Burgenske, Danielle M.

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 Radio-resistant properties of melanomas undermine benefit of radiation therapy (RT). DNA-dependent protein kinase (DNA-PKcs) is essential for the non-homologous end joining (NHEJ) mediated repair DNA double-strand break (DSB). We evaluated radio-sensitizing effects of M3814, a selective oral inhibitor of DNA-PKcs, in patient-derived xenografts (PDXs) of melanoma brain metastases. M3814 (≥300 nM) inhibited RT-induced (5 Gy) auto-phosphorylation of serine-2056 of DNA-PKcs in primary cultures of M12, M15 and M27 PDX lines. Interestingly, inhibition of RT-induced DNA-PKcs by M3814 coincided with increased KAP1 phosphorylation, a DNA damage signaling regulated via ATM. As a measure of lasting DNA damage, persistent γH2AX foci were observed in 28% cells 24 hours after co-treatment with M3814 and RT as compared to RT controls, where only 12% cells had persistent γH2AX foci. In a clonogenic survival assay, M3841 augmented RT-induced killing of M12 cells in a dose dependent manner. However, a minimal 16 h exposure with ~300 nM M3814 was most effective treatment. Pharmacokinetics (PK) after single oral dose of 20 mg/kg M3814, showed considerably short half-life (~2.44 hours) and poor brain distribution in wildtype (WT) FBV mice (Kpuu, 0.027). Suggesting liability to efflux transporters, brain distribution of M3814 in TKO mice (triple knockouts for efflux transporters Mdr1a, Mdr1b and BCRP1) was ~11 fold higher than WT animals (kpuu, 0.215). Using this preliminary PK data, simulations were performed using simBiology software to define dosing regimen and schedule to maximize drug exposures in brain. Based on this in silico modeling, two regimens- (A: 125 mg/kg twice a day (at 0 and 7 hours), and B: 50 mg/kg dose followed by three additional doses of 35 mg/kg per day at 4 hour intervals), combined with a single fraction RT (3 Gy) delivered 10 min after the first M3814 dose in each regimen, were tested in athymic nude mice carrying M12 flank tumors. In this study, drug levels achieved in brain and plasma (121±95, 1914±1661 nM with regimens A and 79±51, 1205±664 nM with regimen B, respectively at 24 h), exceeded predictions. Consistent with a much higher accumulation of M3814 in flank tumor tissues (1.5 to 3.4 fold higher than plasma), therapy with M3814/RT had robust pharmacodynamics effects on DNA damage signaling both at 6 and 24 hours of treatment as compared to RT alone. In summary, M3814 is a promising radio-sensitizer in melanoma brain metastases. Further studies will determine efficacy and pharmacodynamics effects of M3814/RT regimens in relevant orthotopic models of brain metastases and address potential concerns of normal tissue toxicity. Citation Format: Jianxiong Ji, Emily J. Smith, Paige Sarkaria, Ann C. Mladek, Surabhi Talele, Katelyn Swanson, Afroz S. Mohammad, Lihong He, Zeng Hu, Katrina K. Bakken, Shiv K. Gupta, Danielle M. Burgenske, Gaspar J. Kitange, William F. Elmquist, Jann N. Sarkaria. Inhibition of DNA-PKcs by M3814 potentiates efficacy of ionizing radiation in patient derived xenografts of melanoma brain metastases [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 1381.

Abstract The Mayo Clinic collection of patient-derived xenograft (PDX) models has been developed from the direct implantation of fresh glioblastoma samples into immunodeficient mice. Over 95 models have been extensively characterized with regards to histology, invasion, flank and orthotopic growth rates, and molecular profiling. Evaluation of orthotopic tumors demonstrated clear infiltration of tumor cells into the brain parenchymal in 90% of PDXs with contralateral hemisphere involvement in 64%. Extensive molecular profiling confirmed that our collection of PDXs captures the genetic heterogeneity of GBM, with the majority of known alterations represented at a frequency similar to the TCGA including gain of chromosome 7 and loss of chromosome 10. TERT promoter mutations were the most frequent overall (86%) followed by homozygous deletion of CDKN2A (70%). Multiple alterations were identified in the p53 pathway, including TP53 mutations (36%) and MDM2/4 (10%/2%) amplification. PI3-kinase pathway alterations mainly involved PTEN (48%) while MAP kinase pathway alterations were present in a subset of PDXs (NF1 alterations 17%, BRAF 4%). IDH mutations were found in only two models. Gene expression analysis revealed that our PDX collection includes all GBM subtypes with classical being the most represented (46% vs mesenchymal 31% and proneural 23%). MGMT promoter methylation was also observed in 45% of PDXs. The high fidelity of PDX models to their human counterpart makes these models ideal for testing standard of care and novel targeted therapies. To assess if PDXs are predictive of patient treatment response, 37 orthotopic PDX lines were treated with radiation (RT), temozolomide (TMZ), or concurrent RT/TMZ. Across all PDXs, RT and TMZ demonstrated median survival extension of 90 and 46 days relative to vehicle. Concurrent RT/TMZ further extended survival by 146 days. MGMT promoter methylation was strongly associated with response to TMZ-based strategies but not RT alone.This observation suggests that our PDX collection possesses clinically relevant predictive value. To date, we have characterized treatment responses to over 135 regimens including both GBM standards of care and targeted strategies in our PDX models. Recent lab efforts strive to define the effect of blood brain barrier disruption on drug delivery. With over 20 years of experience, our group has extensive expertise in the development of clinically relevant study designs focused on examining efficacy, tolerability, and pharmacokinetic/pharmacodynamic assessments. Since 2016, we have shared over 5200 individual samples (tumor tissue, cells, DNA/RNA, protein extracts, tissue microarray slides) in both internal and external collaborators. Our models have been utilized in over 41 NIH funded grants, including 33 funded RO1s, as well as by the pharmaceutical community with 50 working relationships to date (20 contracts, 30 collaborations). Finally, our PDX collection has been used in over 120 peer-reviewed published manuscripts. Citation Format: Danielle Burgenske, Ann C. Mladek, Rachael A. Vaubel, Shulan Tian, Mark A. Schroeder, Zeng Hu, Brett L. Carlson, Paul A. Decker, Jeanette E. Eckel-Passow, Jann N. Sarkaria. Evaluation of novel therapeutics using models from the Mayo Clinic GBM patient-derived xenograft (PDX) collection [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 1113.

Abstract Lisavanbulin (LIS; BAL101553) is the prodrug of BAL27862, a microtubule-binding, tumor checkpoint controller and potential radiosensitizer. These studies evaluated optimal integration of LIS with standard of care radiation therapy (RT) and/or temozolomide (TMZ) using GBM PDX models. Distribution across the blood brain barrier was evaluated after a single 30 mg/kg oral LIS dose, and concentrations of the active metabolite BAL27862 were measured by liquid chromatography-tandem mass spectrometry. Similar BAL27862 concentrations were detected in the brain (B) and plasma (P) at both two (B:P ratio 1.29) and six hours (B:P ratio 1.64) post-dose. An in vivo screen of LIS monotherapy across 14 orthotopic GBM PDX models showed significant survival benefit (p<0.01) in seven models (median survival extension 24-87%). Extending from these results, LIS was evaluated in several of the sensitive models in combination with RT +/- TMZ. Two MGMT unmethylated PDXs, GBM6 and GBM150, were treated with vehicle or two weeks of RT +/- LIS. LIS dosing during the RT dosing period did not significantly improve median survival in either line (GBM6 survival with RT 54 days vs RT/LIS 58 days, p=0.16; GBM150 RT 86 days vs RT/LIS 101 days, p=0.21). However, prolonged LIS dosing from the start of RT until mice reached a moribund state demonstrated added benefit (GBM6 median 90 days vs RT 69 days, p=0.0001; GBM150 median 143 days vs RT 73 days, p=0.06). In GBM6, prolonged LIS dosing also significantly extended survival when combined with 2 weeks of RT/TMZ (median 101 days vs 66 days, p<0.0001), while LIS alone or RT/TMZ resulted in similar median survivals (63 days vs 66 days, respectively; p=0.68). This same RT/TMZ/LIS benefit was not seen in the MGMT methylated GBM12. Subsequent experiments were performed to evaluate integration of prolonged LIS dosing with concurrent RT/TMZ followed by 3 cycles of adjuvant TMZ (‘Stupp’ regimen). In MGMT methylated GBM39, LIS alone did not significantly extend survival, but LIS addition to the Stupp regimen doubled median survival (Stupp 249 days vs Stupp/LIS 502 days, p=0.0001). GBM150 demonstrated equal benefit from LIS alone or Stupp regimen (median 118 days vs 123 days, p=0.49). Stupp/LIS showed no additional survival benefit (median 98 days, p=0.97). In a second MGMT unmethylated, TMZ-resistant GBM26 PDX, LIS alone or combined with the Stupp regimen provided significant survival benefit: median survival 53 days for vehicle vs. 80 days for LIS (p=0.0001), 114 days for RT only (p<0.0001), 147 days for RT/LIS (p=0.30 relative to RT), 121 days for ‘Stupp’ regimen alone (p=0.57 relative to RT), and 172 days for Stupp/LIS (p=0.04 relative to Stupp). A follow-up GBM39 study revealed a significant increase in the mitotic marker phospho-histone H3 with LIS treatment relative to vehicle-treated controls (p=0.01) while Ki67 levels were similar (p=0.15). This suggests that LIS induces a mitotic arrest associated with microtubule deregulation. Collectively, these data provide a strong rationale to evaluate lisavanbulin (BAL101553) with RT +/- TMZ in GBM and provided the basis for an ongoing Phase I clinical trial. Citation Format: Danielle M Burgenske, Ann C Mladek, Jenny L Pokorny, Heidi A Lane, Felix Bachmann, Rachael A Vaubel, Mark A Schroeder, Katrina K Bakken, Lihong He, Zeng Hu, Brett L Carlson, Surabhi Talele, Gautham Gampa, Matthew L Kosel, Paul A Decker, Jeanette E Eckel-Passow, William F Elmquist, Jann Sarkaria. Modeling the clinical paradigm of lisavanbulin (BAL101553) deployment in patient-derived xenografts (PDX) of glioblastoma (GBM) [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr C096. doi:10.1158/1535-7163.TARG-19-C096

Abstract Cell survival following radiation therapy (RT) is critically modulated by multiple DNA damage response pathways including the p53 tumor suppressor, which promotes cell cycle arrest and/or apoptosis in the face of DNA damage. Murine Double Minute 2 (MDM2) targets p53 for degradation and suppresses its functions. The MDM2 locus is amplified in approximately 14% of glioblastoma (GBM) tumors and is a promising target for individualized therapy. In these studies, we tested the MDM2 small molecule inhibitor KRT-232, alone and in combination with RT, in MDM2-amplified and/or p53 wildtype patient-derived xenograft (PDX) models of GBM in vitro and in vivo. KRT-232 suppressed GBM PDX cell explant culture viability in three MDM2-amplified lines (GBM46, 108, and 148) and two non-amplified but p53 wildtype lines (GBM10 and 39) with similar IC50s ranging from 300-800nM in FBS culture conditions. As expected, little suppression was observed in a p53 mutant (GBM43) line. Many drugs have limited distribution in the brain, and to understand limitations surrounding KRT-232, LCMS-MS was used to compare brain and plasma levels of drug in mice. Preliminary pharmacokinetic analysis using FVB wildtype mice compared to mice which maintain triple knockout of the efflux transporters Mdr1a, Mdr1b and Bcrp1–/– (TKO) treated with a single 10 mg/kg oral dose of KRT-232 and harvested 2 hours later demonstrate an 8% brain/plasma ratio of KRT-232 in the TKO mice, while levels were undetectable in WT mice. In light of these data, the efficacy of KRT-232 was initially tested in vivo in an MDM2-amplified GBM108 PDX with an artificially disrupted BBB afforded by VEGFA lentiviral expression. In this model, daily dosing with KRT-232 alone produced a 100 day prolongation in survival as compared to vehicle. In a subsequent experiment, just one week of daily KRT-232 dosing, either alone or in combination with RT, in the control GBM108 PDX with a relatively intact BBB, was remarkably effective; median survival for placebo 22 days, KRT-232 alone 46 days, RT alone 31 days, and the KRT-232/RT combination 77 days. Despite low BBB penetration as determined by pharmacokinetic studies, KRT-232 alone and in combination with RT is an effective treatment in a pre-clinical model of glioblastoma. Future studies will evaluate KRT-232 pharmacodynamic responses as well as PDX orthotopic combination therapy in additional MDM2 amplified and non-amplified/p53 wildtype lines to understand if this therapy can be used in a larger population of GBM tumors. Citation Format: Ann C Mladek, Shiv Gupta, Minjee Kim, Afroz Shareef Mohammad, Katrina K Bakken, Helen He, Zeng Hu, Danielle M Burgenske, Brett L Carlson, William F Elmquist, Jann Sarkaria. MDM2 inhibitor KRT-232 extends survival in glioblastoma patient-derived xenograft models [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr C051. doi:10.1158/1535-7163.TARG-19-C051

Abstract Background: IDH mutated gliomas are critically dependent on glutaminase for glutamate biosynthesis. CB-839 is a novel glutaminase-1 inhibitor which has demonstrated efficacy in both genetically engineered and patient derived IDH mutated glioma cells, especially in combination with radiation. These preclinical studies evaluate the pharmacodynamic and pharmacokinetic impact of combining CB-839 with TMZ in patient derived xenograft (PDX) glioma models. Methods: GBM164 is a PDX model derived from an IDH1 mutant, 1p/19q non-codeleted glioma. D- and L-2HG levels in untreated GBM164 tumors were compared to GBM6 tumors (IDH wildtype glioma PDX) by GC/MS. Athymic nude mice bearing GBM164 flank tumors were treated with CB-839 (200 mg/kg PO BID x 9 doses) and/or temozolomide (50 mg/kg PO daily x 5 doses) and sacrificed four hours after the last dose to harvest plasma, normal brain and flank tumor. Tumor metabolomics were assessed by GC/MS. DNA damage signaling in tumors was assessed by Western blotting for KAP1 / Chk1 / Chk2 phosphorylation and H2AX / Rad51. Tumor, plasma and brain pharmacokinetics were assessed by LC/MS/MS. Results: Total 2HG levels in GBM164 were 18-fold higher than GBM6. CB-839 monotherapy reduced tumor glutamate (18% reduction, p = 0.15) and aspartate (30% reduction, p = 0.06) when compared to vehicle, however these changes did not reach statistical significance. The combination of CB-839 and TMZ more significantly reduced both tumor glutamate (30% reduction, p = 0.03) and aspartate (34% reduction, p < 0.001) when compared to TMZ monotherapy. CB-839 did not significantly increase DNA damage compared to vehicle, and the combination of CB-839 and TMZ did not significantly increase DNA damage compared to TMZ monotherapy. The mean tumor: plasma ratios of CB-839 concentrations were 0.68 and 0.58 in mice treated with CB-839 monotherapy and CB-839/TMZ, respectively. The mean brain: plasma ratios of CB-839 concentrations were 0.07 and 0.12 in mice treated with CB-839 monotherapy and CB-839/TMZ, respectively. Conclusion: The addition of CB-839 to TMZ significantly reduces glioma aspartate and glutamate in an IDH1 mutant PDX glioma model, without any impact on DNA damage. Survival studies are in progress to assess the efficacy of CB-839 when used in combination with RT and/or TMZ. Citation Format: Sani Haider Kizilbash, Danielle M. Burgenske, Samuel McBrayer, Sandhya Devarajan, Shiv K. Gupta, Taro Hitosugi, Lihong He, Mark A. Schroeder, Brett L. Carlson, Marina Gelman, Charles A. Kunos, Joel M. Reid, Alex A. Adjei, Jann N. Sarkaria. The addition of CB-839 to temozolomide significantly reduces glioma aspartate and glutamate in an IDH mutated patient derived glioma xenograft model [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 3870.

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.

AbstractBackgroundGlioblastoma (GBM) represents an aggressive cancer type with a median survival of only 14 months. With fewer than 5% of patients surviving 5 years, comprehensive profiling of these rare patients could elucidate prognostic biomarkers that may confer better patient outcomes. We utilized multiple molecular approaches to characterize the largest patient cohort of isocitrate dehydrogenase (IDH)–wildtype GBM long-term survivors (LTS) to date.MethodsRetrospective analysis was performed on 49 archived formalin-fixed paraffin embedded tumor specimens from patients diagnosed with GBM at the Mayo Clinic between December 1995 and September 2013. These patient samples were subdivided into 2 groups based on survival (12 LTS, 37 short-term survivors [STS]) and subsequently examined by mutation sequencing, copy number analysis, methylation profiling, and gene expression.ResultsOf the 49 patients analyzed in this study, LTS were younger at diagnosis (P = 0.016), more likely to be female (P = 0.048), and MGMT promoter methylated (UniD, P = 0.01). IDH-wildtype STS and LTS demonstrated classic GBM mutations and copy number changes. Pathway analysis of differentially expressed genes showed LTS enrichment for sphingomyelin metabolism, which has been linked to decreased GBM growth, invasion, and angiogenesis. STS were enriched for DNA repair and cell cycle control networks.ConclusionsWhile our findings largely report remarkable similarity between these LTS and more typical STS, unique attributes were observed in regard to altered gene expression and pathway enrichment. These attributes may be valuable prognostic markers and are worth further examination. Importantly, this study also underscores the limitations of existing biomarkers and classification methods in predicting patient prognosis.

Abstract O6-methylguanine-DNA methyltransferase (MGMT) is an enzyme that removes alkyl groups at the O6-position of guanine in DNA. MGMT expression is reduced or absent in many tumor types derived from a diverse range of tissues, most notably in glioma. Low MGMT expression confers significant sensitivity to DNA alkylating agents such as temozolomide, providing a natural therapeutic index over normal tissue. In this study, we sought to identify novel approaches that could maximally exploit the therapeutic index between tumor cells and normal tissues based on MGMT expression, as a means to enhance selective tumor cell killing. Temozolomide, unlike other alkylators, activated the ataxia telangiectasia and Rad3–related (ATR)–checkpoint kinase 1 (Chk1) axis in a manner that was highly dependent on MGMT status. Temozolomide induced growth delay, DNA double-strand breaks, and G2–M cell-cycle arrest, which led to ATR-dependent phosphorylation of Chk1; this effect was dependent on reduced MGMT expression. Treatment of MGMT-deficient cells with temozolomide increased sensitivity to ATR inhibitors both in vitro and in vivo across numerous tumor cell types. Taken together, this study reveals a novel approach for selectively targeting MGMT-deficient cells with ATR inhibitors and temozolomide. As ATR inhibitors are currently being tested in clinical trials, and temozolomide is a commonly used chemotherapeutic, this approach is clinically actionable. Furthermore, this interaction potently exploits a DNA-repair defect found in many cancers. Significance: Monofunctional alkylating agents sensitize MGMT-deficient tumor cells to ATR inhibitors.