Connors, Margaret A.

Abstract Background Most oncology therapeutics have limited distribution into the brain, and developing strategies to overcome this limitation would be clinically impactful. While therapeutic radiation is often cited as a strategy to accomplish this, there are no published studies demonstrating the effect of radiation on drug distribution into the brain or brain tumors. Methods Mice were treated with brain radiation (6 Gy × 5, 4 Gy × 10; 40 Gy × 1) and dosed with drugs (levetiracetam, cefazolin, nedisertib, brigimadlin, apitolisib, or GNE-317) at times ranging from just prior to months after radiation. Plasma and tissue drug concentrations were measured by LC-MS/MS. Results Radiation did not significantly enhance drug delivery into brain tissue for levetiracetam, cefazolin, GNE-317, apitolisib, or nedisertib at any time post-radiation. Even a single, supra-therapeutic dose of radiation (40 Gy) did not significantly affect brain distribution of GNE-317 or apitolisib (P ≥ .07) from 16 to 160 hours post-radiation. For brigimadlin, radiation (6 Gy × 5) was associated with a modest but significant increase in drug accumulation only at 72 hours post-radiation (brain-to-plasma ratio 0.014 ± 0.006 vs. 0.025 ± 0.010, respectively; P = .04), but not at any other timepoint (24 hours, 15, 28, 94, 133, 183 days; P > .05). Similarly, radiation (6 Gy × 5) of orthotopic tumors did not increase levels of brigimadlin in GBM10 or GBM108 or nedisertib in GBM108 (P > .05). Conclusions Radiation had no meaningful impact on drug delivery into brain or brain tumors for the drugs tested.

Abstract Ataxia Telangiectasia–mutated (ATM) inhibitors are being developed as radiosensitizers to improve the antitumor effects of radiotherapy, but ATM inhibition can also radiosensitize normal tissues. Therefore, understanding the elevated risk of normal tissue toxicities is critical for radiosensitizer development. This study focused on modeling the relationship between acute mucosal toxicity, radiation dose, fractionation schedule, and radiosensitizer exposure. The ATM inhibitor WSD0628 was combined with single or fractionated doses of radiation delivered to the oral cavity or esophagus of Friend Leukemia virus B (FVB) mice. The potentiation by WSD0628 was quantified by a sensitizer enhancement ratio (SER), which describes the changes in radiation tolerance for radiation combined with WSD0628 relative to radiation-only regimens. WSD0628 profoundly enhanced radiation-induced acute oral and esophageal toxicities. For oral mucosal toxicity, the enhancement by WSD0628 with 3 fractions of radiation resulted in an SER ranging from 1.3 (0.25 mg/kg) to 3.1 (7.5 mg/kg). For the 7.5 mg/kg combination, the SER increased with increasing number of fractions from 2.2 (1 fraction) to 4.3 (7 fractions) for oral toxicity and from 2.2 (1 fraction) to 3.6 (3 fractions) for esophageal toxicity, which reflects a loss of the normal tissue sparing benefit of fractionated radiation. These findings were used to develop a modified biologically effective dose model to determine alternative radiation schedules with or without WSD0628 that result in similar levels of toxicity. Successful radiosensitizer dose escalation to a maximally effective therapeutic dose will require careful deliberation of tumor site and reduction of radiation dose volume limits for organs at risk.

Abstract Glioblastoma (GBM) remains one of the most therapy-resistant malignancies with frequent local failures despite aggressive surgery, chemotherapy, and ionizing radiation (IR). Small molecule inhibitors of DNA-dependent protein kinase (DNA-PKi) are potent radiosensitizers currently in clinical trials. Determining which patients may benefit from radiosensitization with DNA-PKi is critical to avoid unnecessary increased risk of normal tissue toxicity. In this study, we used GBM patient-derived xenografts (PDX) in orthotopic murine models to study the relationship between molecular features, pharmacokinetics, and the radiosensitizing potential of the DNA-PKi peposertib. We show that peposertib radiosensitizes established and PDX GBM lines in vitro at 300 nmol/L and above, with a significant increase in radiosensitization by maintaining post-IR exposure for >12 hours. Radiosensitization by peposertib is mediated by catalytic inhibition of DNA-PK, and knockdown of DNA-PK by short hairpin RNA (shRNA) largely abolished the radiosensitizing effect. Peposertib decreased auto-phosphorylation of DNA-PKcs after IR in a dose-dependent manner with a delay in resolution of γH2AX foci at 24 hours. The addition of peposertib to IR significantly increased survival in GBM120 orthotopic xenografts, but not in GBM10. There was no difference in plasma or average tumor concentrations of peposertib in the two cohorts. Although the mechanism underpinning this discordant effect in vitro versus in vivo is not clear, there was an association for greater sensitization in TP53 mutant lines. Transfection of a dominant-negative TP53 mutant in baseline TP53 wild-type GBM lines significantly delayed growth and decreased nonhomologous end joining efficiency (but not homologous recombination), after peposertib exposure. See related commentary by Buchsbaum, p. 840

Abstract Radioresistance of melanoma brain metastases limits the clinical utility of conventionally fractionated brain radiation in this disease, and strategies to improve radiation response could have significant clinical impact. The catalytic subunit of DNA-dependent protein kinase (DNA-PKcs) is critical for repair of radiation-induced DNA damage, and inhibitors of this kinase can have potent effects on radiation sensitivity. In this study, the radiosensitizing effects of the DNA-PKcs inhibitor peposertib were evaluated in patient-derived xenografts of melanoma brain metastases (M12, M15, M27). In clonogenic survival assays, peposertib augmented radiation-induced killing of M12 cells at concentrations ≥100 nmol/L, and a minimum of 16 hours exposure allowed maximal sensitization. This information was integrated with pharmacokinetic modeling to define an optimal dosing regimen for peposertib of 125 mpk dosed just prior to and 7 hours after irradiation. Using this drug dosing regimen in combination with 2.5 Gy × 5 fractions of radiation, significant prolongation in median survival was observed in M12-eGFP (104%; P = 0.0015) and M15 (50%; P = 0.03), while more limited effects were seen in M27 (16%, P = 0.04). These data support the concept of developing peposertib as a radiosensitizer for brain metastases and provide a paradigm for integrating in vitro and pharmacokinetic data to define an optimal radiosensitizing regimen for potent DNA repair inhibitors.

Abstract Background EGFR targeting antibody-drug conjugates (ADCs) are highly effective against EGFR-amplified tumors, but poor distribution across the blood–brain barrier (BBB) limits their efficacy in glioblastoma (GBM) when administered systemically. We studied whether convection-enhanced delivery (CED) can be used to safely infuse ADCs into orthotopic patient-derived xenograft (PDX) models of EGFRvIII mutant GBM. Methods The efficacy of the EGFR-targeted ADCs depatuxizumab mafodotin (Depatux-M) and Serclutamab talirine (Ser-T) was evaluated in vitro and in vivo. CED was performed in nontumor and tumor-bearing mice. Immunostaining was used to evaluate ADC distribution, pharmacodynamic effects, and normal cell toxicity. Results Dose-finding studies in orthotopic GBM6 identified single infusion of 2 μg Ser-T and 60 μg Depatux-M as safe and effective associated with extended survival prolongation (>300 days and 95 days, respectively). However, with serial infusions every 21 days, four Ser-T doses controlled tumor growth but was associated with lethal toxicity approximately 7 days after the final infusion. Limiting dosing to two infusions in GBM108 provided profound median survival extension of over 200 days. In contrast, four Depatux-M CED doses were well tolerated and significantly extended survival in both GBM6 (158 days) and GBM108 (310 days). In a toxicity analysis, Ser-T resulted in a profound loss in NeuN+ cells and markedly elevated GFAP staining, while Depatux-M was associated only with modest elevation in GFAP staining. Conclusion CED of Depatux-M is well tolerated and results in extended survival in orthotopic GBM PDXs. In contrast, CED of Ser-T was associated with a much narrower therapeutic window.

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 Introduction: EGFR targeted antibody-drug conjugates (ADCs) show promise as a novel treatment in a subset of glioblastoma (GBM). Two EGFR targeting ADCs include first generation Depatux-M, with an antimitotic toxin monomethyl auristatin F (MMAF), and ABBV-321, with a DNA crosslinking agent pyrrolobenzodiazepine dimer (PBD) toxin. Due to the large molecular weight, poor drug distribution across the blood-brain barrier significantly limits the efficacy in EGFR-amplified GBM. We studied whether convection enhanced delivery (CED) can be used to safely infuse these two EGFR-targeted ADCs in patient-derived xenograft (PDX) models of EGFR-amplified GBM. Methods: The efficacy of Depatux-M and ABBV-321 was evaluated in vitro and in vivo in two EGFRviii-amplified PDXs (GBM6 and GBM108). Immunofluorescence staining was used to evaluate drug distribution along with pharmacodynamics of the ADCs. CED was performed by stereotactic placement of an infusion catheter in the same location as the original tumor implantation. Immunohistochemistry was used to explore mechanisms of normal cell toxicity. Results: Despite potent activity in vitro (high ng/ml IC50), systemic administration of either ADC conferred minimal extension in survival for either GBM6 or GBM108. In contrast, CED significantly enhanced ADC delivery to tumor and peri-tumoral regions and extended survival. In a pilot study in GBM6 with n=3 mice per group, a single CED infusion of 0.002mg ABBV-321 resulted in extended survival beyond 365 days for two mice, while other doses and placebo were associated with shorter median survival (0 mg - 39 days; 0.03 mg - 105 days; 0.3 mg - 49 days). In a subsequent trial evaluating serial infusions performed every 21 days, four infusions at 0.002 mg ABBV-321 resulted in lethal toxicity. In contrast, limiting ABBV-321 to only two serial CED infusions in GBM108 was associated with extended survival of more than 300 days vs. 53 days for AB095 antibody control infusion. In contrast, serial infusion with Depatux-M was much better tolerated. In a single infusion in GBM6, a 0.06 mg dose was well tolerated and associated with a 49-day extension in median survival. Further, four serial infusions at 0.06 mg given every 21 days in GBM6 and GBM108 was associated with 100 day and more than 250-day extension in survival as compared to AB095 antibody control infusion. To investigate the mechanism of toxicity, infusion of non-tumor bearing C57Bl6 mice with ABBV-321 resulted in a marked loss in NeuN staining and elevated CD68 and GFAP staining 7 days later; Depatux-M infusion was only associated with modest elevation in GFAP without loss of NeuN staining or elevated CD68-positive cells. Conclusion: Depatux-M is well tolerated when infused into normal brain and results in extended survival in orthotopic GBM PDXs. In contrast, ABBV-321, with a distinct PBD toxin, had a much narrower therapeutic window when delivered by CED. Citation Format: Kendra A. Porath, Ann Mladek, Rachael A. Vaubel, Michael S. Regan, Sonia Jain, Danielle Burgenske, Katrina Bakken, Brett Carlson, Margaret Connors, Zeng Hu, Lihong He, Paul A. Decker, Gaspar Kitange, Shiv Gupta, Nathalie Y. Agar, Thomas M. Feldsien, Didier R. Lefebvre, Jann N. Sarkaria. Convection enhanced delivery of EGFR-targeting antibody drug conjugates ABBV-321 and Depatux-M [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 334.

Abstract Background RBBP4 activates transcription by histone acetylation, but the partner histone acetyltransferases are unknown. Thus, we investigated the hypothesis that RBBP4 interacts with p300 in a complex in glioblastoma (GBM). Methods shRNA silencing of RBBP4 or p300 and RNAseq was used to identify genes co-regulated by RBBP4 and p300 in GBM43 patient-derived xenograft (PDX). RBBP4/p300 complex was demonstrated using proximity ligation assay (PLA) and ChIPseq delineated histone H3 acetylation and RBBP4/p300 complex binding in promoters/enhancers. Temozolomide (TMZ)-induced DNA double strand breaks (DSBs) were evaluated by γ-H2AX and proliferation by CyQuant and live cell monitoring assays. In vivo efficacy was based on survival of mice with orthotopic tumors. Results shRBBP4 and shp300 downregulated 4768 genes among which 1485 (31%) were commonly downregulated by both shRNAs, while upregulated genes were 2484, including 863 (35%) common genes. The pro-survival genes were the top-ranked among the downregulated genes, including C-MYC. RBBP4/p300 complex was demonstrated in the nucleus, and shRBBP4 or shp300 significantly sensitized GBM cells to TMZ compared to the control shNT in vitro (P < .05). Moreover, TMZ significantly prolonged the survival of mice bearing GBM22-shRBBP4 orthotopic tumors compared with control shNT tumors (median shNT survival 52 days vs. median shRBBP4 319 days; P = .001). CREB-binding protein (CBP)/p300 inhibitor CPI-1612 suppressed H3K27Ac and RBBP4/p300 complex target proteins, including C-MYC, and synergistically sensitized TMZ in vitro. Pharmacodynamic evaluation confirmed brain penetration by CPI-1612 supporting further investigation to evaluate efficacy to sensitize TMZ. Conclusions RBBP4/p300 complex is present in GBM cells and is a potential therapeutic target.

Abstract Radio-resistance mechanisms limit the benefit of radiation therapy (RT) for melanoma brain metastases. A key pathway for radiation-induced DNA double-strand break repair is non-homologous end joining, which is critically mediated by DNA-dependent protein kinase (DNA-PKcs). Here we evaluated radio-sensitizing effects of M3814, a selective oral inhibitor of DNA-PKcs, in patient-derived xenografts (PDXs) of melanoma brain metastases. In a clonogenic survival assay, M3841 augmented RT-induced killing of M12 cells at concentrations of ≥300 nM, and a minimum of 16 h exposure with ~300 nM M3814 was required for effective sensitization. M3814 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. Persistent γH2AX foci were observed in 28% M12 cells at 24 hours after co-treatment with M3814 and RT as compared to 12% cells following RT alone. In vivo pharmacokinetic analysis after single oral dose of 20 mg/kg M3814, showed reasonably short half-life (~2.44 hours) and poor brain distribution in wild-type FBV mice (Kpuu, 0.027). Consistent with an efflux liability, brain distribution of M3814 in triple knockout mice for BCRP/MDR1A/B was ~11 fold higher (Kpuu, 0.215). Compared to normal brain, much higher M3814 concentrations were detected in intracranially implanted M12 tumors (~23 fold and ~20 fold) 2 and 6 hours after a single oral dose of 50mg/kg respectively. The relative exclusion of M3814 from normal brain as compared to brain metastases suggests that this drug may have a favorable toxicity profile when combined with radiation for treatment of melanoma brain metastases, and this hypothesis is being tested in ongoing efficacy studies.