified medulloblastoma we performed a CRISPR-Cas9 essentiality screen targeting 1140 genes annotated as the druggable genome. A strand guide library (4–10 Sg/gene) was transduced along with Cas9 into 3 different Myc amplified medulloblastoma cell lines in triplicate and DNA extracted and sequenced 18 days after transduction. Analysis of the resultant data revealed multiple gene networks as essential for Myc-medulloblastoma. The cyclin-dependent kinase, CDK7, was identified as a top candidate. CDK7 phosphorylates the c-terminal domain of RNA Pol II facilitating transcriptional initiation and elongation. We found that CDK7 expression is elevated in Myc-medulloblastoma patient samples and cell lines compared to other subgroups and normal pediatric cerebellum. Genetic depletion of CDK7 suppressed medulloblastoma cell clonogenicity in vitro. Recently a covalent inhibitor, THZ1, with high potency for CDK7 was identified and shown to efficiently reduce tumor growth in several pediatric cancers. To evaluate whether CDK7 is a therapeutic target in Myc-driven medulloblastoma we treated Myc-amplified medulloblastoma cells with THZ1. We observed a significant inhibition of clonogenic potential and induction of apoptosis in Myc-medulloblastoma cells in vitro. Using RNA-seq-based gene set enrichment analysis we show that THZ1 decreases c-Myc driven transcriptomic programs and enhanced inflammatory and apoptotic responses. Further studies established that chemical inhibition of CDK7 enhanced radiation sensitivity of Myc MB cells by potentiating DNA damage. Furthermore, CDK7 inhibition with THZ2 suppresses medulloblastoma tumor formation in vivo. Our studies are the first demonstration of CDK7 inhibition as a viable therapeutic strategy alone or in combination with radiation for Myc-amplified medulloblastoma.

Mutations in the histone 3 (H3) gene (H3K27M) are the eponymous driver in diffuse intrinsic pontine gliomas (DIPGs), aggressive pediatric brain tumors for which no curative therapy currently exists. In order to identify specific epigenetic dependencies which arise as a consequence of the H3K27M mutation, we performed an shRNA screen targeting 408 genes classified as epigenetic/chromatin-associated molecules in patient-derived DIPG cell lines. This identified AFF4, a component of the super elongation complex (SEC), as both highly expressed and necessary for DIPG cells to maintain growth and self-renewal. We hypothesized that SEC overexpression occurs as a direct consequence of the H3K27M mutation and that this relative abundance overcomes repressive transcriptional regulation in order to suppresses differentiation and promote self-renewal of DIPG tumor stem cells. We found that AFF4 expression is consistently elevated in both DIPG patient samples and established cell lines relative to normal pediatric pons. We interrogated the role of AFF4 in H3K27M-mutant DIPG using an shRNA lentiviral approach. Using live cell imaging, we demonstrate a significant decrease in in vitro clonogenicity and stem cell maintenance following AFF4 depletion. We employed RNA-seq-based gene set enrichment analysis to delineate differentiation programs under AFF4 regulatory control. Finally, we sought to determine whether CDK9, the catalytic subunit of the SEC, represents a therapeutic vulnerability in DIPG. Using ChIP-seq following CDK9 pharmacologic inhibition, we demonstrate that the loss of regulatory transcriptional input in DIPG may be reversed via CDK9 inhibition and that this acts as a potent inhibitor of DIPG cell growth both in vitro and in vivo orthotopic xenograft models. These studies represent the first pre-clinical validation of SEC inhibition as a novel therapeutic approach in DIPG.

Background and Rationale Diffuse intrinsic pontine glioma (DIPG) is one of the most aggressive pediatric brain tumors. Currently, the main treatment for DIPG is radiation and it’s only a palliative care, as the tumor eventually becomes resistant to radiation. In this study we found that radiation leads to an increase in anti-apoptotic BH3 proteins mainly BCL2 in DIPG. Previous studies in other tumor types have shown that increase in these pro-survival BCL2 family members are associated with treatment resistance and poor prognosis. Therefore, we hypothesize that inhibition of BCL2 using a small-molecule inhibitor, venetoclax that crosses the blood-brain barrier, will represent a possible therapeutic strategy to overcome radiation resistance in DIPG. Approach: For in vitro studies, DIPG cells were exposed to different radiation doses (0–10 Gy) and the magnitude of the sensitizing effect of venetoclax (with IC15) was calculated by clonogenic assay. Evaluated BCL2 family proteins by western and cytotoxicity by cleaved caspase incucyte assays. For in vivo studies, NSG mice orthotopically engrafted with a human H3K27M-DIPG luciferase-expressing cells in the pons were exposed to a focal fractionated radiation of 2Gy/day for 3 days. Mice were randomized into 2 groups based on bioluminescence IVIS signal intensity; each group receiving either venetoclax (15 mg/kg, by i.p) 3 days/week for 10 weeks or vehicle. Decrease in tumor burden was measured by IVIS and survival was evaluated compared to vehicle treated mice. Results Single agent venetoclax showed no significant activity against DIPG tumors in in vitro and in vivo DIPG xenografts. Single-agent radiation cleared the tumor burden but only transiently. Combination of radiation with venetoclax showed considerable synergistic anti-tumor effect in vitro and in vivo leading to a significant increase in animal survival beyond either single agent treatments. The metabolic reprogramming that results in this enhanced cell-killing effect will be discussed.

Simple Summary Group 3 medulloblastoma (MB) is often accompanied by MYC amplification and has a poor prognosis. FBXW7, a critical tumor suppressor in many types of cancer, regulates the proteasome-mediated degradation of oncoproteins including MYC. However, the role of FBXW7 in the tumorigenesis of group 3 MB has not been well studied. In this study, we show that FBXW7 is downregulated in group 3 MB patient samples, and FBXW7 stabilization is crucial for inhibiting c-MYC. We identified a FBXW7-MYC-PLK1 regulatory loop in MYC-driven MB, which provides a mechanism of using protein kinase inhibitors for translation in the future. Abstract Polo-like kinase 1 (PLK1) is highly expressed in group 3 medulloblastoma (MB), and it has been preclinically validated as a cancer therapeutic target in medulloblastoma. Here, we demonstrate that PLK1 inhibition with PCM-075 or BI6727 significantly reduces the growth of MB cells and causes a decrease of c-MYC mRNA and protein levels. We show that MYC activates PLK1 transcription, while the inhibition of PLK1 suppresses MB tumor development and causes a decrease in c-MYC protein level by suppressing FBXW7 auto poly-ubiquitination. FBXW7 physically interacts with PLK1 and c-MYC, facilitating their protein degradation by promoting ubiquitination. These results demonstrate a PLK1-FBXW7-MYC regulatory loop in MYC-driven medulloblastoma. Moreover, FBXW7 is significantly downregulated in group 3 patient samples. The overexpression of FBXW7 induced apoptosis and suppressed proliferation in vitro and in vivo, while constitutive phosphorylation mutation attenuated its tumor suppressor function. Altogether, these findings demonstrated that PLK1 inhibition stabilizes FBXW7 in MYC-driven MB, thus revealing an important function of FBXW7 in suppressing medulloblastoma progression.

Myc-driven Medulloblastoma remains a major therapeutic challenge due to frequent metastasis and a poor 5-year survival rate. Myc overexpression results in transcriptional dysregulation, proliferation, and survival of malignant cells. To identify therapeutic targets in Myc-amplified medulloblastoma we performed a CRISPR-Cas9 essentiality screen targeting 1140 genes annotated as the druggable genome. The cyclin-dependent kinase, CDK7, was identified as a top candidate. CDK7 phosphorylates the c-terminal domain of RNA Pol II facilitating transcriptional initiation and elongation. We subjected Myc-amplified cells treated with CDK7 inhibitors to whole transcriptomic analysis. The resultant data revealed gene networks mediating DNA repair were functionally repressed. Consistent with this data, ChIP-sequencing showed the most significant reduction in RNA Pol II and Myc promoter occupancy within a subset of DNA repair genes including BRCA2 and RAD51 but not across the whole genome. These data suggest that inhibition of CDK7 mechanistically limits Myc driven transcriptional processivity of DNA repair networks. Further, evaluation of genes mediating DNA repair show a muted response to DNA damage and increased cell death with CDK7 inhibition. We next evaluated Myc-amplified MB cell response to ionizing radiation in vitro and in vivo with CDK7 inhibition. Inhibition of CDK7 enhanced radiation sensitivity of Myc MB cells by potentiating DNA damage. Further, cotreatment produced decreased MRI T2 tumor volumes and enhanced survival benefit in orthotopic PDX xenografted mice compared to radiation alone. Our studies establish a mechanism for selective inhibition of Myc-driven MB by CDK7 inhibition combined with radiation as a viable therapeutic strategy for Myc-amplified medulloblastoma.

BACKGROUND Diffuse intrinsic pontine gliomas (DIPGs) driven by mutations in the histone 3 (H3) gene (H3K27M) are aggressive pediatric brain tumors for which there is no curative therapy. METHODS To identify novel therapeutic targets we performed a high throughput drug screen combined with an epigenetically targeted RNAi screen using H3K27M and H3.3 WT DIPG cells. RESULTS Chemical and genetic depletion of BMI1 in vitro resulted in inhibition of clonogenicity and cell self-renewal consistent with previous studies. We show for the first time that clinically relevant BMI1 inhibitors attenuates growth of orthotopic DIPG xenografts as measured by MRI and prolong survival in vivo. We found that BMI1 inhibition drives phenotypic cellular senescence and that the senescent cells were able reactivate to form new neurospheres in vitro and tumor growth in vivo. RNA-seq, ChIP-Seq and immuno-proteomic analysis revealed that the senescent cells induced the expression of the Senescence Associated Secretory Phenotype (SASP) cytokines by increasing occupancy of activated histone marks at SASP factor promoters. The SASP results in increased expression of anti-apoptotic BH3 proteins including BCLxl, and BCL2. Treatment of the PTC028 treated senescent DIPG cells with BH3 mimetics induces apoptosis and clears the senescent cells. Combining BH3 mimetics with BMI1 inhibition attenuates tumor growth in vivo synergistically and significantly prolongs survival of DIPG bearing mice compared to BMI1 inhibition alone. CONCLUSION These data inform the current trial of BMI1 inhibition as a monotherapy and predict the need for adding BH3 mimetics to achieve efficacy.

Mutations in the histone 3 gene (H3K27M) are the eponymous driver in diffuse intrinsic pontine gliomas (DIPGs) and other diffuse midline gliomas (DMGs), aggressive pediatric brain tumors for which no curative therapy currently exists. To identify specific epigenetic dependencies within the context of the H3K27M mutation, we performed an shRNA screen targeting 408 genes classified as epigenetic/chromatin-associated molecules in patient-derived DMG cultures. This identified AFF4, a component of the super elongation complex (SEC), as necessary for DMG cells to maintain growth and self-renewal. We hypothesized that aberrant SEC expression occurs as a consequence of the H3K27M mutation and that this dysregulated SEC signaling overcomes repressive transcriptional regulation in order to suppresses differentiation and promote self-renewal of DMG tumor stem cells. We interrogated the role of AFF4 in DMG using an shRNA lentiviral approach. We demonstrate a significant decrease in in vitro clonogenicity and stem cell maintenance following AFF4 depletion. We employed RNA-seq-based gene set enrichment analysis to delineate differentiation programs under SEC regulatory control. Finally, we sought to determine whether CDK9, the catalytic subunit of the SEC, represents a therapeutic vulnerability in DMG. Using RNA polymerase II ChIP-seq, we demonstrate that CDK9 pharmacologic inhibition restores regulatory Pol II pausing, promotes cellular differentiation, and leads to potent anti-tumor effect both in vitro and in patient-derived xenograft models. These studies present a biologic rationale for the translational exploration of CDK9 inhibition as a promising therapeutic approach.

Group 3 medulloblastoma (MB) is often accompanied by MYC amplification and has a higher rate of metastatic disease. So, it is critical to have more effective therapies for high MYC expressing sub-groups. Here we report that FBXW7, a substrate recognition component of the SKP1-CUL1-Fbox (SCF) E3 ligase, interacts with and targets c-MYC for polyubiquitination and proteasomal degradation. FBXW7 shows lower expression level in MYC-driven MB compared with other MB subgroups suggesting activity as a tumor suppressor. Genomic deletion or mutation of Fbxw7 has frequently been identified in many human cancers but not in MB. We demonstrate that overexpression of Fbxw7 in MB cells induces apoptosis and suppresses proliferation in vitro and in vivo. Both phospho-deficient (T205A) and phosphomimetic aspartic acid (T205D) mutants deactivate its tumor suppressor function suggesting a conformational change of its protein structure. Mechanistically, PLK1 kinase specifically phosphorylates FBXW7 and promotes its auto-polyubiquitination and proteasomal degradation, counteracting FBXW7-mediated degradation of oncogene substrates, including c-MYC and PLK1. Chip-Seq results show stabilized c-MYC in turn directly activates PLK1 and FBXW7 transcription, constituting a feedforward regulatory loop. Co-immunoprecipitation demonstrates that FBXW7 directly binds to PLK1 and c-MYC, facilitating their protein degradation by promoting the ubiquitination of both proteins. Furthermore, we show that FBXW7 protein can be stabilized by various kinase inhibitors, proposing a mechanism of kinase-targeted agents to treat MYC-driven MB. These results collectively demonstrate how kinase inhibition stabilizes the tumor suppressor FBXW7 in MYC-driven MB, thus revealing an important function of FBXW7 in suppressing MB progression.

KDM6A and EZH2 are epigenetic regulators that play a key role in embryogenesis and development. Presumably, EZH2 and KDM6A have opposite impact on H3K27me3. KDM6A functions as a demethylase of Histone3 lysine 27 trimethylation and is closely associated with the mixed lineage leukemia 2/3 in creating a transcriptionally active chromatin state through removal of methyl groups. EZH2, on the other hand, is a methyl transferase which catalyzes the trimethylation of Histone3 lysine 27. Several studies including ours have shown that KDM6A is genomically deleted in Group 4 medulloblastoma suggesting it may have a role as a tumor suppressor. The biological role of KDM6A in medulloblastoma is not clear. To better understand the role of KDM6A in MB, in our current study we used the Crispr/Cas9 system to knock out KDM6A in MB cell lines and to determine the effect of KDM6A on MB tumor cell stemness and proliferation. Increased expression of stemcell-associated genes including Nanog, Nestin and Oct4 were observed in cells depleted of KDM6A. Intriguingly, Crispr/Cas9-mediated deletion of KDM6A significantly abolished MB cell neurosphere formation and cell proliferation compared to control cells. As expected, in the knockdown cells there was a concomitant increase in H3K27me3 and decrease in H3K4me3 expression but the overall biological impact of KDM6A deletion was unexpected. This puzzling effect on cells with the knockdown of KDM6A opens a door to investigate the target genes that are affected by the depletion of KDM6A. This study will shine light on new epigenetic therapies in medulloblastoma.

Mutations in the histone 3 (H3) gene (H3K27M) are the eponymous driver in diffuse midline gliomas (DMGs). Emerging understanding of H3K27M biology suggests that secondary cooperating factors are required to promote oncogenesis. We have identified AFF4, a component of the super elongation complex (SEC), as critical for DMG viability. We hypothesized that AFF4 promotes DMG tumorigenesis by co-operating with the H3K27M mutation to drive a gene expression program which suppresses scheduled transcription of pro-differentiation pathways and promotes self-renewal of tumor stem cells. We initially performed an shRNA screen targeting 408 genes classified as epigenetic/chromatin-associated molecules in a patient-derived DMG cell line. We validated initial hits in secondary screens using multiple shRNA constructs. Live cell imaging systems were utilized to assay growth and neurosphere formation. Finally, we employed RNA-seq-based gene expression analysis to delineate downstream transcriptional changes under AFF4 regulatory control. We found that AFF4 expression is consistently elevated in DMG lines relative to normal pons, and AFF4 inhibition results in decreased rates of growth and stem cell maintenance uniquely within the context of the H3K27M mutation. Further mechanistic studies will lay the groundwork for pre-clinical validation of SEC inhibition as a novel therapeutic approach in DMG.

Ionizing Radiation (IR) is a key treatment modality for DIPG, but it provides only temporary relief as the tumor cells develop resistance to radiation. Recently, we and others have shown that inhibition of BMI1 either alone or in combination with radiation attenuates DIPG cell proliferation in vitro. While we are demonstrating the in vivo efficacy of pharmacological inhibition of BMI1 and understanding the mechanism of anti-tumor effect of BMI1 inhibition in DIPG, the existence of treatment-resistant cells remains a major obstacle for a prolonged cure. Both IR and genetic or pharmacological inhibition of BMI1 induces cellular senescence as a mechanism to suppress tumor cell proliferation, implying that senescence can be considered as tumor suppressor. Paradoxically, recent studies have shown that accelerated senescence can mediate tumor recurrence due to the development of pro-oncogenic environment. In line with this, we are investigating whether clearance of treatment-induced senescent cells enhances treatment outcomes. DIPG cells exposed to different doses of radiation followed by treatment with ABT-263 (Navitoclax), a drug which selectively clears the senescent cells, resulted in increased radiosensitization. Treatment of pre-radiated DIPG cells with ABT-263 decreased the activity of senescence-associated beta-galactosidase and anti-apoptotic protein expression. Similarly, chemical inhibition of BMI1 in combination with ABT-263 showed synergistic killing of DIPG cells. The synergy was most pronounced in DIPG cells harboring wildtype p53. Our study highlights the importance of eliminating treatment-induced senescent cells while inhibiting proliferation of DIPG tumors, a combination which can immensely improve therapeutic efficacy in DIPG patients.

// Hamid H. Gari 1 , Christy M. Gearheart 2 , Susan Fosmire 2 , Gregory D. DeGala 1 , Zeying Fan 1 , Kathleen C. Torkko 1 , Susan M. Edgerton 1 , M. Scott Lucia 1 , Rahul Ray 3 , Ann D. Thor 1 , Christopher C. Porter 2 and James R. Lambert 1 1 Department of Pathology, University of Colorado School of Medicine, Aurora, CO, USA 2 Department of Pediatrics, University of Colorado School of Medicine, Aurora, CO, USA 3 Department of Medicine, Boston University School of Medicine, Boston, MA, USA Correspondence to: James R. Lambert, email: // Keywords : AMPI-109, PRL-3, triple-negative breast cancer, phosphatase, functional genomics Received : December 22, 2015 Accepted : February 09, 2016 Published : February 17, 2016 Abstract Triple-negative breast cancers (TNBC) are among the most aggressive and heterogeneous cancers with a high propensity to invade, metastasize and relapse. Here, we demonstrate that the anticancer compound, AMPI-109, is selectively efficacious in inhibiting proliferation and inducing apoptosis of multiple TNBC subtype cell lines as assessed by activation of pro-apoptotic caspases-3 and 7, PARP cleavage and nucleosomal DNA fragmentation. AMPI-109 had little to no effect on growth in the majority of non-TNBC cell lines examined. We therefore utilized AMPI-109 in a genome-wide shRNA screen in the TNBC cell line, BT-20, to investigate the utility of AMPI-109 as a tool in helping to identify molecular alterations unique to TNBC. Our screen identified the oncogenic phosphatase, PRL-3, as a potentially important driver of TNBC growth, migration and invasion. Through stable lentiviral knock downs and transfection with catalytically impaired PRL-3 in TNBC cells, loss of PRL-3 expression, or functionality, led to substantial growth inhibition. Moreover, AMPI-109 treatment, downregulation of PRL-3 expression or impairment of PRL-3 activity reduced TNBC cell migration and invasion. Histological evaluation of human breast cancers revealed PRL-3 was significantly, though not exclusively, associated with the TNBC subtype and correlated positively with regional and distant metastases, as well as 1 and 3 year relapse free survival. Collectively, our study is proof-of-concept that AMPI-109, a selectively active agent against TNBC cell lines, can be used as a molecular tool to uncover unique drivers of disease progression, such as PRL-3, which we show promotes oncogenic phenotypes in TNBC cells.

Inhibition of WEE1 is emerging as a promising chemosensitization strategy in many cancers including acute leukemia. Our lab and others have demonstrated that a small-molecule inhibitor of WEE1, AZD1775, sensitizes acute leukemia cells to cytarabine; however, a mechanism of combinatorial activity has remained elusive. Thus, we sought to determine the relative contribution of WEE1 targets CDK1 and CDK2 to the combinatorial activity of AZD1775 and cytarabine. To accomplish this, we expressed "WEE1 resistant" CDK1 (CDK1-AF) and CDK2 (CDK2-AF) constructs in a T-ALL cell line. Expression of CDK1/2-AF together, but neither alone, enhanced the anti-proliferative effects, DNA damage and apoptosis induced by cytarabine. Furthermore, pharmacologic inhibition of CDK1 alone or CDK1 and CDK2 together reduced the combinatorial activity of AZD1775 and cytarabine. Thus, increased activity of both CDK1 and CDK2 in response to WEE1 inhibition is necessary for the combinatorial activity of AZD1775 and cytarabine. This suggests the role of WEE1 in cells with accumulated DNA damage extends beyond regulation of CDK1 and the G2/M checkpoint and highlights the importance of WEE1 in mediating progression through the cell cycle.

Autophagy is a protein and organelle degradation pathway that is involved in diverse diseases, including cancer. Recent evidence suggests that autophagy is a cell survival mechanism in tumor cells and that its inhibition, especially in combination with other therapy, could be beneficial but it remains unclear if all cancer cells behave the same way when autophagy is inhibited. We inhibited autophagy in a panel of breast cancer cell lines and found that some of them are dependent on autophagy for survival even in nutrient rich conditions without any additional stress, whereas others need autophagy only when stressed. Survival under unstressed conditions is due to cell type–specific autophagy regulation of STAT3 activity and this phenotype is enriched in triple-negative cell lines. This autophagy-dependency affects response to therapy because autophagy inhibition reduced tumor growth in vivo in autophagy-dependent but not in autophagy-independent breast tumors, whereas combination treatment with autophagy inhibitors and other agent was preferentially synergistic in autophagy-dependent cells. These results imply that autophagy-dependence represents a tumor cell–specific characteristic where autophagy inhibition will be more effective. Moreover, our results suggest that autophagy inhibition might be a potential therapeutic strategy for triple-negative breast cancers, which currently lack an effective targeted treatment. Cancer Res; 74(9); 2579–90. ©2014 AACR.

Although some patients with acute leukemia have good prognoses, the prognosis of adult and pediatric patients who relapse or cannot tolerate standard chemotherapy is poor. Inhibition of WEE1 with AZD1775 has been shown to sensitize cancer cells to genotoxic chemotherapies, including cytarabine in acute myeloid leukemia (AML) and T-ALL. Inhibition of WEE1 impairs homologous recombination by indirectly inhibiting BRCA2. Thus, we sought to determine whether AZD1775 could sensitize cells to the PARP1/2 inhibitor olaparib. We found that combined treatment with AZD1775 and olaparib was synergistic in AML and ALL cells, and this combination impaired proliferative capacity upon drug withdrawal. AZD1775 impaired homologous recombination in olaparib-treated cells, resulting in enhanced DNA damage accumulation and apoptosis induction. This combination enhanced disease control and increased survival in a murine AML model. Furthermore, we demonstrated that combined treatment with AZD1775 and olaparib reduces proliferation and colony formation and increases apoptosis in AML patient samples. In aggregate, these studies raise the possibility of rational combinations of targeted agents for leukemia in patients for whom conventional chemotherapeutics may not be effective or well tolerated. Mol Cancer Ther; 16(10); 2058–68. ©2017 AACR.

TRAIL is a potent death-inducing ligand that mediates apoptosis through the extrinsic pathway and serves as an important endogenous tumor suppressor mechanism. Because tumor cells are often killed by TRAIL and normal cells are not, drugs that activate the TRAIL pathway have been thought to have potential clinical value. However, to date, most TRAIL-related clinical trials have largely failed due to the tumor cells having intrinsic or acquired resistance to TRAIL-induced apoptosis. Previous studies to identify resistance mechanisms have focused on targeted analysis of the canonical apoptosis pathway and other known regulators of TRAIL receptor signaling. To identify novel mechanisms of TRAIL resistance in an unbiased way, we performed a genome-wide shRNA screen for genes that regulate TRAIL sensitivity in sublines that had been selected for acquired TRAIL resistance. This screen identified previously unknown mediators of TRAIL resistance including angiotensin II receptor 2, Crk-like protein, T-Box Transcription Factor 2, and solute carrier family 26 member 2 (SLC26A2). SLC26A2 downregulates the TRAIL receptors, DR4 and DR5, and this downregulation is associated with resistance to TRAIL. Its expression is high in numerous tumor types compared with normal cells, and in breast cancer, SLC26A2 is associated with a significant decrease in relapse-free survival. Implication: Our results shed light on novel resistance mechanisms that could affect the efficacy of TRAIL agonist therapies and highlight the possibility of using these proteins as biomarkers to identify TRAIL-resistant tumors, or as potential therapeutic targets in combination with TRAIL. Mol Cancer Res; 15(4); 382–94. ©2017 AACR.

Mutations in the histone 3 (H3) gene (H3K27M) are the eponymous driver in diffuse midline gliomas (DMGs), aggressive pediatric brain tumors for which no curative therapy currently exists. Emerging understanding of H3K27M biology suggests that secondary factors are required to promote oncogenesis. In order to identify specific epigenetic co-operators, we performed an shRNA screen targeting 408 genes classified as epigenetic/chromatin-associated molecules in patient-derived DMG cell lines. This identified AFF4, a component of the super elongation complex (SEC), as critical for DMG viability. We hypothesized that AFF4 promotes DMG tumorigenesis by co-operating with the H3K27M mutation to suppress scheduled transcription of pro-differentiation pathways and promote self-renewal of tumor stem cells. We found that AFF4 expression is consistently elevated in both DMG patient samples and established cell lines relative to the normal pediatric pons. We interrogated the role of AFF4 in H3K27M-mutant DMG using an shRNA lentiviral approach. Using live cell imaging, we demonstrate a significant decrease in in vitro clonogenicity and stem cell maintenance following AFF4 depletion. We employed RNA-seq-based gene set enrichment analysis to delineate downstream transcriptional changes under AFF4 regulatory control. Finally, we sought to determine whether CDK9, the catalytic subunit of the SEC, represents a therapeutic vulnerability in DMG. Using a combination of CDK9 overexpression and pharmacologic inhibition, we demonstrate that the disordered regulatory input of the SEC in DMG is dependent on the kinase activity of CDK9 and that this may be exploited through small molecule inhibition in both in vitro and in vivo patient-derived xenograft models. These studies represent the first pre-clinical validation of SEC inhibition as a novel therapeutic approach in pediatric DMG.

The capacity of nicotine to affect the behavior of non-neuronal cells through neuronal nicotinic acetylcholine receptors (nAChRs) has been the subject of considerable recent attention. Previously, we showed that exposure to nicotine activates the nuclear factor of activated T cells (NFAT) transcription factor in lymphocytes and endothelial cells, leading to alterations in cellular growth and vascular endothelial growth factor production. Here, we extend these studies to document effects of nicotine on lymphocyte survival. The data show that nicotine induces paradoxical effects that might alternatively enforce survival or trigger apoptosis, suggesting that depending on timing and context, nicotine might act both as a survival factor or as an inducer of apoptosis in normal or transformed lymphocytes, and possibly other non-neuronal cells. In addition, our results show that, while having overlapping functions, low and high affinity nAChRs also transmit signals that promote distinct outcomes in lymphocytes. The sum of our data suggests that selective modulation of nAChRs might be useful to regulate lymphocyte activation and survival in health and disease.

Recurrent chromosomal aberrations in solid tumors can reveal the genetic pathways involved in the evolution of a malignancy and in some cases predict biological behavior. However, the role of individual genetic backgrounds in shaping karyotypes of sporadic tumors is unknown. The genetic structure of purebred dog breeds, coupled with their susceptibility to spontaneous cancers, provides a robust model with which to address this question. We tested the hypothesis that there is an association between breed and the distribution of genomic copy number imbalances in naturally occurring canine tumors through assessment of a cohort of Golden Retrievers and Rottweilers diagnosed with spontaneous appendicular osteosarcoma. Our findings reveal significant correlations between breed and tumor karyotypes that are independent of gender, age at diagnosis, and histological classification. These data indicate for the first time that individual genetic backgrounds, as defined by breed in dogs, influence tumor karyotypes in a cancer with extensive genomic instability.

The significance of p16/Rb tumor suppressor pathway inactivation in T-cell non-Hodgkin's lymphoma (NHL) remains incompletely understood. We used naturally occurring canine NHL to test the hypothesis that p16 inactivation has specific pathologic correlates. Forty-eight samples (22 T-cell NHL and 26 B-cell NHL) were included. As applicable, metaphase- or array-based comparative genomic hybridization, Southern blotting, promoter methylation, and Rb phosphorylation were used to determine the presence, expression, and activity of p16. Fisher's exact test was used to test for significance. Deletion of p16 (or loss of dog chromosome 11) was restricted to high-grade T-cell NHL (lymphoblastic T-cell lymphoma and peripheral T-cell lymphoma, not otherwise specified). These were characterized by a concomitant increase of tumor cells with Rb phosphorylation at canonical CDK4 sites. Rb phosphorylation also was seen in high-grade B-cell NHL (diffuse large B-cell lymphoma and Burkitt-type lymphoma), but in those cases, it appeared to be associated with c-Myc overexpression. The data show that p16 deletion or inactivation occurs almost exclusively in high-grade T-cell NHL; however, alternative pathways can generate functional phenotypes of Rb deficiency in low-grade T-cell NHL and in high-grade B-cell NHL. Both morphologic classification according to World Health Organization criteria and assessment of Rb phosphorylation are prognostically valuable parameters for canine NHL.

The outcome for pediatric acute lymphoblastic leukemia (ALL) patients who relapse is dismal. A hallmark of relapsed disease is acquired resistance to multiple chemotherapeutic agents, particularly glucocorticoids. In this study, we performed a genome-scale short hairpin RNA screen to identify mediators of prednisolone sensitivity in ALL cell lines. The incorporation of these data with an integrated analysis of relapse-specific genetic and epigenetic changes allowed us to identify the mitogen-activated protein kinase (MAPK) pathway as a mediator of prednisolone resistance in pediatric ALL. We show that knockdown of the specific MAPK pathway members MEK2 and MEK4 increased sensitivity to prednisolone through distinct mechanisms. MEK4 knockdown increased sensitivity specifically to prednisolone by increasing the levels of the glucocorticoid receptor. MEK2 knockdown increased sensitivity to all chemotherapy agents tested by increasing the levels of p53. Furthermore, we demonstrate that inhibition of MEK1/2 with trametinib increased sensitivity of ALL cells and primary samples to chemotherapy in vitro and in vivo. To confirm a role for MAPK signaling in patients with relapsed ALL, we measured the activation of the MEK1/2 target ERK in matched diagnosis-relapse primary samples and observed increased phosphorylated ERK levels at relapse. Furthermore, relapse samples have an enhanced response to MEK inhibition compared to matched diagnosis samples in xenograft models. Together, our data indicate that inhibition of the MAPK pathway increases chemosensitivity to glucocorticoids and possibly other agents and that the MAPK pathway is an attractive target for prevention and/or treatment of relapsed disease.

Recognition of the domestic dog as a model for the comparative study of human genetic traits has led to major advances in canine genomics. The pathophysiological similarities shared between many human and dog diseases extend to a range of cancers. Human tumors frequently display recurrent chromosome aberrations, many of which are hallmarks of particular tumor subtypes. Using a range of molecular cytogenetic techniques we have generated evidence indicating that this is also true of canine tumors. Detailed knowledge of these genomic abnormalities has the potential to aid diagnosis, prognosis, and the selection of appropriate therapy in both species. We recently improved the efficiency and resolution of canine cancer cytogenetics studies by developing a small-scale genomic microarray comprising a panel of canine BAC clones representing subgenomic regions of particular interest. We have now extended these studies to generate a comprehensive canine comparative genomic hybridization (CGH) array that comprises 1158 canine BAC clones ordered throughout the genome with an average interval of 2 Mb. Most of the clones (84.3%) have been assigned to a precise cytogenetic location by fluorescence in situ hybridization (FISH), and 98.5% are also directly anchored within the current canine genome assembly, permitting direct translation from cytogenetic aberration to DNA sequence. We are now using this resource routinely for high-throughput array CGH and single-locus probe analysis of a range of canine cancers. Here we provide examples of the varied applications of this resource to tumor cytogenetics, in combination with other molecular cytogenetic techniques.

We examined the expression of CD20 in normal canine peripheral blood mononuclear cells, normal canine spleen, and canine non-Hodgkin lymphoma (NHL) to determine the feasibility of using this antigen as a diagnostic aid and as a possible target for therapy. An antibody generated against a C-terminal (intracytoplasmic) epitope of human CD20 recognized proteins of 32-36 kd in normal and malignant canine lymphocytes. This antibody showed restricted membrane binding in a subset of lymphocytes in peripheral blood, in the B-cell regions from a normal canine spleen and lymph node, and in malignant cells from 19 dogs with B-cell NHL, but not from 15 dogs with T-cell NHL. The patterns of CD20 reactivity in these samples overlapped those seen using an antibody that recognizes canine CD79a. This anti-CD20 antibody is therefore suitable as an aid to phenotype canine NHL. In contrast, normal canine B cells were not recognized by any of 28 antibodies directed against the extracellular domains of human CD20 (including the chimeric mouse-human antibody Rituximab) or by any of 12 antibodies directed against the extracellular domains of mouse CD20. Thus, the use of CD20 as a therapeutic target will require the generation of specific antibodies against the extracellular domains of canine CD20.

We used primary peripheral blood T cells, a population that exists in G(0) and can be stimulated to enter the cell cycle synchronously, to define more precisely the effects of nicotine on pathways that control cell cycle entry and progression. Our data show that nicotine decreased the ability of T cells to transit through the G(0)/G(1) boundary (acquire competence) and respond to progression signals. These effects were due to nuclear factor of activated T cells c2 (NFATc2)-dependent repression of cyclin-dependent kinase 4 (CDK4) expression. Growth arrest at the G(0)/G(1) boundary was further enforced by inhibition of cyclin D2 expression and by increased expression and stabilization of p27Kip1. Intriguingly, T cells from habitual users of tobacco products and from NFATc2-deficient mice constitutively expressed CDK4 and were resistant to the antiproliferative effects of nicotine. These results indicate that nicotine impairs T cell cycle entry through NFATc2-dependent mechanisms and suggest that, in the face of chronic nicotine exposure, selection may favor cells that can evade these effects. We postulate that cross talk between nicotinic acetylcholine receptors and growth factor receptor-activated pathways offers a novel mechanism by which nicotine may directly impinge on cell cycle progression. This offers insight into possible reasons that underlie the unique effects of nicotine on distinct cell types and identifies new targets that may be useful control tobacco-related diseases.

We describe here a previously unrecognized property of dendritic cells (DCs), the ability to deacylate the lipid A moiety of gram-negative bacterial LPSs. Both immature DCs of the XS52 cell line and bone marrow–derived DCs produce acyloxyacyl hydrolase, an enzyme that detoxifies LPS by selectively removing the secondary acyl chains from lipid A. Acyloxyacyl hydrolase expression decreased when DCs were incubated with IL-4, IL-1β, TNFα, and an agonistic CD40 antibody (maturation cocktail), and increased after treatment with LPS, CpG oligodeoxynucleotides, or a gram-positive bacterium (Micococcus luteus). Maturation cocktail treatment also diminished, whereas LPS treatment enhanced or maintained the cells' ability to kill Escherichia coli, deacylate LPS, and degrade bacterial protein. Enzymatic deacylation of LPS is an intrinsic, regulated mechanism by which DCs may modulate host responses to this potent bacterial agonist.

Lymphohematopoietic malignancies are common spontaneous diseases of dogs whose clinical presentation and biologic behavior closely resemble their human counterparts. The goal of this study was to define the potential to use canine lymphoma and leukemia as suitable models to refine therapeutic approaches targeting the interleukin-2 receptor (IL-2R). The authors evaluated the patterns of IL-2R expression in 13 dogs with multicentric non-Hodgkin's lymphoma (NHL) and in six dogs with leukemia (acute lymphocytic leukemia, n = 3; chronic lymphocytic leukemia in blast crisis, n = 1; acute monoblastic leukemia, n = 2). The authors first cloned and sequenced the complete coding domains of the wild-type canine IL-2R alpha-chain gene. They next used qualitative reverse transcription polymerase chain reaction (RT-PCR) analysis to examine IL-2R alpha, beta, and gamma(c) subunit expression in the tumors. Messenger RNA (mRNA) for the interleukin-2 receptor alpha, beta, and gammac subunits that comprise the high-affinity receptor was present in samples from all dogs with NHL. Expression of functional surface IL-2R also was observed flow cytometrically in NHL cells from all four dogs tested. Leukemic cells from one dog with B cell acute lymphocytic leukemia and two dogs with acute monoblastic leukemia expressed mRNA for all three subunits, whereas cells from another dog with B cell leukemia and both dogs with T cell leukemia expressed only mRNA for the beta and gammac subunits that comprise the intermediate-affinity receptor. These results indicate that the IL-2R is commonly expressed in canine lymphohematopoietic malignancies, and support the suitability of this large-animal model to evaluate targeted IL-2R cancer therapy using approaches of interest in the treatment of humans with hemolymphatic cancers.

In the recent past, polo-like kinase (PLK1) has gained considerable attention as a potential therapeutic target in medulloblastoma. Volasertib (BI6727), highly specific and potent PLK1 inhibitor, is under clinical trials. However, volasertib exhibits varied clinical efficacies and only few patients respond well to single-agent volasertib. Therefore, we tested whether combination of BI6727 with other standard chemotherapy drugs could show promise in the treatment of highly aggressive Myc driven medulloblastoma. We evaluated the cellular effects of BI6727 in combination with other drugs in a panel of high and low cMyc expressing medulloblastoma cell lines. Of all the drugs tested, we found pairing the kinase inhibitor, BI6727, with the topoisomerase inhibitor, topotecan, showed synergistic effect in inhibiting tumor cell growth and enhanced tumor cell killing. Combination treatment resulted in a significant increase in apoptosis when compared with either drug alone. BI6727 in combination with topotecan induced both S and G2/M phase cell cycle arrest. In addition, BI6727 treatment caused a marked induction of tumor suppressor proteins, p53 and p21, when combined with topotecan. Interestingly we found that high-myc expressing medulloblastoma cell lines displayed increased sensitivity to BI6727 in combination with topotecan treatment when compared to low-myc cell lines. Using ChIP-PCR we found that Myc binds to the PLK1 promoter to induce expression of PLK1 and conversely volesertib suppresses Myc driven transcriptomic programs suggesting a positive feedback loop between PLK1 and Myc. In summary our study suggests that a combination, topotecan, and volasertib will synergize to potentiate cell death in high-Myc medulloblastoma patients.