A High-Throughput Drug Repurposing Strategy to Treat TBX2 and/or TBX3 Dependent Cancers.

BACKGROUND: The highly homologous T-box transcription factors TBX2 and TBX3 are critical for embryonic development, and their overexpression in postnatal tissues contributes to a wide range of malignancies, including melanoma and rhabdomyosarcoma. Importantly, when TBX2 and TBX3 are depleted in cancers where they are overexpressed, the malignant phenotype is inhibited, and they have therefore been regarded as druggable targets. However, the time and costs associated with de novo drug development are challenging and result in drugs that are costly, especially for patients in low- and middle-income countries. In the current study, we therefore combined a targeted and drug repurposing approach to identify drugs that are expected to be more efficacious and cost-effective with significantly reduced side effects. METHODS: A high-throughput cell-based immunofluorescence screen was performed to identify drugs in the Pharmakon 1600 drug library that can negatively regulate TBX2 and/or TBX3 levels. "Hit" drugs were validated for their effect on TBX2/TBX3 levels and cytotoxicity in TBX2/TBX3-dependent melanoma and rhabdomyosarcoma cells. To this end, immunofluorescence, western blotting, quantitative real-time PCR, and MTT cell viability assays were performed. RESULTS: Niclosamide, piroctone olamine, and pyrvinium pamoate, were identified as TBX2 and/or TBX3-targeting drugs, and they exhibited cytotoxicity in a TBX2/TBX3-dependent manner. Furthermore, these "Hit" drugs were shown to induce senescence and/or apoptosis. CONCLUSIONS: Niclosamide, piroctone olamine, and pyrvinium pamoate are promising, cost-effective therapeutic agents for the treatment of TBX2/TBX3-dependent cancers.

Proton Stereotactic Body Radiotherapy for Liver Metastases From Malignant Pancreatic Insulinoma.

Insulin-producing pancreatic tumors are a common subtype of neuroendocrine tumor. Standard of care includes surgical resection of the pancreatic tumor and medical management with somatostatin analogs. For patients with metastatic disease, tumor control and hypoglycemic symptom relief can be achieved through surgical resection of the tumor, hepatic artery embolization, radiofrequency ablation, or radioembolization using radioactive isotopes as well as with systemic therapy such as somatostatin analogs and everolimus. We present the case of a 74-year-old male with metastatic insulin-producing pancreatic carcinoma. After a long history of successfully controlling his hypoglycemic episodes post-liver wedge resection, bland embolizations subsequently failed to maintain control of the frequency and severity of his hypoglycemic symptoms. Stereotactic body radiotherapy (SBRT) with protons was used to achieve symptomatic control and led to partial radiographic response with complete resolution of his hypoglycemic episodes. This case demonstrates the potential utility of proton SBRT in metastatic insulinomas.

Particle Beam Radiobiology Status and Challenges: A PTCOG Radiobiology Subcommittee Report.

Particle therapy (PT) represents a significant advancement in cancer treatment, precisely targeting tumor cells while sparing surrounding healthy tissues thanks to the unique depth-dose profiles of the charged particles. Furthermore, their linear energy transfer and relative biological effectiveness enhance their capability to treat radioresistant tumors, including hypoxic ones. Over the years, extensive research has paved the way for PT's clinical application, and current efforts aim to refine its efficacy and precision, minimizing the toxicities. In this regard, radiobiology research is evolving toward integrating biotechnology to advance drug discovery and radiation therapy optimization. This shift from basic radiobiology to understanding the molecular mechanisms of PT aims to expand the therapeutic window through innovative dose delivery regimens and combined therapy approaches. This review, written by over 30 contributors from various countries, provides a comprehensive look at key research areas and new developments in PT radiobiology, emphasizing the innovations and techniques transforming the field, ranging from the radiobiology of new irradiation modalities to multimodal radiation therapy and modeling efforts. We highlight both advancements and knowledge gaps, with the aim of improving the understanding and application of PT in oncology.

A Practical Primer on Particle Therapy.

PURPOSE: Particle therapy is a promising treatment technique that is becoming more commonly used. Although proton beam therapy remains the most commonly used particle therapy, multiple other heavier ions have been used in the preclinical and clinical settings, each with its own unique properties. This practical review aims to summarize the differences between the studied particles, discussing their radiobiological and physical properties with additional review of the available clinical data. METHODS AND MATERIALS: A search was carried out on the PubMed databases with search terms related to each particle. Relevant radiobiology, physics, and clinical studies were included. The articles were summarized to provide a practical resource for practicing clinicians. RESULTS: A total of 113 articles and texts were included in our narrative review. Currently, proton beam therapy has the most data and is the most widely used, followed by carbon, helium, and neutrons. Although oxygen, neon, silicon, and argon have been used clinically, their future use will likely remain limited as monotherapy. CONCLUSIONS: This review summarizes the properties of each of the clinically relevant particles. Protons, helium, and carbon will likely remain the most commonly used, although multi-ion therapy is an emerging technique.

Targeting NKG2D ligands in glioblastoma with a bispecific T-cell engager is augmented with conventional therapy and enhances oncolytic virotherapy of glioma stem-like cells.

BACKGROUND: Glioblastoma (GBM) almost invariably becomes resistant towards conventional treatment of radiotherapy and temozolomide (TMZ) chemotherapy, partly due to subpopulations of intrinsically resistant glioma stem-like cells (GSC). The oncolytic herpes simplex virus-1 G207 is a promising approach for GBM virotherapy although its efficacy in patients with GBM is often limited. Natural killer group 2 member D ligands (NKG2DLs) are minimally expressed by healthy cells but are upregulated by the DNA damage response (DDR) and in malignant cells with chronic DDR signaling, resulting in innate immune activation. METHODS: We have designed a bispecific T-cell engager (BiTE) capable of cross-linking CD3 on T cells with NKG2DL-expressing GBM cells. We then engineered the G207 virus to express the NKG2D BiTE and secrete it from infected cells. The efficacy of the free BiTE and BiTE delivered by G207 was evaluated in combination with conventional therapies in GBM cells and against patient-derived GSCs in the context of T-cell activation and target cell viability. RESULTS: NKG2D BiTE-mediated cross-linking of GBM cells and T cells causes antigen-independent T-cell activation, pro-inflammatory cytokine release, and tumor cell death, thereby combining direct viral oncolysis with BiTE-mediated cytotoxicity. Surface NKG2DL expression was further elevated on GBM cells following pretreatment with sublethal doses of TMZ and radiation to induce the DDR, increasing sensitivity towards G207-NKG2D BiTE and achieving synergistic cytotoxicity. We also demonstrate a novel strategy for targeting GSCs that are non-permissive to G207 infection but remain sensitive to NKG2D BiTE. CONCLUSIONS: We propose a potential model for targeting GSCs in heterogeneous tumors, whereby differentiated GBM cells infected with G207-NKG2D BiTE produce NKG2D BiTE locally, directing T-cell cytotoxicity towards the GSC subpopulations in the tumor microenvironment.

Accuracy of a Cancer Registry Versus Clinical Care Team Chart Abstraction in Identifying Cancer Recurrence.

Objective: To evaluate the completeness and reliability of recurrence data from an institutional cancer registry for patients with head and neck cancer. Patients and Methods: Recurrence information was collected by radiation oncology and otolaryngology researchers. This was compared with the institutional cancer registry for continuous patients treated with radiation therapy for head and neck cancer at a tertiary cancer center. The sensitivity and specificity of institutional cancer registry data was calculated using manual review as the gold standard. False negative recurrences were compared to true positive recurrences to assess for differences in patient characteristics. Results: A total of 1338 patients who were treated from January 1, 2010, through December 31, 2017, were included in a cancer registry and underwent review. Of them, 375 (30%) had confirmed cancer recurrences, 45 (3%) had concern for recurrence without radiologic or pathologic confirmation, and 31 (2%) had persistent disease. Most confirmed recurrences were distant (37%) or distant plus locoregional (29%), whereas few were local (11%), regional (9%), or locoregional (14%) alone. The cancer registry accuracy was 89.4%, sensitivity 61%, and specificity 99%. Time to recurrence was associated with registry accuracy. True positives had recurrences at a median of 414 days vs 1007 days for false negatives. Conclusion: Currently, institutional cancer registry recurrence data lacks the required accuracy for implementation into studies without manual confirmation. Longer follow-up of cancer status will likely improve sensitivity. No identified differences in patients accounted for differences in sensitivity. New, ideally automated, data abstraction tools are needed to improve detection of cancer recurrences and minimize manual chart review.

Optimized protocol for CRISPR knockout of human iPSC-derived macrophages.

Here, we present a protocol for lentiviral delivery of CRISPR-Cas9 to human induced pluripotent stem cell (iPSC)-derived macrophages using co-incubation with VPX virus-like particles (VPX-VLPs). We describe steps for producing polybrene and puromycin kill curves, VPX viral production, and VPX-VLP titration by western blotting. We then detail procedures for iPSC macrophage precursor lentiviral transduction and lentiviral CRISPR-Cas9-based knockout in iPSC-derived macrophages. This protocol uses efficient genome-editing techniques to explore macrophage involvement in immune response, chronic inflammation, neurodegenerative disease, and cancer progression. For complete details on the use and execution of this protocol, please refer to Navarro-Guerrero et al.1.

The Radiosurgery Society Case-Based Discussion of the Management of Head and Neck or Skull Base Paragangliomas with Stereotactic Radiosurgery and Radiotherapy.

Stereotactic radiosurgery (SRS) and fractionated stereotactic radiotherapy (FSRT) have been used for the treatment of head and neck or skull base paraganglioma for a considerable time, demonstrating promising local control rates and a favorable safety profile compared with surgical approaches. Nevertheless, the choice of treatment must be carefully tailored to each patient's preferences, tumor location, and size, as well as anticipated treatment-related morbidity. This case-based review serves as a practical and concise guide for the use of SRS and FSRT in the management of head and neck or skull base paragangliomas, providing information on the diagnosis, treatment, follow-up considerations, and potential pitfalls.

Assessment of bladder filling during prostate cancer radiation therapy with ultrasound and cone-beam CT.

Prostate cancer patients undergoing external beam radiation therapy (EBRT) benefit from a full bladder to decrease bowel and bladder toxicity. Ultrasound may offer a proxy metric for evaluation, sparing CBCT dosing. Patients were prospectively enrolled pre-simulation from January 2017 to February 2018. Bladder volume was evaluated prior to RT using US daily and CBCT for three daily treatments and then weekly unless otherwise indicated. 29 patients completed median 40 days of RT, resulting in 478 CBCT and 1,099 US bladder volumes. 21 patients were treated to intact glands and 8 to the post-prostatectomy bed. Median patient age was 70 years. Bladder volume on CBCT and US positively correlated (r = 0.85), with average bladder volume for all patients of 162 mL versus 149 mL, respectively. Bladder volume during treatment was consistently lower than the volume at CT simulation (153 mL vs 194 mL, p<0.01) and progressively declined during treatment. Patients older than 70 years presented with lower average bladder volumes than those < 70 years (122 mL vs 208 mL, respectively, p<0.01). Patients with the highest agreement between CBCT and US (<10% variability) had higher average bladder volumes (192 mL vs 120 mL, p=0.01). US was found to be an accurate measure of bladder volume and may be used to monitor daily bladder volumes in patients being treated with radiation for prostate cancer.

C16orf72/HAPSTR1/TAPR1 functions with BRCA1/Senataxin to modulate replication-associated R-loops and confer resistance to PARP disruption.

While the toxicity of PARP inhibitors to cells with defects in homologous recombination (HR) is well established, other synthetic lethal interactions with PARP1/PARP2 disruption are poorly defined. To inform on these mechanisms we conducted a genome-wide screen for genes that are synthetic lethal with PARP1/2 gene disruption and identified C16orf72/HAPSTR1/TAPR1 as a novel modulator of replication-associated R-loops. C16orf72 is critical to facilitate replication fork restart, suppress DNA damage and maintain genome stability in response to replication stress. Importantly, C16orf72 and PARP1/2 function in parallel pathways to suppress DNA:RNA hybrids that accumulate at stalled replication forks. Mechanistically, this is achieved through an interaction of C16orf72 with BRCA1 and the RNA/DNA helicase Senataxin to facilitate their recruitment to RNA:DNA hybrids and confer resistance to PARP inhibitors. Together, this identifies a C16orf72/Senataxin/BRCA1-dependent pathway to suppress replication-associated R-loop accumulation, maintain genome stability and confer resistance to PARP inhibitors.

Chemogenomic library design strategies for precision oncology, applied to phenotypic profiling of glioblastoma patient cells.

Designing a targeted screening library of bioactive small molecules is a challenging task since most compounds modulate their effects through multiple protein targets with varying degrees of potency and selectivity. We implemented analytic procedures for designing anticancer compound libraries adjusted for library size, cellular activity, chemical diversity and availability, and target selectivity. The resulting compound collections cover a wide range of protein targets and biological pathways implicated in various cancers, making them widely applicable to precision oncology. We characterized the compound and target spaces of the virtual libraries, in comparison with a minimal screening library of 1,211 compounds for targeting 1,386 anticancer proteins. In a pilot screening study, we identified patient-specific vulnerabilities by imaging glioma stem cells from patients with glioblastoma (GBM), using a physical library of 789 compounds that cover 1,320 of the anticancer targets. The cell survival profiling revealed highly heterogeneous phenotypic responses across the patients and GBM subtypes.

Simulation study of comparative dosimetric analysis of coplanar horizontal-port scanned carbon-ion beam therapy in the head and neck.

OBJECTIVE: Carbon-ion radiotherapy (CIRT) has demonstrated success in treating radioresistant disease within the head and neck, owing to its unique physical and radiobiological properties. Construction cost remains prohibitive; a center offering only a horizontal port may bridge this difficulty, but removal of the vertical port may prohibit treatment of disease near critical organs-at-risk. Building a center only containing a horizontal treatment port has been proposed as one method for cost savings. METHODS: 20 complex cases of head and neck cancer previously treated with conventional CIRT were retrospectively planned using horizontal-port-only treatment incorporating non-coplanar treatment angles to achieve greater degrees of freedom. These were dosimetrically compared with the previous plans. RESULTS: Comparable D95 coverage of both planning target volume and gross tumor volume with ability to meet organ-at-risk constraints were feasible with horizontal-port-only treatment. Collectively differences were noted in PTV D95, brain stem Dmax, contralateral eye Dmax and V10 Gy (RBE); further qualitative differences were noted on a plan-by-plan basis dependent on disease location. CONCLUSION: Horizontal-port-only treatment employing non-coplanar angles was feasible for complicated head and neck disease typically treated with CIRT, though careful consideration is necessary on a plan-by-plan basis. ADVANCES IN KNOWLEDGE: It is worth noting that non-coplanar approaches are not typically used with the current treatment gantry and may extend further the difference between horizontal port planning and a gantry-based gold-standard.

Comparing Oncologic Outcomes and Toxicity for Combined Modality Therapy vs. Carbon-Ion Radiotherapy for Previously Irradiated Locally Recurrent Rectal Cancer.

No standard treatment paradigm exists for previously irradiated locally recurrent rectal cancer (PILRRC). Carbon-ion radiotherapy (CIRT) may improve oncologic outcomes and reduce toxicity compared with combined modality therapy (CMT). Eighty-five patients treated at Institution A with CIRT alone (70.4 Gy/16 fx) and eighty-six at Institution B with CMT (30 Gy/15 fx chemoradiation, resection, intraoperative electron radiotherapy (IOERT)) between 2006 and 2019 were retrospectively compared. Overall survival (OS), pelvic re-recurrence (PR), distant metastasis (DM), or any disease progression (DP) were analyzed with the Kaplan-Meier model, with outcomes compared using the Cox proportional hazards model. Acute and late toxicities were compared, as was the 2-year cost. The median time to follow-up or death was 6.5 years. Median OS in the CIRT and CMT cohorts were 4.5 and 2.6 years, respectively (p ≤ 0.01). No difference was seen in the cumulative incidence of PR (p = 0.17), DM (p = 0.39), or DP (p = 0.19). Lower acute grade ≥ 2 skin and GI/GU toxicity and lower late grade ≥ 2 GU toxicities were associated with CIRT. Higher 2-year cumulative costs were associated with CMT. Oncologic outcomes were similar for patients treated with CIRT or CMT, although patient morbidity and cost were lower with CIRT, and CIRT was associated with longer OS. Prospective comparative studies are needed.

Small-Molecule Polθ Inhibitors Provide Safe and Effective Tumor Radiosensitization in Preclinical Models.

PURPOSE: DNA polymerase theta (Polθ, encoded by the POLQ gene) is a DNA repair enzyme critical for microhomology mediated end joining (MMEJ). Polθ has limited expression in normal tissues but is frequently overexpressed in cancer cells and, therefore, represents an ideal target for tumor-specific radiosensitization. In this study we evaluate whether targeting Polθ with novel small-molecule inhibitors is a feasible strategy to improve the efficacy of radiotherapy. EXPERIMENTAL DESIGN: We characterized the response to Polθ inhibition in combination with ionizing radiation in different cancer cell models in vitro and in vivo. RESULTS: Here, we show that ART558 and ART899, two novel and specific allosteric inhibitors of the Polθ DNA polymerase domain, potently radiosensitize tumor cells, particularly when combined with fractionated radiation. Importantly, noncancerous cells were not radiosensitized by Polθ inhibition. Mechanistically, we show that the radiosensitization caused by Polθ inhibition is most effective in replicating cells and is due to impaired DNA damage repair. We also show that radiosensitization is still effective under hypoxia, suggesting that these inhibitors may help overcome hypoxia-induced radioresistance. In addition, we describe for the first time ART899 and characterize it as a potent and specific Polθ inhibitor with improved metabolic stability. In vivo, the combination of Polθ inhibition using ART899 with fractionated radiation is well tolerated and results in a significant reduction in tumor growth compared with radiation alone. CONCLUSIONS: These results pave the way for future clinical trials of Polθ inhibitors in combination with radiotherapy.

Automated 96-well format high throughput colony formation assay for siRNA library screen.

The colony formation assay is the gold-standard technique to assess cell viability after treatment with cytotoxic reagents, ionizing radiation, and cytotoxic combinatorial treatments. This protocol describes a high-throughput automated and high-content imaging approach to screen siRNA molecular libraries in HeLa cervical cancer cells in 96-well format. We detail reverse transfection of cells with siRNAs, followed by ionizing radiation, fixing, and staining of the plates for automated colony counting. This protocol can be used across a broad range of cell types. For complete details on the use and execution of this protocol, please refer to Tiwana et al. (2015).

Cost-Effectiveness of Treatment Strategies for Spinal Metastases.

PURPOSE: We analyzed the cost-effectiveness of standard palliative external beam radiation (EBRT, 8 Gy in 1 fraction), stereotactic body radiation therapy (SBRT, 24 Gy in 2 fractions), and radiofrequency ablation for painful spinal metastases. Single-fraction SBRT (delivering 24 Gy) was also assessed. METHODS AND MATERIALS: A Markov state transition model was constructed. Key model parameters were derived from prospective clinical trial data. Strategies were compared using the incremental cost-effectiveness ratio (ICER), with effectiveness in quality-adjusted life-years (QALYs) and a willingness-to-pay threshold of $100,000 per QALY gained. Costs included both hospital and professional costs using 2020 Medicare reimbursement. RESULTS: The base case demonstrated that 2-fraction SBRT was not cost-effective compared with single-fraction EBRT, with an ICER of $194,145 per QALY gained. Radiofrequency ablation was a more costly and less effective strategy in this model. Probabilistic sensitivity analysis demonstrated that EBRT was favored in 66% of model iterations. If median survival were improved after SBRT, 2-fraction SBRT became cost-effective, with ICERs of $80,394, $57,062, and $47,038 for 3-, 6-, and 9-month improvements in survival, respectively. Because 2-fraction SBRT data reported that 18% of patients had an indeterminant pain response at 3 months and 2-fraction SBRT is infrequently used in clinical practice, single-fraction SBRT data were also assessed. Single-fraction SBRT delivering 24 Gy was cost-effective compared with single-fraction EBRT, with an ICER of $92,833 per QALY gained. CONCLUSIONS: For appropriately chosen patients, single-fraction SBRT was more cost-effective than conventional EBRT or radiofrequency ablation. Conventional EBRT remains a cost-effective treatment for patients with poor expected survival.