Uday Bhanu Maachani

Targeting MPS1 Enhances Radiosensitization of Human Glioblastoma by Modulating DNA Repair Proteins

To ensure faithful chromosome segregation, cells use the spindle assembly checkpoint (SAC), which can be activated in aneuploid cancer cells. Targeting the components of SAC machinery required for the growth of aneuploid cells may offer a cancer cell–specific therapeutic approach. In this study, the effects of inhibiting Monopolar spindle 1, MPS1 (TTK), an essential SAC kinase, on the radiosensitization of glioblastoma (GBM) cells were analyzed. Clonogenic survival was used to determine the effects of the MPS1 inhibitor NMS-P715 on radiosensitivity in multiple model systems, including GBM cell lines, a normal astrocyte, and a normal fibroblast cell line. DNA double-strand breaks (DSB) were evaluated using γH2AX foci, and cell death was measured by mitotic catastrophe evaluation. Transcriptome analysis was performed via unbiased microarray expression profiling. Tumor xenografts grown from GBM cells were used in tumor growth delay studies. Inhibition of MPS1 activity resulted in reduced GBM cell proliferation. Furthermore, NMS-P715 enhanced the radiosensitivity of GBM cells by decreased repair of DSBs and induction of postradiation mitotic catastrophe. NMS-P715 in combination with fractionated doses of radiation significantly enhanced the tumor growth delay. Molecular profiling of MPS1-silenced GBM cells showed an altered expression of transcripts associated with DNA damage, repair, and replication, including the DNA-dependent protein kinase (PRKDC/DNAPK). Next, inhibition of MPS1 blocked two important DNA repair pathways. In conclusion, these results not only highlight a role for MPS1 kinase in DNA repair and as prognostic marker but also indicate it as a viable option in glioblastoma therapy. Implications: Inhibition of MPS1 kinase in combination with radiation represents a promising new approach for glioblastoma and for other cancer therapies. Mol Cancer Res; 13(5); 852–62. ©2015 AACR.

FOXM1 and STAT3 interaction confers radioresistance in glioblastoma cells

Glioblastoma multiforme (GBM) continues to be the most frequently diagnosed and lethal primary brain tumor. Adjuvant chemo-radiotherapy remains the standard of care following surgical resection. In this study, using reverse phase protein arrays (RPPAs), we assessed the biological effects of radiation on signaling pathways to identify potential radiosensitizing molecular targets. We identified subsets of proteins with clearly concordant/discordant behavior between irradiated and non-irradiated GBM cells in vitro and in vivo. Moreover, we observed high expression of Forkhead box protein M1 (FOXM1) in irradiated GBM cells both in vitro and in vivo. Recent evidence of FOXM1 as a master regulator of metastasis and its important role in maintaining neural, progenitor, and GBM stem cells, intrigued us to validate it as a radiosensitizing target. Here we show that FOXM1 inhibition radiosensitizes GBM cells by abrogating genes associated with cell cycle progression and DNA repair, suggesting its role in cellular response to radiation. Further, we demonstrate that radiation induced stimulation of FOXM1 expression is dependent on STAT3 activation. Co-immunoprecipitation and co-localization assays revealed physical interaction of FOXM1 with phosphorylated STAT3 under radiation treatment. In conclusion, we hypothesize that FOXM1 regulates radioresistance via STAT3 in GBM cells. We also, show GBM patients with high FOXM1 expression have poor prognosis. Collectively our observations might open novel opportunities for targeting FOXM1 for effective GBM therapy.

Dual Inhibition of PI3K/AKT and MEK/ERK Pathways Induces Synergistic Antitumor Effects in Diffuse Intrinsic Pontine Glioma Cells

Diffuse intrinsic pontine glioma (DIPG) is a devastating disease with an extremely poor prognosis. Recent studies have shown that platelet-derived growth factor receptor (PDGFR) and its downstream effector pathway, PI3K/AKT/mTOR, are frequently amplified in DIPG, and potential therapies targeting this pathway have emerged. However, the addition of targeted single agents has not been found to improve clinical outcomes in DIPG, and targeting this pathway alone has produced insufficient clinical responses in multiple malignancies investigated, including lung, endometrial, and bladder cancers. Acquired resistance also seems inevitable. Activation of the Ras/Raf/MEK/ERK pathway, which shares many nodes of cross talk with the PI3K/AKT pathway, has been implicated in the development of resistance. In the present study, perifosine, a PI3K/AKT pathway inhibitor, and trametinib, a MEK inhibitor, were combined, and their therapeutic efficacy on DIPG cells was assessed. Growth delay assays were performed with each drug individually or in combination. Here, we show that dual inhibition of PI3K/AKT and MEK/ERK pathways synergistically reduced cell viability. We also reveal that trametinib induced AKT phosphorylation in DIPG cells that could not be effectively attenuated by the addition of perifosine, likely due to the activation of other compensatory mechanisms. The synergistic reduction in cell viability was through the pronounced induction of apoptosis, with some effect from cell cycle arrest. We conclude that the concurrent inhibition of the PI3K/AKT and MEK/ERK pathways may be a potential therapeutic strategy for DIPG.

Advances in Molecular Imaging of Locally Delivered Targeted Therapeutics for Central Nervous System Tumors

Thanks to the recent advances in the development of chemotherapeutics, the morbidity and mortality of many cancers has decreased significantly. However, compared to oncology in general, the field of neuro-oncology has lagged behind. While new molecularly targeted chemotherapeutics have emerged, the impermeability of the blood–brain barrier (BBB) renders systemic delivery of these clinical agents suboptimal. To circumvent the BBB, novel routes of administration are being applied in the clinic, ranging from intra-arterial infusion and direct infusion into the target tissue (convection enhanced delivery (CED)) to the use of focused ultrasound to temporarily disrupt the BBB. However, the current system depends on a “wait-and-see” approach, whereby drug delivery is deemed successful only when a specific clinical outcome is observed. The shortcomings of this approach are evident, as a failed delivery that needs immediate refinement cannot be observed and corrected. In response to this problem, new theranostic agents, compounds with both imaging and therapeutic potential, are being developed, paving the way for improved and monitored delivery to central nervous system (CNS) malignancies. In this review, we focus on the advances and the challenges to improve early cancer detection, selection of targeted therapy, and evaluation of therapeutic efficacy, brought forth by the development of these new agents.

Biomarker based PET Imaging of Diffuse Intrinsic Pontine Glioma in Mouse Models.

Diffuse intrinsic pontine glioma (DIPG) is a childhood brainstem tumor with a universally poor prognosis. Here, we characterize a positron emission tomography (PET) probe for imaging DIPG in vivo In human histological tissues, the probes target, PARP1, was highly expressed in DIPG compared to normal brain. PET imaging allowed for the sensitive detection of DIPG in a genetically engineered mouse model, and probe uptake correlated to histologically determined tumor infiltration. Imaging with the sister fluorescence agent revealed that uptake was confined to proliferating, PARP1-expressing cells. Comparison with other imaging technologies revealed remarkable accuracy of our biomarker approach. We subsequently demonstrated that serial imaging of DIPG in mouse models enables monitoring of tumor growth, as shown in modeling of tumor progression. Overall, this validated method for quantifying DIPG burden would serve useful in monitoring treatment response in early phase clinical trials. Cancer Res; 77(8); 2112-23. ©2017 AACR.

Abstract 190: Modulation of MiR-21 signaling by MPS1 in human glioblastoma

Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PA Monopolar spindle 1 (MPS1) is an essential spindle assembly checkpoint (SAC) kinase involved in determining spindle integrity. Beyond its mitotic functions, it has been implicated in several other signaling pathways. Its overexpression in cancer cells is known to contribute to genomic instability and tumorigenesis. Our earlier studies have elaborated on role of MPS1 in glioblastoma (GBM) radiosensitization. In this study using reverse phase protein arrays (RPPA), we assessed MPS1 mediated cell signaling pathways and demonstrated that inhibiting MPS1 could upregulate the expression of the tumor suppressor genes PDCD4 and MSH2 by downregulating micro RNA-21 (MiR-21). In GBMs MiR-21 expression is significantly elevated and is associated with chemo and radioresistance. Here, we show that MPS1 inhibition significantly repressed MiR-21 levels and elevated PDCD4 and MSH2 expression in GBM cells both in vitro and in vivo. Both MPS1 and MiR-21 depletion suppressed GBM cell proliferation, whereas, ectopic expression of MiR-21 rescued GBM cell growth from MPS1 inhibition. Further, we demonstrate that MPS1 mediates phosphorylation of SMAD3 but not SMAD2 in GBM cells; A possible mechanism behind MiR-21 modulation by MPS1. Collectively, our results shed light onto an important role of MPS1 in TGF-β/SMAD signaling via MiR-21 regulation. We also, evaluated the prognostic effect of MiR-21, PDCD4 and MSH2 expression on survival in GBM patients of different molecular subtypes from The Cancer Genome Atlas (TCGA) database, showing tumors with high MiR-21 expression and low PDCD4 and MSH2 expression were associated with significantly shorter survival times. To our knowledge this is the first report to demonstrate the role of MPS1 in MiR-21 modulation. This scenario in which MiR-21 is modulated by MPS1 inhibition may be exploited as a potential target for effective GBM therapy. Citation Format: Uday Bhanu Maachani, Anita Tandle, Uma Shankavaram, Tamalee Kramp, Kevin A. Camphausen. Modulation of MiR-21 signaling by MPS1 in human glioblastoma. [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 190. doi:10.1158/1538-7445.AM2015-190

18F-Radiolabeled Panobinostat Allows for Positron Emission Tomography Guided Delivery of a Histone Deacetylase Inhibitor

Histone deacetylase (HDAC) inhibition is becoming an increasingly popular approach to treat cancer, as HDAC overexpression is common in many malignancies. The blood-brain barrier (BBB) prevents systemically delivered drugs from reaching brain at effective concentration, making small-molecule-HDAC inhibition in brain tumors particularly challenging. To circumvent the BBB, novel routes for administering therapeutics are being considered in the clinic, and a need exists for drugs whose deliveries can be directly imaged, so that effective delivery across the BBB can be monitored. We report chemistry for radiolabeling the HDAC inhibitor, panobinostat, with fluoride-18 (compound-1). Like panobinostat, compound 1 retains nanomolar efficacy in diffuse intrinsic pontine glioma (DIPG IV and XIII) cells (IC50 = 122 and 108 nM, respectively), with lesser activity against U87 glioma. With a favorable therapeutic ratio, 1 is highly selective to glioma and demonstrates considerably less toxicity toward healthy astrocyte controls (IC50 = 5265 nM). Compound 1 is stable in aqueous solution at physiological pH (>7 days, fetal bovine serum), and its delivery can be imaged by positron emission tomography (PET). Compound 1 is synthesized in two steps, and employs rapid, late-stage aqueous isotopic exchange 18F-radiochemistry. PET is used to image the in vivo delivery of [18F]-1 to the murine central nervous system via convection enhanced delivery.

Abstract 2986: Targeting multiple nodes in the RTK- PI3K/AKT/mTOR signaling pathway in a p53-/- mouse model of DIPG induces G2/M phase cell cycle arrest

Introduction: Diffuse intrinsic pontine glioma (DIPG) is the most common pediatric brainstem tumor, but its prognosis is dismal, with a median survival time of less than one year. Prior studies have implicated amplifications in the receptor tyrosine kinase (RTK)-PI3K/AKT/mTOR signaling pathway in DIPG gliomagenesis, and platelet-derived growth factor receptor (PDGFR) is the most commonly over-expressed RTK. One treatment strategy is thus to inhibit kinases in this pathway with drugs such as dasatinib (PDGFR inhibitor), perifosine (AKT inhibitor), and everolimus (mTOR inhibitor). In this study, we aim to show that combinatorial therapy with dasatinib, perifosine, and everolimus is more effective in impeding tumor cell growth than each drug individually. Methods: Mouse brainstem glioma cells (mBSG) were derived from a transgenic mouse model of DIPG driven by PDGFB overexpression and p53 loss. Cells were treated for 72 hours with dasatinib, perifosine, and everolimus individually and in combination, and cell viability was assayed with MTS. Western blot with cleaved-caspase 3 antibody was used to assess apoptosis. Cell cycle analysis was performed by propidium iodide flow cytometry. Results: GI50 concentrations of each individual drug were as follows: 50 nM dasatinib, 50 μM perifosine, and 10 μM everolimus. With combined dasatinib and perifosine treatment, 31.6% of cells survived (p = 0.016 vs. dasatinib alone). The addition of everolimus to dasatinib and perifosine further reduced cell survival to 27.0% (p = 0.011 vs. combinatorial treatment with dasatinib and perifosine). Cell cycle analysis revealed that dasatinib treatment alone prominently arrested cells in G0/G1 phase while both 2-drug combinatorial treatment with dasatinib and perifosine and 3-drug combinatorial treatment with dasatinib, perifosine, and everolimus caused substantial cell cycle arrest in the G0/G1 and G2/M phases. However, Western blot analysis found that no drug treatment group effectively induced apoptosis. Conclusion: Three-drug combinatorial therapy with dasatinib, perifosine, and everolimus was more effective in reducing DIPG cell viability than dasatinib alone, primarily through the induction of cell cycle arrest at G0/G1 and G2/M. This effect and the lack of apoptosis is consistent with previous observations that inhibition of the PI3K/AKT/mTOR signaling pathway is cytostatic rather than cytotoxic. Moreover, mutations in p53 are seen in up to 50% of patients with DIPG, which allow tumor cells to evade apoptosis despite treatment with chemotherapeutic agents. Taken together, targeting the PI3K/AKT/mTOR pathway alone appears to be insufficient. Ongoing studies aim to understand resistance mechanisms to inhibitors of this pathway and to improve therapeutic efficacy in DIPG by identifying potential synergistic drug combinations targeting alternate survival pathways. Citation Format: Yue Linda Wu, Uday Bhanu Maachani, Melanie Schweitzer, Oren J. Becher, Melinda Wang, Ranjodh Singh, Zhiping Zhou, Mark M. Souweidane. Targeting multiple nodes in the RTK- PI3K/AKT/mTOR signaling pathway in a p53-/- mouse model of DIPG induces G2/M phase cell cycle arrest. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2986.

RT-18TARGETING MPS1 ENHANCES RADIOSENSITIZATION OF HUMAN GLIOBLASTOMA BY MODULATING DNA REPAIR PROTEINS

During the cell cycle, genomic stability requires accurate chromosome segregation. Errors in this process can cause aneuploidy, leading to tumorigenesis. To ensure faithful chromosome segregation, cells use the spindle assembly checkpoint (SAC) mechanism. However, in aneuploid cancer cells components of the SAC machinery are frequently altered. Thus, targeting the components of SAC machinery required for the growth of aneuploid cells may thus offer a cancer cell specific therapeutic approach. Monopolar spindle 1 (MPS1) is an essential SAC kinase involved in determining spindle integrity. MPS1 is overexpressed in a wide range of tumors and is required for tumor cell proliferation; there is thus increased interest in targeting MPS1 for cancer treatment. In this study, we have analyzed the mechanistic basis of the effects of inhibiting MPS1 in glioblastoma multiforme (GBM). We show that the inhibition of MPS1 in conjunction with radiation reduces GBM cell growth, and clonogenic survival and that this is associated with mitotic cell arrest and induction of mitotic catastrophe. MNS-P715 not alone, but along with fractionated doses of radiation significantly enhanced the tumor growth delay. Using gene expression profiling of GBM cells in which MPS1 was silenced we identified altered expression of genes associated with DNA damage, DNA repair, and DNA replication, including PRKDC, the DNA-dependent protein kinase (DNAPK). Inhibition of MPS1 blocked two important DNA repair pathways, non-homologous end joining and homologous recombination. To our knowledge, this is the first report demonstrating a role for MPS1 in DNA repair. Data mining of publically available databases showed the clinical relevance of MPS1 as an important cancer target in GBM. We conclude that inhibiting MPS1 kinase in combination with DNA damage, including irradiation, could represent a promising new approach to cancer therapy.

Abstract 849: Profiling signaling networks using reverse phase protein arrays: validating FOXM1 as a potential target to radiosensitize glioblastoma (GBM) stem cells

Glioblastoma multiforme (GBM) continues to be the most frequently diagnosed and lethal primary brain tumor. Adjuvant chemo-radiotherapy remains the standard of care following surgical resection. In this study, using reverse phase protein arrays (RPPAs), we assessed the biological effects of radiation on signaling pathways to identify potential radiosensitizing molecular targets. We examined levels of 172 phosphorylated and non-phosphorylated proteins under conditions of Ionizing radiation (IR) in patient derived GBM stem cells and established U251, U87 GBM cell lines in vitro and in an in vivo orthotropic mouse model. We identified subsets of proteins with clearly concordant/discordant behavior between GBM cells in vitro and in vivo. In general, molecules involved in anti-apoptotic, cell-cycle, survival pathways, tumor metastasis and DNA repair were affected. Comparing in vivo and in vitro samples after IR, 9 proteins were commonly elevated; phospho(p)-STAT3, CDC2, CyclinB1, BAX, pEIF4BP1, pAKT, pRB, pMEK1, and FOXM1. Conversely, 4 other proteins were commonly decreased; pPRKCA, pPRKCD, pNDRG1 and pRPS6. Recent evidence of FOXM1 as a master regulator of metastasis and its important role in maintaining neural, progenitor, and GBM stem cells intrigued us to validate it as a radiosensitizing target. We show high expression of FOXM1 across different patient derived stem cells. When GBM stem cells (NSC11, GBAM1) were differentiated in serum, we observed a decrease in FOXM1 levels, attaining more differentiation markers. In both differentiated and un-differentiated GBM stem cells, treatment with IR resulted in an increase of FOXM1 expression. However, inhibition of FOXM1 was only seen to have an effect on un-differentiated GBM stem cells, and resulted in reduced cell viability, a significant reduction in clonogenicity, and anchorage-independent growth, along with enhanced radiosenstivity with IR. Importantly, the combination of IR with FOXM1 inhibition showed these same effects irrespective of serum-differentiation. These results clearly suggest, inhibition of FOXM1 leads to radiosensitization. Since GBM stem cells, which comprise a subpopulation of tumor cells, maybe responsible for therapeutic resistance, we show that FOXM1 inhibition stands as a potential cancer stem-cell specific chemo-radio therapeutic target for GBM. Citation Format: Uday Bhanu Maachani, Anita T. Tandle, Uma Shankavaram, Tamalee Meushaw, Philip J. Tofilon, Kevin A. Camphausen. Profiling signaling networks using reverse phase protein arrays: validating FOXM1 as a potential target to radiosensitize glioblastoma (GBM) stem cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 849. doi:10.1158/1538-7445.AM2014-849