scholarly journals PARP Inhibitor Veliparib and Busulfan in a Xenograft Model of Myeloproliferative Neoplasm

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 3319-3319
Author(s):  
Pritesh Patel ◽  
Vitalyi Senyuk ◽  
Natalie S. Rodriguez ◽  
Dolores Mahmud ◽  
Annie Lee Oh ◽  
...  
Keyword(s):  
High Risk ◽  
Combination Treatment ◽  
Xenograft Model ◽  
Replication Forks ◽  
Cd34 Cells ◽  
Synergistic Cytotoxicity ◽  
Hel Cells ◽  
Stalled Replication Forks ◽  
Parp 1

Abstract Despite new targeted therapies, patients with high risk myeloproliferative neoplasms (MPNs) have an increased chance of transforming into acute myeloid leukemia (AML) and can only be cured with allogeneic hematopoietic stem cell transplant (HSCT). JAK2V617F mutation is common in MPNs and is associated with genomic instability, with baseline DNA double strand breaks and homologous recombination (HR) activity as a result of stalled replication forks. Poly(ADP-ribose) polymerase-1 (PARP-1) detects disrupted replication forks and recruits HR repair enzymes to restart DNA replication. Because busulfan, which is used in preparative regimens for HSCT, also leads to stalled replication forks through DNA strand crosslinking, we hypothesized that the PARP-1 inhibitor veliparib and busulfan may lead to synergistic cytotoxicity in MPN cells. We first treated two JAK2V617F positive MPN cell lines with increasing doses (from 0.1 to 100μM) of veliparib in liquid cultures and measured cell proliferation. SET2 and HEL cells were relatively sensitive to veliparib (IC50 of 11.3μM and 74.2μM respectively). We next treated the cell lines with increasing doses of busulfan in combination with a fixed subtherapeutic dose of veliparib (4μM) for 48 hours. With combination treatment, the busulfan IC50 decreased from 27μM to 4μM in SET2 cells and from 45.1μM to 28.1μM in HEL cells. The combination was synergistic with a mean combination index (CI) of 0.55 for SET2 and 0.40 for HEL cells. Combination treatment resulted in a higher fraction of cells in G2M arrest than with veliparib alone (p=0.03) or busulfan alone (p=0.04). Similarly, when the combination treatment was tested on CD34+ cells obtained from 5 patients with JAK2V617F mutated and 2 CALR mutated PMF in a standard clonogenic (CFU) assay, it caused a greater inhibition of colony formation than busulfan or veliparib alone (p=0.001). Veliparib alone did not show any effect on colony formation from healthy control CD34+ cells. Finally we utilized a xenograft model with NOD/SCID/IL-2Rγnull (NSG) mice to test the in-vivo effect of combined low doses of busulfan and veliparib in JAK2V617F MPNs. In order to establish disease, mice were injected with 5x106 SET2 cells via tail vein 12 hours after sub-lethal irradiation. Then, 14 days after SET-2 injection 4 groups of mice (n=5 each) were treated for up to 3 weeks with intra-peritoneal vehicle (control); or a subtherapeutic dose of veliparib (3mg/kg daily) for 5 days a week; or busulfan (25mg/kg weekly); or a combination of both drugs. Death from leukemia was documented by marrow and spleen cell immunophenotype using non-cross reactive anti-human CD33 and CD34 antibodies. Veliparib alone did not improve survival vs control. On the contrary, survival was increased by the combination treatment vs busulfan alone (p=0.02). Here we show that treatment with the PARP-1 inhibitor veliparib and busulfan elicits a synergistic cytotoxicity in MPN cells both in-vitro and in-vivo. Our data provide the rationale for testing novel pre-transplant conditioning regimens by combining veliparib with alkylating agents, such as busulfan or melphalan, in HSCT for high risk MPNs or MPN-AML. Disclosures Patel: Janssen: Honoraria; Celgene: Consultancy, Honoraria; Amgen: Consultancy, Honoraria.

scholarly journals Interactions and Localization of Escherichia coli Error-Prone DNA Polymerase IV after DNA Damage

2015 ◽  
Vol 197 (17) ◽  
pp. 2792-2809 ◽  
Author(s):  
Sarita Mallik ◽  
Ellen M. Popodi ◽  
Andrew J. Hanson ◽  
Patricia L. Foster
Keyword(s):  
Dna Damage ◽  
Dna Polymerase ◽  
Dna Helicase ◽  
Dna Lesions ◽  
Replication Forks ◽  
Content Type ◽  
Pol Iv ◽  
Dna Polymerase Iv ◽  
Stalled Replication Forks

ABSTRACTEscherichia coli's DNA polymerase IV (Pol IV/DinB), a member of the Y family of error-prone polymerases, is induced during the SOS response to DNA damage and is responsible for translesion bypass and adaptive (stress-induced) mutation. In this study, the localization of Pol IV after DNA damage was followed using fluorescent fusions. After exposure ofE. colito DNA-damaging agents, fluorescently tagged Pol IV localized to the nucleoid as foci. Stepwise photobleaching indicated ∼60% of the foci consisted of three Pol IV molecules, while ∼40% consisted of six Pol IV molecules. Fluorescently tagged Rep, a replication accessory DNA helicase, was recruited to the Pol IV foci after DNA damage, suggesting that thein vitrointeraction between Rep and Pol IV reported previously also occursin vivo. Fluorescently tagged RecA also formed foci after DNA damage, and Pol IV localized to them. To investigate if Pol IV localizes to double-strand breaks (DSBs), an I-SceI endonuclease-mediated DSB was introduced close to a fluorescently labeled LacO array on the chromosome. After DSB induction, Pol IV localized to the DSB site in ∼70% of SOS-induced cells. RecA also formed foci at the DSB sites, and Pol IV localized to the RecA foci. These results suggest that Pol IV interacts with RecAin vivoand is recruited to sites of DSBs to aid in the restoration of DNA replication.IMPORTANCEDNA polymerase IV (Pol IV/DinB) is an error-prone DNA polymerase capable of bypassing DNA lesions and aiding in the restart of stalled replication forks. In this work, we demonstratein vivolocalization of fluorescently tagged Pol IV to the nucleoid after DNA damage and to DNA double-strand breaks. We show colocalization of Pol IV with two proteins: Rep DNA helicase, which participates in replication, and RecA, which catalyzes recombinational repair of stalled replication forks. Time course experiments suggest that Pol IV recruits Rep and that RecA recruits Pol IV. These findings providein vivoevidence that Pol IV aids in maintaining genomic stability not only by bypassing DNA lesions but also by participating in the restoration of stalled replication forks.

scholarly journals Combination of proteasome inhibitors bortezomib and NPI-0052 trigger in vivo synergistic cytotoxicity in multiple myeloma

Blood ◽  
2008 ◽  
Vol 111 (3) ◽  
pp. 1654-1664 ◽  
Author(s):  
Dharminder Chauhan ◽  
Ajita Singh ◽  
Mohan Brahmandam ◽  
Klaus Podar ◽  
Teru Hideshima ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Proteasome Inhibitors ◽  
Xenograft Model ◽  
Er Stress Response ◽  
Synergistic Cytotoxicity ◽  
Proteolytic Activities ◽  
Dose Combination ◽  
Inhibition Of Tumor Growth

AbstractOur recent study demonstrated that a novel proteasome inhibitor NPI-0052 triggers apoptosis in multiple myeloma (MM) cells, and importantly, that is distinct from bortezomib (Velcade) in its chemical structure, effects on proteasome activities, and mechanisms of action. Here, we demonstrate that combining NPI-0052 and bortezomb induces synergistic anti-MM activity both in vitro using MM cell lines or patient CD138+ MM cells and in vivo in a human plasmacytoma xenograft mouse model. NPI-0052 plus bortezomib–induced synergistic apoptosis is associated with: (1) activation of caspase-8, caspase-9, caspase-3, and PARP; (2) induction of endoplasmic reticulum (ER) stress response and JNK; (3) inhibition of migration of MM cells and angiogenesis; (4) suppression of chymotrypsin-like (CT-L), caspase-like (C-L), and trypsin-like (T-L) proteolytic activities; and (5) blockade of NF-κB signaling. Studies in a xenograft model show that low dose combination of NPI-0052 and bortezomib is well tolerated and triggers synergistic inhibition of tumor growth and CT-L, C-L, and T-L proteasome activities in tumor cells. Immununostaining of MM tumors from NPI-0052 plus bortezomib–treated mice showed growth inhibition, apoptosis, and a decrease in associated angiogenesis. Taken together, our study provides the preclinical rationale for clinical protocols evaluating bortezomib together with NPI-0052 to improve patient outcome in MM.

CD34+CD19−, CD34+CD19+ and CD34−CD19+ Cells May All Have Leukemia-Initiating Potential in NOD/Scid Mice.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 2542-2542
Author(s):  
Christoph Le Viseur ◽  
Marc Hotfilder ◽  
Annegret Rosemann ◽  
Ronald Stam ◽  
Andre Schrauder ◽  
...  
Keyword(s):  
Bone Marrow ◽  
High Risk ◽  
Lymphoblastic Leukemia ◽  
Xenograft Model ◽  
Current Data ◽  
Scid Mice ◽  
Functional Assay ◽  
Childhood All ◽  
Mouse Xenograft Model

Abstract Current data on the leukemic stem cell (LSC) compartment in childhood acute lymphoblastic leukemia (ALL) are conflicting. The traditional hypothesis supposed that childhood ALL originates in a lymphoid progenitor cell and this is assumed to be consistent with the overall good treatment responses in pediatric patients. In accordance with this hypothesis, our previous studies failed to detect involvement of immature CD34+CD19− progenitor cells in ALL/t(12;21) (Hotfilder et al., Blood 2002) while high-risk ALL/t(9;22) and t(4;11) appears to originate in a more primitive CD34+CD19− cell (Hotfilder et al., Cancer Res 2005). In order to characterize the leukemia-initiating cell in vivo, we established a mouse xenograft model by serial intrafemoral transplantation of NOD/scid mice with flow sorted subpopulations from childhood ALL. Samples were taken from the bone marrow of children with ALL/t(12;21) (n=1), t(4;11) (n=3) and t(11;19) (n=1) and B-cell precursor ALL without a marker translocation (n=2). Primary transplantations were performed with freshly thawed unsorted cells, followed by secondary, tertiary and quaternary transplantations with flow sorted populations. Human leukemic engraftment was defined by a proportion of >5% human CD45+ cells in the murine bone marrow that simultaneously express CD34 and/or CD19. From the bone marrow of leukemic mice, we isolated different leukemic populations and successfully re-transplanted 2×103 − 1×105 CD34+CD19− cells, 2×104 − 6×106 CD34+CD19+ lymphoid progenitors and 3×104 − 2×106 more differentiated CD34−CD19+ blasts onto secondary, tertiary and quaternary mice (average purity after flow sorting: >96%). So far, we detected leukemic engraftment in 60 of 161 (37%) transplanted mice (with many mice - having only recently been transplanted - still alive). These include 7 of 36 (19%) mice engrafted with CD34+CD19− cells, 33 of 72 (46%) mice engrafted with CD34+CD19+ cells and 20 of 53 (38%) mice engrafted with CD34−CD19+ cells. With as few as 2 × 103 CD34+CD19− cells being sufficient to re-initiate the leukemia, this intrafemoral ALL-NOD/scid mouse model represents a very sensitive functional assay for candidate LSC in childhood ALL. We have initiated limiting dilution experiments with the different subpopulations to quantify LSC frequency in the different compartments and to exclude that low levels of contaminating blasts with an immunophenotype different from the main transplanted cell population blurred the results. We are also currently investigating whether there is heterogeneity in the CD34+CD19− compartment in respect to standard and high-risk ALL. Altogether, our data indicate that all three subpopulations, CD34+CD19−, CD34+CD19+ and CD34−CD19+ cells, may have the capacity to transfer the leukemia onto NOD/scid mice and that lymphatic LSC may not loose their self-renewal potential with differentiation.

scholarly journals Regulation of Rtt107 Recruitment to Stalled DNA Replication Forks by the Cullin Rtt101 and the Rtt109 Acetyltransferase

2008 ◽  
Vol 19 (1) ◽  
pp. 171-180 ◽  
Author(s):  
Tania M. Roberts ◽  
Iram Waris Zaidi ◽  
Jessica A. Vaisica ◽  
Matthias Peter ◽  
Grant W. Brown
Keyword(s):  
Dna Damage ◽  
Dna Damage Response ◽  
Replication Forks ◽  
Chromatin Binding ◽  
Direct Role ◽  
Repair Enzymes ◽  
Damage Response ◽  
Stalled Replication Forks

RTT107 (ESC4, YHR154W) encodes a BRCA1 C-terminal domain protein that is important for recovery from DNA damage during S phase. Rtt107 is a substrate of the checkpoint kinase Mec1, and it forms complexes with DNA repair enzymes, including the nuclease subunit Slx4, but the role of Rtt107 in the DNA damage response remains unclear. We find that Rtt107 interacts with chromatin when cells are treated with compounds that cause replication forks to arrest. This damage-dependent chromatin binding requires the acetyltransferase Rtt109, but it does not require acetylation of the known Rtt109 target, histone H3-K56. Chromatin binding of Rtt107 also requires the cullin Rtt101, which seems to play a direct role in Rtt107 recruitment, because the two proteins are found in complex with each other. Finally, we provide evidence that Rtt107 is bound at or near stalled replication forks in vivo. Together, these results indicate that Rtt109, Rtt101, and Rtt107, which genetic evidence suggests are functionally related, form a DNA damage response pathway that recruits Rtt107 complexes to damaged or stalled replication forks.

scholarly journals Ablation of PARP-1 does not interfere with the repair of DNA double-strand breaks, but compromises the reactivation of stalled replication forks

Oncogene ◽  
2004 ◽  
Vol 23 (21) ◽  
pp. 3872-3882 ◽  
Author(s):  
Yun-Gui Yang ◽  
Ulrich Cortes ◽  
Srinivas Patnaik ◽  
Maria Jasin ◽  
Zhao-Qi Wang
Keyword(s):  
Double Strand Breaks ◽  
Replication Forks ◽  
Dna Double Strand Breaks ◽  
Strand Breaks ◽  
Stalled Replication Forks ◽  
Parp 1

scholarly journals Colocalization of Mec1 and Mrc1 is sufficient for Rad53 phosphorylation in vivo

2012 ◽  
Vol 23 (6) ◽  
pp. 1058-1067 ◽  
Author(s):  
Theresa J. Berens ◽  
David P. Toczyski
Keyword(s):  
Dna Replication ◽  
Double Strand Breaks ◽  
Sensor Kinase ◽  
Replication Forks ◽  
Checkpoint Activation ◽  
Replication Checkpoint ◽  
Strand Breaks ◽  
Crucial Step ◽  
Stalled Replication Forks

When DNA is damaged or DNA replication goes awry, cells activate checkpoints to allow time for damage to be repaired and replication to complete. In Saccharomyces cerevisiae, the DNA damage checkpoint, which responds to lesions such as double-strand breaks, is activated when the lesion promotes the association of the sensor kinase Mec1 and its targeting subunit Ddc2 with its activators Ddc1 (a member of the 9-1-1 complex) and Dpb11. It has been more difficult to determine what role these Mec1 activators play in the replication checkpoint, which recognizes stalled replication forks, since Dpb11 has a separate role in DNA replication itself. Therefore we constructed an in vivo replication-checkpoint mimic that recapitulates Mec1-dependent phosphorylation of the effector kinase Rad53, a crucial step in checkpoint activation. In the endogenous replication checkpoint, Mec1 phosphorylation of Rad53 requires Mrc1, a replisome component. The replication-checkpoint mimic requires colocalization of Mrc1-LacI and Ddc2-LacI and is independent of both Ddc1 and Dpb11. We show that these activators are also dispensable for Mec1 activity and cell survival in the endogenous replication checkpoint but that Ddc1 is absolutely required in the absence of Mrc1. We propose that colocalization of Mrc1 and Mec1 is the minimal signal required to activate the replication checkpoint.

scholarly journals PrimPol is required for replication reinitiation after mtDNA damage

2017 ◽  
Vol 114 (43) ◽  
pp. 11398-11403 ◽  
Author(s):  
Rubén Torregrosa-Muñumer ◽  
Josefin M. E. Forslund ◽  
Steffi Goffart ◽  
Annika Pfeiffer ◽  
Gorazd Stojkovič ◽  
...  
Keyword(s):  
Dna Polymerase ◽  
Translesion Synthesis ◽  
Replication Forks ◽  
Mtdna Damage ◽  
Replicative Stress ◽  
Mtdna Replication ◽  
Stalled Replication Forks ◽  
Lagging Strand

Eukaryotic PrimPol is a recently discovered DNA-dependent DNA primase and translesion synthesis DNA polymerase found in the nucleus and mitochondria. Although PrimPol has been shown to be required for repriming of stalled replication forks in the nucleus, its role in mitochondria has remained unresolved. Here we demonstrate in vivo and in vitro that PrimPol can reinitiate stalled mtDNA replication and can prime mtDNA replication from nonconventional origins. Our results not only help in the understanding of how mitochondria cope with replicative stress but can also explain some controversial features of the lagging-strand replication.

scholarly journals Dimerization of the ATRIP Protein through the Coiled-Coil Motif and Its Implication to the Maintenance of Stalled Replication Forks

2005 ◽  
Vol 16 (12) ◽  
pp. 5551-5562 ◽  
Author(s):  
Eisuke Itakura ◽  
Isao Sawada ◽  
Akira Matsuura
Keyword(s):  
Mammalian Cells ◽  
Coiled Coil ◽  
Genotoxic Stress ◽  
Replication Forks ◽  
Cycle Arrest ◽  
Coiled Coil Domain ◽  
Functional Complex ◽  
Stalled Replication Forks

ATR (ATM and Rad3-related), a PI kinase-related kinase (PIKK), has been implicated in the DNA structure checkpoint in mammalian cells. ATR associates with its partner protein ATRIP to form a functional complex in the nucleus. In this study, we investigated the role of the ATRIP coiled-coil domain in ATR-mediated processes. The coiled-coil domain of human ATRIP contributes to self-dimerization in vivo, which is important for the stable translocation of the ATR-ATRIP complex to nuclear foci that are formed after exposure to genotoxic stress. The expression of dimerization-defective ATRIP diminishes the maintenance of replication forks during treatment with replication inhibitors. By contrast, it does not compromise the G2/M checkpoint after IR-induced DNA damage. These results show that there are two critical functions of ATR-ATRIP after the exposure to genotoxic stress: maintenance of the integrity of replication machinery and execution of cell cycle arrest, which are separable and are achieved via distinct mechanisms. The former function may involve the concentrated localization of ATR to damaged sites for which the ATRIP coiled-coil motif is critical.

Overcoming Apoptosis Resistance In High Risk Acute Lymphoblastic Leukemia By Smac Mimetics In a Preclinical ALL Xenograft Model In Vivo

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 1433-1433
Author(s):  
Melanie Schirmer ◽  
Manon Queudeville ◽  
Luca Trentin ◽  
Sarah M Eckhoff ◽  
Lueder H Meyer ◽  
...  
Keyword(s):  
Cell Death ◽  
Acute Lymphoblastic Leukemia ◽  
High Risk ◽  
Small Molecule ◽  
Lymphoblastic Leukemia ◽  
Xenograft Model ◽  
Apoptosis Resistance ◽  
Smac Mimetics ◽  
Smac Mimetic

Abstract Intensified treatment of pediatric acute lymphoblastic leukemia (ALL) has lead to increased survival rates of about 80%, however therapy fails in the remaining patients leading to relapse of the disease associated with inferior prognosis. Because treatment failure is, at least in part, due to defects in apoptosis programs, novel therapeutic strategies that counter apoptosis resistance are needed. “Inhibitor of Apoptosis” (IAP) proteins block the apoptosis machinery at a central point and are highly expressed in acute leukemias, thereby providing a target structure for therapeutic intervention. Molecules antagonizing these apoptosis inhibitors, so called SMAC-mimetics, therefore provide a promising strategy to overcome apoptosis deficiency and effectively treat high risk ALL. In this study, we investigated the effects of the small molecule SMAC-mimetic BV6 (kindly provided by Genentech) in B cell precursor- (BCP-) ALL. BV6 showed a clear induction of cell death at nanomolar concentrations in ALL cell lines. ALL cells sensitive for SMAC-mimetic induced cell death showed rapid cIAP degradation, NFkB activation and secretion of TNF-alpha (TNF-a). Interestingly, mitochondrial perturbation and caspase activation could be inhibited by the soluble TNF-a receptor Etanercept indicating the induction of a TNF-a dependent feed forward loop by the SMAC-mimetic BV6. In addition to cell lines, we investigated the effects of BV6 on a series of 42 primary ALL samples isolated from ALL bearing mice of established patient derived NOD/SCID/huALL xenograft leukemias. Intriguingly, upon treatment with the small molecule SMAC mimetic BV6, induction of cell death was observed in a majority of 70% of all individual patient-derived leukemias and BV6 induced cell death was inhibited by Etanercept demonstrating TNF-a dependency also in primary ALL. We previously described that rapid engraftment of ALL cells transplanted onto NOD/SCID mice (short Time To Leukemia, TTLshort) is associated with deficient apoptosis signaling in the ALL cells and indicative for early patient relapse. Importantly, primary xenograft ALL samples with a TTLshort/early relapse phenotype showed increased cell death upon treatment with SMAC-mimetic BV6 and activation of the constitutive deficient apoptosis signaling pathway, demonstrating that SMAC-mimetics induce intact apoptosis signaling in former apoptosis resistant primary ALL cells. Based on theses findings, we further evaluated the in vivo effectivity of the SMAC-mimetic BV6 on high risk ALL using our NOD/SCID/huALL xenograft model in a preclinical setting. ALL bearing recipients were treated with either BV6 or solvent for a given time of two weeks and further investigated for the presence of leukemia. Most interestingly, a significant delay of post-treatment leukemia reoccurrence was observed upon BV6 in vivo treatment along with a profound reduction of tumor load in the recipients compared to solvent treated animals. In a clinical setting, high-risk disease is unlikely to be treated by one compound alone. Therefore, we combined BV6 with multidrug chemotherapy resembling ALL induction treatment and observed a significant delay of ALL reoccurrence and prolonged survival of animals treated with the combination of the SMAC-mimetic and chemotherapy in contrast to chemotherapy alone. Most importantly, concomitant in vivo therapy with Etanercept revoked the cell death sensitizing effect of BV6, both in single treatment and in combination with chemotherapy. This indicates that BV6 induced apoptosis sensitization involves signaling via TNF-a and thereby provides a potential biomarker for the identification of patients who would benefit from SMAC-mimetic treatment. Taken together, we show that the small molecule SMAC-mimetic BV6 induces cell death via a TNF-a loop ex vivo and in vivo in primary patient-derived ALL. Moreover, BV6 is able to overcome apoptosis deficiency of high risk ALL leading to prolonged in vivo survival in a preclinical therapy model of patient-derived ALL xenograft ALL. Thus, induction of cell death by new generation small molecule SMAC-mimetics provides a promising novel strategy for targeted therapy of high-risk acute lymphoblastic leukemia and involvement of TNF-a signaling in BV6-sensitive patients points to its potential use as biomarker indicating effective cell death sensitization. Disclosures: No relevant conflicts of interest to declare.

Utilizing metformin as a radiosensitizing agent in the treatment of prostate cancer.

2011 ◽  
Vol 29 (7_suppl) ◽  
pp. 89-89
Author(s):  
L. Klotz ◽  
N. Venier ◽  
A. Vandersluis ◽  
R. Besla ◽  
N. Fleshner ◽  
...  
Keyword(s):  
Prostate Cancer ◽  
Tumor Growth ◽  
Combination Treatment ◽  
Colony Formation ◽  
Xenograft Model ◽  
External Beam Radiation ◽  
Diabetic Patients ◽  
Beam Radiation

89 Background: External beam radiation therapy (EBRT) is a well recognized curative prostate cancer (PCa) treatment modality utilizing ionizing radiation (IR). In addition to mediating DNA damage, IR upregulates several intracellular pro-survival pathways including the insulin- like growth factor (IGR) signaling network. This may contribute to the intrinsic radioresistance exhibited by certain tumors. Diabetic patients with PCa experience poorer outcomes following EBRT than their non-diabetic counterparts. Some attribute this to diabetes-induced chronic hyperinsulinemia with consequent upregulation of pro-survival insulin/IGF signalling. Previous work by our group showed diet-induced hyperinsulinemia to enhance PCa tumor growth in vivo. Metformin, a diabetic treatment, alleviates hyperinsulinemia, and also exhibits anti-neoplastic properties. We postulate that pre-treatment with metformin to correct hyperinsulinemia may protect cells from radiation-mediated pro-survival insulin/IGF signaling. Thus we assessed the radiosensitizing potential of metformin using in vitro and in vivo PCa models. Methods: The effect of IR and/or metformin on colony formation rates was assessed in LNCaP, PC3, DU145 and PC3AR2 PCa cell lines using clonogenic assay. The combination treatment regimen was assessed in vivo using a murine xenograft model. Western blot and cell cycle analyses are ongoing to try and elucidate any mechanisms of interaction between metformin and IR. Results: Monotherapy with IR (1-8Gy) or metformin (0.01-10.0mM) caused significant dose-dependent reduction in colony formation rates (p<0.001). Combination treatment further significantly reduced colony formation rates (p<0.03). Preliminary results from our in vivo study show diminished tumor growth in response to combination treatment (p<0.0001), and are currently subject to ongoing statistical analyses. Conclusions: Our in vitro findings confirm combining metformin with IR significantly reduces PCa cell colony formation rates further than either monotherapy. Recapitulation of these results in vivo would provide justification for translating this work into a phase II clinical trial of metformin as a radiosensitizing agent. No significant financial relationships to disclose.

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