Citations

Abstract Background Follow-up blood cultures (FUBCs) for streptococcal bacteremia are infrequently positive and may not provide sufficient clinical impact to justify the time and resources expended for their routine use. We aimed to evaluate the frequency of and risk factors for positive FUBCs in patients with streptococcal bacteremia and to compare clinical outcomes among patients with or without positive FUBCs. Methods We retrospectively reviewed hospitalized patients with streptococcal bacteremia from September 2023 to June 2024 within a multisite health system. Patients ≥18 years old with ≥1 positive blood culture for a Streptococcus species were included. Patients with polymicrobial bacteremia, those who died or were discharged to hospice within 48 hours of their index culture, and Minnesota residents who declined research authorization were excluded. Results We included 454 patients, with 48 (10.6%) having positive FUBCs. The rate of positivity ranged from 0% for Streptococcus pyogenes to 18.5% for Streptococcus anginosus. Endocarditis and osteomyelitis were more common in the positive than in the negative FUBC group (22.9% vs 12.1%, respectively, for endocarditis [P = .04] and 16.7% vs 5.4% [P = .005] for osteomyelitis). Positive FUBCs were associated with a higher incidence of infection relapse (8.5% vs 2.7%; P = .047) and longer duration of antibiotics (30 vs 16 days; P = .01), but no differences in mortality rate, length of stay, or infection-related readmission were observed. Conclusions Positive FUBCs were infrequent for S pyogenes, Streptococcus agalactiae, Streptococcus pneumoniae, and group C and G streptococci. It may be reasonable to forego FUBC for these streptococcal species if endocarditis or osteomyelitis are not clinically suspected.

Abstract Glioblastoma (GBM) are inherently resistant to radiation therapy (RT), and development of radiosensitizers is one strategy to overcome this limitation. Repair of DNA double strand breaks induced by RT are mediated by the protein kinase Ataxia Telangiectasia mutated (ATM). In this study, the novel ATM inhibitor WSD-0628 was evaluated in combination with RT using GBM and melanoma models. In vitro evaluation of 10uM WSD-0628 binding to a panel of potential drug targets, including receptors, ion channels, enzymes, and transporters, indicated a satisfactory safety profile with low risk for off-target liability. WSD-0628 potently inhibits ATM-mediated phosphorylation of the DNA damage response protein KAP1 in MCF-7 cells at sub-nanomolar (nM) concentrations (IC50 0.42nM) in comparison to much less potent inhibition of the related kinases ATR (phosphorylation of CHK1, IC50 742nM) or DNA-PKcs (auto-phosphorylation of DNA-PK, IC50 169nM) in HT29 cells assessed by ELISA. In U251 GBM cells, 30 nM WSD-0628 potently inhibited RT-induced phospho-KAP1 and robustly reduced clonogenic survival by 5-fold when combined with 5 Gy irradiation (combination vs RT alone, p<0.01). Similar potent radiosensitizing effects were seen in a melanoma brain metastasis PDX line M12 (10nM WSD-0628+IR-5Gy 1% survival vs 5% survival with IR-5Gy alone. p<0.01), and the SV-40 transformed astrocyte line SVG-A (10nM WSD-0628 + IR-2.5Gy survival 0.2% vs 15% with IR-2.5Gy alone. p<0.01). Evaluation of the pharmacokinetic profile of WSD-0628 in mice 2h after a single 5 mg/kg oral dose reveals a high level of free drug availability in the brain (34nM) and in the CSF (50nM) with little to no Pgp/BCRP substrate liability. An initial in vivo dose finding study in orthotopic GBM43 PDX yielded significant benefit with WSD-0628 at either 5 or 10 mg/kg PO daily when combined with radiation (2Gy QD for 5 days); Median survival for sham RT (29d) or RT alone (34d) were significantly different from RT combinations with 5 mg/kg (54d) and 10 mg/kg (73d; p<0.01 for both dose levels), although the higher dose combination was poorly tolerated with body weight loss between 15-20% one week after RT completion. Dosing of WSD-0628 (7.5 mg/kg PO, QD) given just before and 24h after a single dose of RT (12.5Gy) in mice with orthotopic M12 was well tolerated and provided robust radiosensitizing effects with median survival for combination treatment of over 180d vs 17d for control and 49d with RT alone groups (combination vs RT alone, p=0.04). Collectively, these results suggest a promising role for WSD-0628 in combination with RT in GBM and melanoma metastatic to the brain. Citation Format: Ann Mladek Tuma, Wei Zhong, Lily Liu, Danielle M. Burgenske, Brett L. Carlson, Katrina K. Bakken, Zeng Hu, Margaret A. Connors, Jann N. Sarkaria. WSD-0628, a novel brain penetrant ATM inhibitor, radiosensitizes GBM and melanoma patient derived xenografts [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3305.

Abstract The ATM inhibitor AZD1390 disrupts cellular responses to ionizing radiation (IR) and is a potent radiosensitizer being tested in clinical trials. Here, we evaluated, the effects of AZD1390 on radiation sensitivity and DNA damage repair pathways in glioblastoma (GBM) cells and patient derived xenografts (PDXs). AZD1390 (30 nM and higher) suppressed IR (5 Gy)-induced phosphorylation of ATM-Serine1981 and downstream phosphorylation sites on Kap1, Chk2 and H2AX in U251 cells and multiple PDXs. Consistent with enhanced DNA damage, AZD1390 increased IR induced G2/M arrest in U251 (80.6% with AZD1390/IR vs. 64.6% with IR, p= 0.01), GBM43 (61.9% vs. 25.7%, p= 0.01) and GBM39 (40.9% vs. 25.4%, p= 0.01). Moreover, in a clonogenic survival assay, AZD1390 sensitized U251 cells to 5 Gy IR (0.24% survival with AZD1390/IR vs. 2.3% with IR alone, p=0.01). In a reporter-based analysis of DNA repair capacity, ATM inhibition resulted in a 40 to 60% reduction in homologous recombination (HR) and modest but significant decrease in micro-homology mediated end joining (MMEJ) and gap fill-in synthesis in U251 cells but had no effect on non-homologous end joining, translesion synthesis, nucleotide or base excision repair pathways. Comparing effects of AZD1390 on repair in GBM14 (TP53-wt) and GBM43 (TP53-mutant), similar results were observed except that decreased MMEJ was seen only in GBM43 (0.03± 0.01% vs. 0.07± 0.01% in control, p=0.002). Intriguingly, RAD51 knockdown (to disrupt HR) sensitized U251 and GBM43 but not GBM14. The efficacy of AZD1390 ± IR was studied in vivo in 10 PDXs. IR was delivered to orthotopic tumors using opposed lateral 225 kVp beams. AZD1390 (20 mg/kg PO) was given just prior to each radiation dose (2 Gy x 5 fractions). AZD1390 monotherapy was mostly ineffective, IR alone was reasonably efficacious with an average 1.8 ± 0.1-fold-increase in survival relative to sham radiation (survival ratio) across all 10 models. IR/AZD1390 treatment resulted in significant survival extension relative to IR alone in 6 of 10 models. Analysis of the survival benefit of combination therapy compared to IR alone across the entire cohort of PDXs was statistically marginal (average survival ratio 2.3± 0.3 vs. 1.8 ± 0.1 with IR, p=0.08). However, when stratified by TP53 status, combination therapy was significantly more effective than IR (mean survival ratio 2.3 ± 0.1 vs. 1.6 ± 0.2 with IR alone, p=0.02) in TP53-mutant PDXs, where all 5 models benefited. In contrast, TP53-wt group had no benefit (mean survival ratio 2.2 ± 0.5 vs. 2.0 ± 0.2, p=0.61), GBM39 was only TP53-wt PDX that benefited from the combination. In conclusion, AZD1390 is an effective radio-sensitizer that causes disruption in HR and potentially other DNA repair pathways. Interestingly, in vivo radiosensitizing effects are mostly restricted to TP53-mutant GBM PDXs. Understandingmechanism of resistance in the context of different TP53 backgrounds remains an important future direction. Citation Format: Shiv K. Gupta, Jiajia Chen, Daniel J. Laverty, Surabhi Talele, Brett L. Carlson, Ann C. Mladek Tuma, Danielle Burgenske, Gaspar J. Kitange, Petra Hamerlik, Zachary D. Nagel, Stephen T. Durant, William F. Elmquist, Jann N. Sarkaria. AZD1390 radio-sensitizes p53-mutant GBM via disrupting homology directed DNA repair [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2598.

Abstract Robust function of the p53 tumor suppressor pathway is critical when treating with DNA-damage inducing agents such as radiation therapy (RT), which is a key component of standard care for GBM. MDM2 is an important negative regulator of p53 stability and MDM2 is amplified in approximately 14% of GBM. Based on the concept that suppression of MDM2 can reactivate p53 function and potentially have single agent or combinatorial effects, multiple MDM2 inhibitors have been developed. Here we report in vitro and in vivo efficacy and pharmacodynamic (PD) effects of a BBB-penetrant MDM2-p53 antagonist, BI-MDM2, in GBM patient-derived xenograft (PDX) models. In vitro studies in p53 wild-type (WT) lines with or without MDM2 amplification demonstrate IC50 values in cell viability assays of 2-12 nM in serum-free culture and 5-35 nM in serum-containing culture after seven days of treatment. In vivo studies were performed in p53 WT lines: the MDM2-amplified GBM108, and the non-amplified GBM14, grown as orthotopic tumors in nude mice. Weekly oral treatment at 2 mg/kg of BI-MDM2 doubled median survival (placebo, 28 days (d) vs 2 mg/kg BI-MDM2, 57 d. p<0.0001) while weekly dosing at 10 mg/kg extended the median over 5 fold (176 d. p<0.0001) with half of the mice still living at 223 days post-inoculation. To assess the PD properties of this compound, mice bearing orthotopic GBM108 were treated three weeks after inoculation with a single dose of 2 or 10 mg/kg BI-MDM2. Normal brain, tumor, and plasma were collected at 24 and 48 hours (h) after dosing and downstream p53 transcriptional targets, p21 and PUMA were evaluated by qRT-PCR. p21 mRNA relative quantification detection at 24h increased by 1.5-10.5-fold in the 2 mg/kg group of animals compared to vehicle while the 10 mg/kg dose led to an increase by 2-25 fold compared to vehicle. PUMA was minimally affected by the 2 mg/kg dose but increased 1.2-17-fold over vehicle in the 10 mg/kg group at 24h. The observed increases in p21 and PUMA varied little between the 24 and 48h timepoints. In an orthotopic GBM14 PDX efficacy study, BI-MDM2 was combined with 20 Gy RT delivered in 10 fractions over 2 weeks with dosing limited to 2 weeks of therapy or until mice reached a moribund state. While two doses of 10 mg/kg BI-MDM2 alone had a modest effect on survival (41 d vs 31 d with placebo; p=0.002), continued dosing until moribund further extended median survival (82 d with BI-MDM2; p=0.001). The combination of two doses of BI-MDM2 with 2 weeks of RT extended survival as compared to RT alone (107 d vs. 69 d, respectively; p=0.019), while with extended drug dosing combined with 2 weeks of RT, median survival has not yet been reached (>125 d). Taken together, these results suggest that BI-MDM2 is a promising therapeutic agent that may provide significant anti-tumor efficacy either alone or in combination with RT in both MDM2 amplified and non-amplified p53 WT patients. Citation Format: Ann C. Mladek Tuma, Shiv Gupta, Surabhi Talele, Afroz Shareef Mohammad, Katrina K. Bakken, Helen He, Zeng Hu, Margaret A. Connors, Danielle M. Burgenske, Brett L. Carlson, William F. Elmquist, Ulrike Weyer-Czernilofsky, Jann N. Sarkaria. A promising blood-brain-barrier penetrant MDM2-p53 antagonist, BI-MDM2, increases survival in orthotopic, glioblastoma patient-derived xenograft models [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4190.

Abstract Glioblastoma (GBM) is an aggressive and infiltrative primary brain tumor with a median survival of 14.6 months following the current treatment strategy of radiation and chemotherapy. Therefore, there is a need to develop strategies to enhance the efficacy of chemo-radiation treatments for GBM. DNA damage response signaling pathways play a critical role in DNA repair and cell survival following radiation therapy and the inhibition of these pathways could augment the cytotoxicity associated with radiation providing a radiosensitizing effect. Ataxia Telangiectasia and Rad3-Related Protein (ATR) is a key regulator of the DNA damage response network and VX-970 is the first potent and selective inhibitor of ATR to enter clinical trials. Preliminary in vitro studies from our lab to determine a dose dependent effect of VX-970 in combination with a radiation dose of 5 Gy on the cell survival indicated that administration of radiation led to an enhancement in the cell death with an increasing dose of VX-970 in the U251 human GBM cell line. We evaluated the BBB penetration of VX-970 and studied the role of efflux transporters on the brain exposure of VX-970 in preparation for efficacy studies in PDX models of GBM. Brain distribution studies were performed in wild-type and Mdr1a/b-/- Bcrp1-/- (triple knockout) FVB mice (n=4) following intravenous administration of 20 mg/kg VX-970. Plasma and brain samples were collected at 7 time points post dosing and were analyzed using LC-MS. The brain-to-plasma (B/P) ratio in transporter (Pgp and Bcrp) knockout mice was 17.5 as opposed to 0.8 in transporter intact wild-type mice. This 22-fold increase in the B/P ratio in the triple knockout mice indicates that Pgp and/or Bcrp play a significant role in the efflux of VX-970 from the brain thereby limiting its brain penetration. Oral administration of VX-970 at a dose of 20mg/kg in transporter intact wild-type mice indicated an oral bioavailability of 38%. Steady state brain distribution studies were also performed in wild-type and triple knockout FVB mice following intraperitoneal implantation of the Alzet osmotic pumps to release VX-970 at a rate of 10ul/hr for 48 hours. We observed that the B/P ratio in transporter (Pgp and Bcrp) knockout mice was 12.6 as opposed to 0.12 in transporter intact wild-type mice We are in the process of conducting additional studies in Pgp and Bcrp single knockout mice to determine a more precise status of the role of efflux transporters in the brain delivery of VX-970. The free fraction of VX-970 was found to be 6.1% in plasma indicating that it has a relatively high unbound fraction as compared to other chemotherapeutics for brain delivery. We are in the process of determining free fraction in the brain and relating the concentration of free drug to a dose range associated with effective radiosensitization through efficacy studies in the PDX models of GBM. This PK-PD relationship will help guide future clinical trials. Citation Format: Surabhi Talele, Afroz Mohammad, Minjee Kim, Danielle Burgenske, Ann C. Mladek Tuma, Jann N. Sarkaria, William F. Elmquist. CNS delivery of VX-970: A selective ATR inhibitor for radiosensitization in GBM [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4858.

The liver performs numerous vital functions, including the detoxification of blood before access to the brain while simultaneously secreting and internalizing scores of proteins and lipids to maintain appropriate blood chemistry. Furthermore, the liver also synthesizes and secretes bile to enable the digestion of food. These diverse attributes are all performed by hepatocytes, the parenchymal cells of the liver. As predicted, these cells possess a remarkably well-developed and complex membrane trafficking machinery that is dedicated to moving specific cargos to their correct cellular locations. Importantly, while most epithelial cells secrete nascent proteins directionally toward a single lumen, the hepatocyte secretes both proteins and bile concomitantly at its basolateral and apical domains, respectively. In this Beyond the Cell review, we will detail these central features of the hepatocyte and highlight how membrane transport processes play a key role in healthy liver function and how they are affected by disease.

Abstract Background: Bioluminescence imaging (BLI) of firefly luciferase expressing cells is widely used for analysis of growth, metastasis and drug response in tumor xenografts. D-luciferin, a commonly used luciferase substrate, has limited brain distribution that may undermine BLI for intracranial xenografts. The synthetic luciferin cyclic alkylamino-luciferin (CycLuc1) is a purported brain penetrant luciferase substrate not yet tested in intracranial xenografts. We compared BLI with CycLuc1 and D-luciferin in a glioblastoma (GBM) xenograft model. Methods: GBM6 cells were lentivirally transduced to express LUC2=tdTomato fusion protein. In vivo BLI were performed in orthotopic and heterotopic xenografts established in athymic nude mice. Concentrations of D-luciferin and CycLuc1 were measured by LC-MS/MS. Results: In a dose-response analysis, CycLuc1 at 5 mg/kg provided robust and reproducible imaging; however, an increased photon flux was observed with 10 mg/kg, while escalation to 15 or 20 mg/kg provided marginal additional gains in photon flux. In comparison of CycLuc1 (5 or 25 mg/kg) and D-luciferin (30 or 150 mg/kg, respectively) with serial, crossover BLI at both dose levels, CycLuc1 resulted in less variability and significantly higher photon flux as compared to D-luciferin in GBM6 intracranial xenografts (15 days after implantation). On average BLI with 25 mg/kg CycLuc1 resulted in ~8 fold greater photon flux from these early-stage xenografts as compared to 150 mg/kg D-luciferin (2.9±0.6x106 vs. 3.3±2.8x105 p/sec/cm2, p<0.001). Interestingly, bioluminescence emission with CycLuc1 and D-luciferin was comparable in xenografts imaged 28 days after implantation. In samples harvested after last imaging session, there was no significant difference in substrate distribution in intracranial tumors (tumor to plasma ratios for CycLuc1 and D-luciferin were 0.012 ± 0.020 and 0.012 ± 0.015, respectively; p=0.89). These results suggest that intracranial tumors at early stage may comprise relatively intact blood-brain barrier, which is disrupted at later stage. Consistent with notion that suboptimal delivery may interfere with D-luciferin mediated BLI in intracranial xenografts, photon flux in heterotopic tumors with D-luciferin or CycLuc1 was comparable (4.9±4.6x108 vs. 1.3±0.8x108 p/sec/cm2, p=0.10). Conclusions: These findings demonstrate that CycLuc1 may be a superior BLI substrate as compared to D-luciferin for monitoring tumor growth of intracranial GBM xenografts. Citation Format: Shiv K. Gupta, Minjee Kim, Bianca-Maria Marin, Lihong He, Katelyn Swanson, Tuma Tuma, Brett L. Carlson, Mark A. Schroeder, William F. Elmquist, Jann N. Sarkaria. Synthetic luciferin, CycLuc1, improves bioluminescence imaging for intracranial glioblastoma xenografts [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4112.

Abstract Mismatch repair (MMR) is essential for temozolomide (TMZ) sensitivity, and MMR deficiency causes TMZ resistance and disease recurrence. However, the role MMR in TMZ response has been linked with Msh2/Msh6 (MutSα), while role of Msh3, a component of MutSβ remains unclear. To delineate role of MutSα and MutSβ in TMZ response, we used siRNA to disrupt MSH6 and MSH3 expression. As expected, silencing of MSH6 led to resistance in a TMZ-sensitive U251 cell line. In contrast, silencing of MSH3 enhanced TMZ sensitivity as compared to the control cells. These results support established role of MutSα in TMZ response via replication stress and DNA double strand breaks (DSB), and reveal that MutSβ may potentially influence TMZ response by modulating DSB repair. Consistently, host cell reactivation (HCR) assays in U251 expressing DRGFP reporter showed decreased homologous recombination (HR) after MSH3 knockdown (KD), while Msh6 KD had no effect on HR efficiency. Supporting the concept of synthetic lethality of HR with PARP inhibition, clinically relevant dose of veliparib (1 µM) showed a modest but consistent increase in TMZ sensitization measured in terms of potentiation factor (PF, ratio of relative growth with TMZ alone/ TMZ and veliparib) in MSH3 KD (PF 1.6±0.2) as compared to control cells (PF 1.1±0.2). Sensitizing effect of 1 µM veliparib in MSH6 KD (PF 1.7±0.5) was comparable to that in MSH3 KD cells. Interestingly, supra-therapeutic dose of veliparib (10 µM) induced more pronounced sensitization in MSH6 KD cells (PF 2.6±0.3) as compared to MSH3 KD (PF 2.4±0.3) or control cells (PF 1.6±0.2). Consistently, veliparib (10 µM) alone had no impact on DNA damage signaling, while TMZ induced prominent increase in phosphorylation of KAP1, Chk1 and Chk2 in control and MSH3 KD cells, but not in MSH6 KD cells; veliparib/TMZ co-treatment led a robust signaling in all 3 types of cells. Increased sensitization by veliparib after MSH3 KD was also examined in a TMZ-resistant (GBM22TMZ) PDX model carrying resistance due to a homozygous mutation in MSH6, and a 4-fold increase in HR efficiency. In clonogenic assays silencing of MSH3 or BRCA1 had no effect on TMZ sensitivity in GBM22TMZ cells. However, veliparib alone (10 µM) decreased colony formation with relative colony formation 0.54±0.03 and 0.32±0.06 in MSH3 and BRCA1 KD cells, respectively as compared to 0.82±0.04 in controls. TMZ/veliparib co-treatment in GBM22TMZ led to further decrease in colony formation in MSH3 KD and BRCA1 KD cells with relative colony formation of 0.30±0.04 and 0.19±0.02, respectively versus 0.46±0.03 in control. Taken together, our results suggest that MutSα and MutSβ differentially regulate cytotoxic effects of TMZ. Decreased HR efficiency and enhanced sensitizing effects of veliparib in MSH3 KD cells partially define synthetic lethality of MMR deficiency with PARP, and may help development of PARP inhibitors as sensitizer of TMZ therapy in GBM. Citation Format: Shiv K. Gupta, Emily J. Smith, Ann M. Tuma, Brett L. Carlson, Mark A. Schroeder, Gasper J. Kitange, Jann N. Sarkaria. MutSα and MutSβ mismatch repair complexes differentially regulate temozolomide efficacy & sensitizing effects of veliparib in glioblastoma cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1109. doi:10.1158/1538-7445.AM2017-1109

Lipid droplets (LDs), the organelles central to alcoholic steatosis, are broken down by lipophagy, a specialized form of autophagy. Here, we hypothesize that ethanol administration retards lipophagy by down‐regulating dynamin 2 (Dyn2), a protein that facilitates lysosome re‐formation, contributing to hepatocellular steatosis. Primary hepatocytes were isolated from male Wistar rats fed Lieber–DeCarli control or ethanol (EtOH) liquid diets for 6‐8 weeks. Hepatocytes were incubated in complete medium (fed) or nutrient‐free medium (fasting) with or without the Dyn2 inhibitor dynasore or the Src inhibitor SU6656. Phosphorylated (active) forms of Src and Dyn2 and markers of autophagy were quantified using western blot analysis. Colocalization of LDs with autophagic machinery was determined using confocal microscopy. In hepatocytes from pair‐fed rats, LD breakdown was accelerated during fasting, as judged by smaller LDs and lower triglyceride (TG) content when compared with hepatocytes in complete media. Fasting‐induced TG loss in control hepatocytes was significantly blocked by either SU6656 or Dynasore. Compared with controls, hepatocytes from EtOH‐fed rats had 66% and 40% lower content of phosphorylated Src (pSrc) and phosphorylated Dyn2 (pDyn2), respectively, coupled with a lower rate of fasting‐induced TG loss. This slower rate of fasting‐induced TG loss was blocked in cells coincubated with Dynasore. Microscopic examination of EtOH‐fed rat hepatocytes revealed increased colocalization of the autophagosome marker LC3 on LDs with a concomitant decrease in lysosome marker LAMP1. Whole livers and LD fractions of EtOH‐fed rats exhibited simultaneous increase in LC3II and p62 over that of controls, indicating a block in lipophagy. Conclusion: Chronic ethanol administration slowed the rate of hepatocyte lipophagy, owing in part to lower levels of phosphorylated Src kinase available to activate its substrate, Dyn2, thereby causing depletion of lysosomes for LD breakdown. (Hepatology Communications 2017;1:501–512)

Abstract Glioblastoma multiforme (GBM) is an aggressive and fatal disease commonly treated with radiation and temozolomide (TMZ), but emergence of therapy resistance invariably limits treatment efficacy. Inhibition of Poly-ADP ribose polymerases 1 and 2 (PARP1/2) sensitizes GBM cells, and inhibitors of PARP are emerging as combination partners with TMZ. However, limited brain penetration precludes use of several promising PARP inhibitors in GBM. BGB-290 is a relatively new and potent PARP inhibitor that crosses the blood brain barrier with a brain to plasma ratio of ∼0.2. The focus of the present study was to evaluate antitumor effects of BGB-290, as single agent or in combination with TMZ, in patient derived GBM xenografts and glioma cell lines. In a biochemical assay, low doses (30-100 nM) of BGB-290 suppressed PARP activity, while single agent anti-tumor activity of BGB-290 was modest and restricted to concentrations above 3 μM. Combinations of 0.1 μM BGB-290 with 10 μM TMZ significantly diminished primary neurosphere formation in a patient-derived xenograft line, GBM12, decreasing relative neurosphere count to 3.1 ± 1.3% as compared to 9.2 ± 3.1% with TMZ alone, p = 0.03. Consistent with a mechanism involving disruption of DNA repair, treatment of GBM12 cells with TMZ and BGB-290 combination resulted in robust γH2AX foci formation and a synergistic increase in DNA damage signaling as compared to TMZ or BGB-290 alone. TMZ-sensitizing effects of BGB-290 were heterogeneous in TMZ-resistant (GBM12TMZ) sublines that were developed previously by in vivo selection of parental GBM12 with cyclical TMZ therapy. The average neurosphere formation across 4 resistant sublines lacking MGMT expression after 100 μM TMZ only was 75.3 ± 9.0% versus 14.9 ± 7.4% with TMZ and 0.1 μM BGB-290, p<0.01. In contrast, 2 MGMT overexpressing sublines were much less sensitive to the combination with relative neurospheres 91.9 ± 5.2% versus 73.2 ± 9.2% respectively, p< 0.01. Next Gen Sequencing revealed discrete non-synonymous somatic mutations in the mismatch repair pathway of GBM12TMZ sublines lacking MGMT, which led us to examine the relationship between mismatch repair and sensitizing effects of PARP inhibition. Silencing Msh2 resulted in marked TMZ resistance in U251 cells (94.7± 5.4% relative growth with 100 μM TMZ), while co-treatment with 0.1 μM BGB-290 and 100 μM TMZ decreased growth to 23.5 ± 9.6% (p<0.01), which is similar sensitivity to TMZ alone in U251 parental cells. In an initial in vivo study with orthotopic GBM12 xenografts, administration of BGB-290 at 3 mg/kg twice per day combined with 25 mg/kg TMZ extended survival of animals beyond 300 days (80% survival at day 320), while in the group treated with TMZ alone 80% animals were moribund before day 150. In conclusion, BGB-290 is a promising PARP inhibitor with high potency, cytotoxicity and favorable in vivo efficacy in TMZ sensitive and possibly a subset of recurrent TMZ resistant GBM. Citation Format: Shiv K. Gupta, Brett L. Carlson, Mark A. Schroeder, Katrina K. Bakken, Ann C. Tuma, Jann N. Sarkaria. Inhibition of PARP activity by BGB-290 potentiates efficacy of temozolomide in patient derived xenografts of glioblastoma multiforme. [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 3505. doi:10.1158/1538-7445.AM2015-3505