Dragojevic, Sonja

Background/Objectives: We performed a systematic review of preclinical literature on the use of high-LET particle therapy, DDRi, and/or immunotherapy specifically in pMMR colorectal cancer. Methods: A systematic review of the literature published between 2014 and 2025 was conducted across major databases. Studies were included if they examined particle radiotherapy (e.g., proton, alpha, and carbon) or X-ray radiation either alone or in combination with DDRi and/or immune checkpoint inhibitors (ICIs) in pMMR colorectal cancer models. Results: In total, 131 studies met the inclusion criteria, including 70 preclinical studies. These studies consistently demonstrate that high-LET radiation amplifies immunogenic cell death, increases cGAS-STING pathway activation, and enhances tumor antigen presentation, thereby fostering greater immune infiltration and systemic antitumor responses. Concurrent irradiation with DDRi enhances persistent DNA damage and cytosolic DNA accumulation. In murine models, high-LET therapies show excellent local control, with manageable toxicity profiles. Combination regimens with ICIs exhibit improved local control and elicit systemic antitumor immune responses. Conclusions: High-LET particle radiation and/or the use of concurrent DDRi with ICI have significant preclinical evidence of immunostimulatory effects in pMMR rectal adenocarcinoma and increased response rates to immunotherapy. The clinical evidence will be reviewed in the companion manuscript.

Background/Objectives: We performed a systematic review of the current clinical and preclinical literature on the use of particle therapy, DDRi, and/or immunotherapy, specifically for pMMR colorectal cancer. Methods: A systematic review of the literature published between 2014 and 2025 was conducted across major databases. Studies were included if they examined particle radiotherapy (e.g., proton, alpha, carbon) either alone or in combination with DDRi and/or immune checkpoint inhibitors (ICI), or X-ray radiotherapy (XRT) in combination with DDRi/ICI. Results: In total, 133 studies met the inclusion criteria, including 62 clinical studies. Clinically, particle therapies show excellent local control and normal tissue sparing, with manageable toxicity profiles. Trials with any form of radiation and DDRi are few, potentially owing to toxicity concerns. ICI combinations showed promising efficacy with XRT, with no randomized trials comparing them to particle radiation. Conclusions: Particle radiation and/or DDRi have significant preclinical evidence of immunostimulatory effects in pMMR rectal adenocarcinoma and increase response rates to immunotherapy (presented in the companion manuscript). Despite strong preclinical evidence and rationale, clinical trials including all three modalities are scarce. Existing evidence suggests a potential benefit based on extrapolation from photon-based studies and supports prospective evaluation with careful attention to treatment-related toxicity, which remains a concern.

Abstract Background The ataxia-telangiectasia mutated (ATM) protein coordinates the cellular response to therapeutic radiation, and ATM inhibitors can potentially enhance the efficacy of radiation in otherwise radiation-resistant tumors. Methods The small molecule ATM kinase inhibitor WSD0628 was specifically designed for enhanced distribution across the blood-brain barrier to more effectively treat glioblastoma (GBM) and brain metastases in combination with radiation therapy (RT). GBM and brain metastasis patient-derived xenograft models were used to understand target inhibition, radiosensitization, inhibition of the DNA damage response, and in vivo efficacy. Results Initial in vitro characterization of WSD0628 demonstrates a high level of selectivity across kinase families, limited aldehyde oxidase liability, and low risk of hERG interactions. Consistent with a central role for ATM in radiation response, WSD0628 blocked radiation-induced signaling and enhanced radiosensitivity in U251 glioma cells and brain tumor PDXs GBM120 and M12. In comparison to control or RT alone in orthotopic PDXs, the combination of WSD0628 with RT markedly prolonged median survival—GBM12 (19, 55, and 408 days, respectively); GBM43 (26, 44, and 143 days, respectively); GBM120 (51, 89, and 231 days, respectively); M12 (17, 39, and 190 days, respectively). Pharmacokinetic and pharmacodynamic testing after treatment in orthotopic GBM43 tumors showed inhibitory levels of WSD0628 and a reduction of γH2AX foci in the combination-treated tumors. Conclusions Collectively, these results suggest a promising role for WSD0628 in combination with RT in brain tumors and provide the rationale for an ongoing Phase 0/1A clinical trial testing this combination in recurrent GBM.

259 Background: Rectal cancer patients with a complete response (CR) after chemotherapy and radiotherapy may opt for non-operative management. Currently only 20-40% of patients reach a CR. Escalating the dose of radiation may increase the risk of toxicity. ATM inhibitors (ATMi) are potent radiosensitizers in colorectal cancer (CRC). Proton irradiation exhibits an entrance portion of the beam (ENT) where dose is deposited sparsely and a Bragg Peak (BP) with virtually no ‘exit dose’ thereafter. ATMi renders cells uniquely hypersensitive to proton BP, and BP+ATMi may increase the response of Mismatch Repair proficient (pMMR) rectal cancer to immunotherapy. Methods: Tumor efficacy and immunogenicity was assessed using clonogenic survival, γH2AX foci, micronuclei, and cytoplasmic cGas assays after indicated IR modalities +/-ATMi. Normal tissue toxicity was assessed via murine models of non-tumor bearing BALB/c after whole-abdominal irradiation (WAI) after indicated IR modalities +/- ATMi via acute weight loss, FITC/Dextran assay, and Radiation Injury Score (RIS, Scale 0-18) in jejunal and rectal samples at 4 days post-IR. Results: ATMi decreased clonogenic survival across all types of radiation, but the effect was most pronounced with BP protons. BP+ATMi also significantly increased unresolved γH2AX foci, micronuclei, and cytoplasmic cGas compared to ENT+ATMi. Normal tissue toxicity studies demonstrated that BP+ATMi did not significantly increase acute weight loss, RIS in jejunal or rectal tissue; but did increase FITC/Dextran intestinal permeability. Conclusions: Inhibition of ATM preferentially radiosensitized BP protons compared to x-rays or ENT protons in CRC but not in normal tissue based on weight loss and histopathological evaluation. Differential tumor efficacy and normal tissue toxicity after x-ray, entrance and Bragg Peak Protons +/- ATM inhibitor. A B C D E F G H I J DMSO 1 1 1 2.9 1.5 8764 96.4 10 0.3 0.2 ATMi 0.873 0.842 0.723 8.2 3.0 16300 98.5 5.8 0.7 0.8 x-ray 0.615 0.751 0.109 82.4 6.2 4.8 1.0 x-ray+ATMi 0.123 0.344 0.011 80.0 14.4 5.2 1.5 ENT protons 0.293 0.515 0.080 10.2 5.8 10429 78.5 6 2.9 1.5 ENT+ATMi 0.091 0.066 0.013 15.1 7.2 12274 73.0 23.8 5.2 3.8 BP protons 0.213 0.340 0.032 12.0 12.6 7192 75.6 24.6 4.1 3.7 BP+ATMi 0.074 0.038 0.004 22.7 16.2 43677 73.6 310.4 5.3 4.3 Clonogenic survival of pMMR colorectal cancer cell lines after IR (4 Gy) +/- 10nM AZD1390 (ATM inhibitor) ofA) HT29, B) CT26, and C) SW480 cell lines. D) γH2AX foci at 24 hours post-IR in HT29. E) Micronuclei, and F) cytoplasmic cGas integrated density per cell via immunofluorescence in SW480 72 hours post-IR. WAI (10Gy/1 fraction) was delivered to non-tumor bearing BALB/c mice which were followed for G) acute weight loss (% from baseline), and H) FITC/Dextran permeability assay. Tissue samples were graded for Radiation Injury Score in I) jejunal, and J) rectal samples at 4 days post-IR (n=5 per cohort, 3 samples per mouse). Figures are means of triplicate data.

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 Just 20% of patients with LARC attain a complete response to preoperative chemoradiotherapy which portends an excellent prognosis and opportunity to preserve the rectum through nonoperative surveillance. Moreover, normal tissue radiation toxicity can lead to significant morbidity, and becomes an even more important consideration in non-operative approaches where irradiated tissue is not resected. The geometry of conventional proton beams is already being used clinically to spare patient’s normal tissue, but the biology of entrance (ENT) vs. Bragg Peak (BP) protons has yet to be fully exploited to enhance tumor efficacy. Protons, unlike conventional x-ray radiation, spare normal tissue by lacking an exit dose - but they also exhibit a Bragg Peak (BP), which causes more complex DNA damage and is more biologically effective. ATM is critical in the repair of double strand DNA breaks (DSBs) and our lab has demonstrated that inhibitors of ATM (iATM) preferentially radiosensitize BP protons in CRC (Fig. 1A). Overactivation of the Wnt/β-catenin pathway via loss of function of APC is the most common genetic mutation in colorectal cancer (75-80%) and β-catenin over-expression negatively correlates with prognosis. WSB1 has been discovered by our lab to regulate ATM ubiquitination and degradation, it decreases metastates-free survival in CRC, and also forms a positive feedback loop with β-catenin and c-Myc that drives oncogenic progression. It is unknown if this upregulation of WSB1 promotes ATM degredation in LARC specifically, and a deubiquitinase (DUB) for ATM (which would counter WSB1) has yet to be identified. We preformed a comprehensive database search (UbiBrowser 2.0) for the most likely predicted DUBs for ATM (Fig. 1B), several of which have been identified as regulators of β-catenin/c-Myc, DSB repair pathways, and correlated with poor prognosis in CRC. We have also performed a screen of DUBs predicted to interact with ATM, and have preliminary evidence suggesting USP14 as the worlds first identified DUB of ATM (Fig 1C). USP14 expression is upregulated in CRC compared to surrounding normal tissue, promotes radioresistance, and promotes Wnt/β-catenin signalling. Higher USP14 expression in CRC is associated with liver and lymph node metastases and worse overall survival. USP14 regulates β-catenin by deubiquitinating K63-linked polyubiquitin chains of Dvl, and several small molecule inhibitors of USP14 have been identified. Our lab has preliminary data demonstrating USP14 is preferentially activated after BP proton irradiation (Fig. 1D), suggesting ATM may be necessary for repair of complex DSBs. While more work will be necessary to confirm these findings, many of the other predicted DUBs of ATM have overlapping functions regulating DSB repair and/or β-catenin/c-Myc signalling. We hypothesize that targeting ATM ubiquitination will open a therapeutic window in LARC by disrupting β-catenin/c-Myc signalling and selectively radiosensitizing BP protons. Citation Format: Sonja Dragojevic, Cameron M. Callaghan. Enhancing the therapeutic gain of proton radiotherapy in Locally Advanced Rectal Cancer (LARC) through manipulation of WSB1, ATM, and β-Catenin/c-Myc pathway Ubiquitination [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr B065.

Glioblastoma (GBM) is a primary brain tumor that carries a dismal prognosis, which is primarily attributed to tumor recurrence after surgery and resistance to chemotherapy. Since the tumor recurrence appears near the site of surgical resection, a concept of immediate and local application of chemotherapeutic after initial tumor removal could lead to improved treatment outcome. With the ultimate goal of developing a locally-applied, injectable drug delivery vehicle for GBM treatment, we created elastin-like polypeptide (ELP) hydrogels. The ELP hydrogels can be engineered to release anti-cancer drugs over an extended period. The purpose of this study was to evaluate the biomechanical properties of ELP hydrogels, to characterize their ability to release doxorubicin over time, and to investigate, in vitro, the anti-proliferative effect of Dox-laden ELP hydrogels on GBM. Here, we present microstructural differences, swelling ratio measurements, drug release characteristics, and in vitro effects of different ELP hydrogel compositions. We found that manipulation of the ELP–collagen ratio allows for tunable drug release, that the released drug is taken up by cells, and that incubation with a small volume of ELP-Dox hydrogel drastically reduced survival and proliferation of GBM cells in vitro. These results underscore the potential of ELP hydrogels as a local delivery strategy to improve prognosis for GBM patients after tumor resection.

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.