Deepa Kushwaha

Targeting EGFR Induced Oxidative Stress by PARP1 Inhibition in Glioblastoma Therapy

Despite the critical role of Epidermal Growth Factor Receptor (EGFR) in glioblastoma pathogenesis [1], [2], EGFR targeted therapies have achieved limited clinical efficacy [3]. Here we propose an alternate therapeutic strategy based on the conceptual framework of non-oncogene addiction [4], [5]. A directed RNAi screen revealed that glioblastoma cells over-expressing EGFRvIII [6], an oncogenic variant of EGFR, become hyper-dependent on a variety of DNA repair genes. Among these, there was an enrichment of Base Excision Repair (BER) genes required for the repair of Reactive Oxygen Species (ROS)-induced DNA damage, including poly-ADP ribose polymerase 1 (PARP1). Subsequent studies revealed that EGFRvIII over-expression in glioblastoma cells caused increased levels of ROS, DNA strand break accumulation, and genome instability. In a panel of primary glioblastoma lines, sensitivity to PARP1 inhibition correlated with the levels of EGFR activation and oxidative stress. Gene expression analysis indicated that reduced expression of BER genes in glioblastomas with high EGFR expression correlated with improved patient survival. These observations suggest that oxidative stress secondary to EGFR hyper-activation necessitates increased cellular reliance on PARP1 mediated BER, and offer critical insights into clinical trial design.

Proteasome Inhibitors Block DNA Repair and Radiosensitize Non-Small Cell Lung Cancer

Despite optimal radiation therapy (RT), chemotherapy and/or surgery, a majority of patients with locally advanced non-small cell lung cancer (NSCLC) fail treatment. To identify novel gene targets for improved tumor control, we performed whole genome RNAi screens to identify knockdowns that most reproducibly increase NSCLC cytotoxicity. These screens identified several proteasome subunits among top hits, including the topmost hit PSMA1, a component of the core 20 S proteasome. Radiation and proteasome inhibition showed synergistic effects. Proteasome inhibition resulted in an 80–90% decrease in homologous recombination (HR), a 50% decrease in expression of NF-κB-inducible HR genes BRCA1 and FANCD2, and a reduction of BRCA1, FANCD2 and RAD51 ionizing radiation-induced foci. IκBα RNAi knockdown rescued NSCLC radioresistance. Irradiation of mice with NCI-H460 xenografts after inducible PSMA1 shRNA knockdown markedly increased murine survival compared to either treatment alone. Proteasome inhibition is a promising strategy for NSCLC radiosensitization via inhibition of NF-κB-mediated expression of Fanconi Anemia/HR DNA repair genes.

Dynamic epigenetic regulation of glioblastoma tumorigenicity through LSD1 modulation of MYC expression

Significance Glioblastoma is the most common type of adult brain cancer, with near-uniform fatality within 2 y of diagnosis. Therapeutic failure is thought to be related to small subpopulations of cells that exhibit tumorigenicity, the cellular capacity to reconstitute the entire tumor mass. One fundamental issue is whether tumorigenicity exists within a static subpopulation of cells or whether the capacity is stochastically acquired. We provide evidence that tumorigenicity is a cellular property that is durable yet undergoes low-frequency stochastic changes. We showed that these changes are driven by lysine-specific demethylase 1 (LSD1)-mediated epigenetic (heritable non-DNA sequence-altering) modifications that impact expression of key transcription factors, which in turn govern transitions between tumorigenic states. These findings harbor implications for glioblastoma therapeutic development. The available evidence suggests that the lethality of glioblastoma is driven by small subpopulations of cells that self-renew and exhibit tumorigenicity. It remains unclear whether tumorigenicity exists as a static property of a few cells or as a dynamically acquired property. We used tumor-sphere and xenograft formation as assays for tumorigenicity and examined subclones isolated from established and primary glioblastoma lines. Our results indicate that glioblastoma tumorigenicity is largely deterministic, yet the property can be acquired spontaneously at low frequencies. Further, these dynamic transitions are governed by epigenetic reprogramming through the lysine-specific demethylase 1 (LSD1). LSD depletion increases trimethylation of histone 3 lysine 4 at the avian myelocytomatosis viral oncogene homolog (MYC) locus, which elevates MYC expression. MYC, in turn, regulates oligodendrocyte lineage transcription factor 2 (OLIG2), SRY (sex determining region Y)-box 2 (SOX2), and POU class 3 homeobox 2 (POU3F2), a core set of transcription factors required for reprogramming glioblastoma cells into stem-like states. Our model suggests epigenetic regulation of key transcription factors governs transitions between tumorigenic states and provides a framework for glioblastoma therapeutic development.

USP9X inhibition promotes radiation-induced apoptosis in non-small cell lung cancer cells expressing mid-to-high MCL1

Background and Purpose: Radiotherapy (RT) is vital for the treatment of locally advanced non-small cell lung cancer (NSCLC), yet its delivery is limited by tolerances of adjacent organs. We sought therefore to identify and characterize gene targets whose inhibition may improve RT. Materials and Methods: Whole genome pooled shRNA cytotoxicity screens were performed in A549 and NCI-H460 using a retroviral library of 74,705 sequences. Cells were propagated with or without daily radiation Monday–Friday. Radiosensitization by top differential dropout hits was assessed by clonogenic assays. Apoptosis was assessed using a caspase 3/7 cell-based activity assay and by annexin V-FITC and PI staining. MCL1 expression was assessed by qPCR and Western blotting. Results: USP9X, a deubiquitinase, was a top hit among druggable gene products. WP1130, a small molecule USP9X inhibitor, showed synergistic cytotoxicity with IR. MCL1, an anti-apoptotic protein deubiquitinated by USP9X, decreased with USP9X inhibition and IR. This was accompanied by increases in caspase 3/7 activity and apoptosis. In a panel of NSCLC lines, MCL1 and USP9X protein and gene expression levels were highly correlated. Lines showing high levels of MCL1 expression were the most sensitive to USP9X inhibition. Conclusions: These data support the use of MCL1 expression as a predictive biomarker for USP9X inhibitors in NSCLC therapy.

Abstract 979: Dynamic epigenetic regulation of glioblastoma tumorigenicity through LSD1 modulation of MYC expression

Glioblastoma is the most common form of primary brain cancer and remains one of the deadliest of human cancers with near-uniform fatality. Increasing evidence suggests that the lethality of glioblastoma is driven by small subpopulations of cells with self-renew ability and tumorigenicity, termed as tumor initiating cells. The mechanism how the tumor initiating cells maintain and gain tumorigenicity in glioblastoma still remains unclear. Here, we used sphere formation and tumor propagating potential to measure the tumorigenicity in established cell line and primary glioblastoma cells. The results indicated that glioblastoma tumorigenicity appears largely deterministic, though spontaneous gain and loss of this property occur at low frequency. Mechanically, this dynamic transition in tumorigenicity was governed by MYC level which was modulated epigenetically by the lysine-specific demethylase 1 (LSD1). Elevated MYC expression, in turn, regulates OLIG2, SOX2 and POU3F2, a core set of transcription factors required for reprogramming glioblastoma cells into stem-like states. Our model suggests epigenetic regulation of key transcription factors facilitates transitions between tumorigenic states and provides a framework for glioblastoma therapeutic development. Importantly, the effect of LSD1 on tumorigenity is “Janus”-like; partial depletion of LSD1 caused increased MYC expression and a pro-tumorigenic state. In contrast, complete suppression of LSD1 induced cell death. As such, therapeutic strategies targeting LSD1 and other targets manifesting similar “Janus” effect should be designed to prevent unintended induction of tumorigenesis during treatment. Citation Format: Jie Li, David Kozono, Masayuki Nitta, Oltea Sampetrean, David Gonda, Deepa S. Kushwaha, Dmitry Merzon, Valya Ramakrishnan, Shan Zhu, Kaya Zhu, Hiroko Matsui, Olivier Harismendy, Wei Hua, Ying Mao, Chang-Hyuk Kwon, Hideyuki Saya, Bob S. Carter, Donald P. Pizzo, Scott R. VandenBerg, Clark C. Chen. Dynamic epigenetic regulation of glioblastoma tumorigenicity through LSD1 modulation of MYC expression. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 979. doi:10.1158/1538-7445.AM2015-979

Abstract PR02: Dynamic epigenetic regulation of glioblastoma tumorigenicity through a LSD1-MYC-OLIG2 axis

Glioblastoma is one of the most devastating of human cancers, with near-uniform fatality within two years of diagnosis. Therapeutic failure is thought to be related to small subpopulation of cells that exhibit the properties of self-renewal and tumorigenicity. Understanding how such subpopulations attain and retain these properties remains a central question in oncology. One fundamental issue is whether tumorigenicity exists within a static population of elite cells or whether the capacity is stochastically acquired. To test these models, we assayed the tumorigenicity of single-cell subclones derived from long-terms passaged and primary patient-derived xenograft (PDX) glioblastoma lines. Our findings were best described by a hybrid model that is largely deterministic (elite) but with opportunities for dynamic (stochastic) interchange between non-tumorigenic and tumorigenic states. To identify molecular determinants of tumorigenicity, we performed gene expression profiling of the subclones. Analysis of the data suggested that tumorigenicity in glioblastoma is a dynamic property driven by variation in MYC expression, which in turn regulates Olig2 expression, a neural stem cell marker. Ectopic expression of MYC conferred tumorigenicty and MYC silencing abolished tumorigenicity in vitro and in vivo for multiple PDX and GEMM models. Transition between tumorigenic and non-tumorigenic cell states was associated with changes in histone modification at the MYC locus mediated by expression of lysine-specific demethylase 1 (LSD1). The model suggests a critical LSD1-MYC-OLIG2 axis that regulates the dynamic transition between glioblastoma cell states of differing tumorigenicity and unveils a novel framework for glioblastoma therapeutic development. Citation Format: Clark Chen, David Kozono, Jie Li, Masayuki Nitta, Oltea Sampetrean, Kimberly Ng, David Gonda, Deepa S. Kushwaha, Dmitry Merzon, Valya Ramakrishnan, Shan Zhu, Kaya Zhu, Hiroko Matsui, Olivier Harismendy, Wei Hua, Ying Mao, Chang-Hyuk Kwon, Keith L. Ligon, Hideyuki Saya, Bob S. Carter, Donald P. Pizzo, Scott R. VandenBerg, Frank Furnari, Webster Cavenee. Dynamic epigenetic regulation of glioblastoma tumorigenicity through a LSD1-MYC-OLIG2 axis. [abstract]. In: Proceedings of the AACR Special Conference on Myc: From Biology to Therapy; Jan 7-10, 2015; La Jolla, CA. Philadelphia (PA): AACR; Mol Cancer Res 2015;13(10 Suppl):Abstract nr PR02.

Abstract PR13: Epigenetic regulation of MYC drives dynamic transition between tumor initiating states in glioblastoma

Glioblastoma is the most common form of brain cancer and remains a devastating disease. Recent studies revealed significant intra-tumoral heterogeneity in the genetic and epigenetic make-up of glioblastomas. One level of heterogeneity involves subpopulations of cells capable of tumor initiation (TI). Here we provide data suggesting that the transition between TI- and non-TI is a dynamic process governed by spontaneous fluctuation in the level of MYC. In vitro culturing of sub-clones derived from long-term passaged and primary glioblastoma lines revealed that only a subset of the sub-clones possess TI capacity. The property of TI for each individual sub-clone appeared stable through serial passages. However, a small fraction of the non-TI clones will spontaneously acquire the TI ability. This phenomenon was observed both in vitro and in vivo. Transcriptome profiling of the sub-clones with varied TI capacity revealed a gene signature enriched for genes regulated by MYC. Among various sub-clones, MYC expression levels correlated with TI capacity tightly. More direct evidence was provided by MYC overexpression which augmented TI capacity in both xenograft and genetic murine models. Reversely, MYC silencing abolished glioblastoma capacity for TI. Importantly, the sub-clones that spontaneously acquired capacity exhibited enhanced MYC expression. In freshly resected glioblastoma specimens, overall MYC expression levels of the specimens correlated with their xenograft-forming ability. When these specimens were sub-fractionated by A2B5, a cell surface marker enriched in TI glioblastoma populations, the MYC level was significantly elevated in the A2B5+ fraction relative to the A2B5- fraction. In The Cancer Genome Atlas (TCGA) glioblastoma specimens, the expression level of a MYC signature directly correlated with mRNA signatures associated with the Cancer Stem Cell states. In dual immunofluorescence staining of clinical glioblastoma specimens, C-MYC co-stained with MIB1, suggesting that MYC expression support in vivo tumor proliferation. Since the various sub-clones were genetically identical based on SNP array profiling, we hypothesized that fluctuations in MYC expression was regulated through epigenetic regulation. Supporting our hypothesis, the primary glioblastoma tumor lines that demonstrated high MYC levels showed higher ratios of H3K4me3 to H3K27me3 at the MYC locus. Culturing conditions that enhanced TI capacity of glioblastoma cell lines also increased the ratio of H3K4me3 to H3K27me3 and induced MYC expression. In sum, our results suggest a threshold model in which TI capacity is driven by epigenetic regulation of MYC. MYC inhibition constitutes an attractive therapeutic target since this inhibition reduces dynamic cell state transition and reduce the complexity of the tumor heterogeneity. This abstract is also presented as Poster B75. Citation Format: David Kozono, Jie Li, Masayuki Nitta, Oltea Sampetrean, Kimberly Ng, David Gonda, Deepa Kushwaha, Matsui Hiroko, Olivier Harismendy, Oren Becher, Chang-Hyuk Kwon, Keith L. Ligon, Hideyuki Saya, Bob S. Carter, Donald Pizzo, Scott Vandenberg, Clark C. Chen. Epigenetic regulation of MYC drives dynamic transition between tumor initiating states in glioblastoma. [abstract]. In: Abstracts: AACR Special Conference on Cellular Heterogeneity in the Tumor Microenvironment; 2014 Feb 26-Mar 1; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2015;75(1 Suppl):Abstract nr PR13. doi:10.1158/1538-7445.CHTME14-PR13

Abstract 4366: ShRNA-based cellular proliferation signaling analysis revealed DRD2 as a novel therapeutic target for glioblastoma.

Glioblastoma remains one of the deadliest of human cancers, with most patients succumbing to the disease within one year of diagnosis. Our best current understanding of its etiology is that glioblastoma involves genetic and epigenetic changes that subvert the inherent molecular circuitry of glial cells to drive uncontrolled cellular proliferation. Unfortunately, this molecular circuitry in glioblastoma appears sufficiently complex and redundant that disruption at any particular point tends to redirects signaling through the remaining circuit. In this context, meaningful therapeutic gains will require the identification of therapeutic agents that target critical nodes within this circuit. To identify novel therapeutic agents that target critical nodes which regulate cellular proliferation, a genome-wide retroviral shRNA library screen was conducted in two glioblastoma cell lines (U87MG and A172) and two lung carcinoma lines (A549 and NCI-H460). Pathway analysis of the pro-proliferative genes using both PANTHER and Ingenuity software revealed an over-representation of G-protein coupled neurotransmitter receptors (GPCR), including dopamine receptor subtype 2 (DRD2), which was also highly expressed in glioblastoma specimens relative to the matched normal parentchymal. Further investigation demonstrated that DRD2 signal regulates cell growth by GNAI2-induced ERK activation. Moreover, Haloperidol, one of DRD2 antagonists, enhances significantly anti-tumor efficacy of EGFR inhibitor AG1478 using U87 and mesenchymal neuroshpere xenografy mouse models. Given that both AG1478 and DRD2 antagonists have been approved for the treatment of glioblastoma and psychotics, separtately, Haloperidol combined with chemotherapy represents a novel and likely conducted strategy for the treatment of glioblastoma. Citation Format: Jie Li, Shan Zhu, David Kozono, Diahnn Futulan, David Gonda, Deepa Kushwaha, Kristopher Kahle, Stephen Elledge, Clark C. Chen. ShRNA-based cellular proliferation signaling analysis revealed DRD2 as a novel therapeutic target for glioblastoma. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4366. doi:10.1158/1538-7445.AM2013-4366

Abstract 1583: Proteasome inhibition as a strategy for non-small cell lung carcinoma via inhibition of DNA double strand break repair.

Background: Each year, ∼60,000 US patients are diagnosed with locally advanced non-small cell lung carcinoma (NSCLC). Despite optimal radiation therapy (RT), chemotherapy +/- surgery, over 30% of patients fails locoregionally. The addition of concurrent chemotherapy to RT adds only ∼5% absolute survival benefit. Novel radiosensitizers may improve the therapeutic index. To identify the best potential targets, we performed whole genome RNAi screens that identified several proteasome subunits among top genes whose knockdown increased NSCLC cytotoxicity. Materials and Methods: Cytotoxicity was assessed by luminescent cell viability and clonogenic assays. Homologous recombination (HR) and non-homologous end-joining (NHEJ) were assessed using NSCLC cell lines transduced with reporter constructs that express GFP upon repair of I-SceI-induced DNA double strand breaks (DSBs). DNA damage-induced focus formation was assessed by immunofluorescence, scoring % of cells showing ≥5 foci. In vivo radiosensitization by proteasome gene knockdown was assessed using NCr nude mice injected with 1x106 NCI-H460 cells stably transfected with inducible PSMA1 shRNA. Once tumors reached ≥3 mm, knockdown was induced with doxycycline, and then one week later, RT to a dose of 20 Gy in 5 fractions was initiated. Results: Radiation and proteasome inhibition showed synergistic cytotoxicity. Irradiation of A549 cells with 1 Gy x 3 decreased clonogenic survival by 58% compared to control, while ionizing radiation (IR) plus 10 nM bortezomib decreased survival by 74% compared to bortezomib alone. Similar results were seen in NCI-H460. Proteasome inhibition via bortezomib or PSMA1 siRNA knockdown resulted in 80-90% decreased HR and NHEJ. Additionally, bortezomib or PSMA1 shRNA knockdown resulted in ≥50% decreases in BRCA1, FANCD2 or RAD51 IR-induced focus formation. Treatment of NCI-H460 xenografts with RT in the setting of PSMA1 knockdown showed marked improvements in survival; at 100 days post treatment initiation, only 20% of RT-only treated mice and 30% of doxycycline-treated mice (for PSMA1 shRNA knockdown in tumors) survived, compared to 100% following dual treatment (n = 10 per arm). There was a statistically significant difference in survival between mice with tumors treated with RT alone vs. RT + PSMA1 knockdown, with median survivals of 43 days vs. not reached, P = .0003 by the log-rank test. Conclusions: Proteasome inhibition has emerged as a promising target in NSCLC radiosensitization with evidence indicating the mechanism is through inhibition of DNA DSB repair pathways HR and NHEJ. Citation Format: Kyle R. Cron, Kaya Zhu, Deepa Kushwaha, Grace Hsieh, Dmitry Merzon, Jack Monahan, Clark C. Chen, Alan D9Andrea, David Kozono. Proteasome inhibition as a strategy for non-small cell lung carcinoma via inhibition of DNA double strand break repair. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1583. doi:10.1158/1538-7445.AM2013-1583

Abstract 5218: A c-Myc driven threshold model of tumor initiating in glioblastoma

Recent evidence suggests that glioblastoma is driven by a subset of Tumor Initiating (TI) cells characterized by their capacity to form tumors in xenograft models and self-renew in vitro. These TI cells share many properties of neural stem/progenitor cells, including the expression of certain cell surface markers. With serial passage, many cells lose their capacity to TI. The transition between TI proficient and deficient states remains poorly understood. There are two theoretic models for the maintenance of TI states. In the “elite” model, TI activity is restricted to a predetermined subpopulation of cells. The alternative “stochastic” model suggests that any tumor cell has a finite chance of acquiring TI capacity through random fluctuations in cell physiology. To distinguish between these two models, we established culturing conditions capable of sustaining a single cell line in both low and high TI states. For both primary and long-term passaged glioblastoma lines, culturing under sphere forming conditions lead to increased in vivo tumorigenicity relative to serum based conditions. Further, cells cultured under sphere forming conditions exhibited increased expression of neural stem cell markers, including Nestin, Musashi, Olig2, and Sox2. Moreover, the ability of individual subclones to form xenografts closely correlated with their ability to grow under sphere forming conditions. In this context, the “elite” model predicts that only a subset of subclones (derived from a single line) cultured under serum conditions would possess high TI capacity and grow under sphere forming conditions. Moreover, individual cells derived from the same subclone would exhibit similar capacity for growth under sphere forming conditions. Surprisingly, single cells derived from a single glioblastoma subclone exhibited a wide range of growth capacity under sphere forming conditions, suggesting the TI state is in part driven by a stochastic process. On the other hand, only a subset of subclones from a single glioblastoma line displayed capacity for growth under sphere forming conditions, as predicted by the elite model. Transcriptome profiling of the different subclones revealed a gene signature associated with TI capacity. Analysis of this signature showed enrichment for genes regulated by c-Myc. Indeed, clones with increased TI capacity tend to harbor increased c-Myc expression. Additionally, over-expression of c-myc increased the TI capacity of glioblastoma cells in xenograft models and led to the formation of malignant brain tumor in an Ink4a/ARF null transgenic murine model. Finally, analysis of The Cancer Genome Atlas Project (TCGA) glioblastoma database revealed c-Myc over-expression in glioblastoma cells, particularly in the pro-neural and mesenchymal subtypes. Our results are most consistent with a threshold model in which TI states in glioblastomas are driven by expression levels of critical factors such as c-Myc. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5218. doi:1538-7445.AM2012-5218