Radiation and anti-PD-L1 synergize by stimulating a stem-like T cell population in the tumor-draining lymph node.

Radiotherapy (RT) and anti-PD-L1 synergize to enhance local and distant (abscopal) tumor control. However, clinical results in humans have been variable. With the goal of improving clinical outcomes, we investigated the underlying synergistic mechanism focusing on a CD8+ PD-1+ Tcf-1+ stem-like T cell subset in the tumor-draining lymph node (TdLN). Using murine melanoma models, we found that RT + anti-PD-L1 induces a novel differentiation program in the TdLN stem-like population which leads to their expansion and differentiation into effector cells within the tumor. Our data indicate that optimal synergy between RT + anti-PD-L1 is dependent on the TdLN stem-like T cell population as either blockade of TdLN egress or specific stem-like T cell depletion reduced tumor control. Together, these data demonstrate a multistep stimulation of stem-like T cells following combination therapy which is initiated in the TdLN and completed in the tumor.

ATR signaling controls the bystander responses of human chondrosarcoma cells by promoting RAD51-dependent DNA repair.

PURPOSE: Radiation-induced bystander effect (RIBE) frequently is seen as DNA damage in unirradiated bystander cells, but the repair processes initiated in response to that DNA damage are not well understood. RIBE-mediated formation of micronuclei (MN), a biomarker of persistent DNA damage, was previously observed in bystander normal fibroblast (AG01522) cells, but not in bystander human chondrosarcoma (HTB94) cells. The molecular mechanisms causing this disparity are not clear. Herein, we investigate the role of DNA repair in the bystander responses of the two cell lines. METHODS: Cells were irradiated with X-rays and immediately co-cultured with un-irradiated cells using a trans-well insert system in which they share the same medium. The activation of DNA damage response (DDR) proteins was detected by immunofluorescence staining or Western blotting. MN formation was examined by the cytokinesis-block MN assay, which is a robust method to detect persistent DNA damage. RESULTS: Immunofluorescent foci of γH2AX and 53BP1, biomarkers of DNA damage and repair, revealed a greater capacity for DNA repair in HTB94 cells than in AG01522 cells in both irradiated and bystander populations. Autophosphorylation of ATR at the threonine 1989 site was expressed at a greater level in HTB94 cells compared to AG01522 cells at the baseline and in response to hydroxyurea treatment or exposure to 1 Gy of X-rays. An inhibitor of ATR, but not of ATM, promoted MN formation in bystander HTB94 cells. In contrast, no effect of either inhibitor was observed in bystander AG01522 cells, indicating that ATR signaling might be a pivotal pathway to preventing the MN formation in bystander HTB94 cells. Supporting this idea, we found an ATR-dependent increase in the fractions of bystander HTB94 cells with pRPA2 S33 and RAD51 foci. A blocker of RAD51 facilitated MN formation in bystander HTB94 cells. CONCLUSION: Our results indicate that HTB94 cells were likely more efficient in DNA repair than AG01522 cells, specifically via ATR signaling, which inhibited the bystander signal-induced MN formation. This study highlights the significance of DNA repair efficiency in bystander cell responses.

A retrospective study on the investigation of potential dosimetric benefits of online adaptive proton therapy for head and neck cancer.

PURPOSE: Proton therapy is sensitive to anatomical changes, often occurring in head-and-neck (HN) cancer patients. Although multiple studies have proposed online adaptive proton therapy (APT), there is still a concern in the radiotherapy community about the necessity of online APT. We have performed a retrospective study to investigate the potential dosimetric benefits of online APT for HN patients relative to the current offline APT. METHODS: Our retrospective study has a patient cohort of 10 cases. To mimic online APT, we re-evaluated the dose of the in-use treatment plan on patients' actual treatment anatomy captured by cone-beam CT (CBCT) for each fraction and performed a templated-based automatic replanning if needed, assuming that these were performed online before treatment delivery. Cumulative dose of the simulated online APT course was calculated and compared with that of the actual offline APT course and the designed plan dose of the initial treatment plan (referred to as nominal plan). The ProKnow scoring system was employed and adapted for our study to quantify the actual quality of both courses against our planning goals. RESULTS: The average score of the nominal plans over the 10 cases is 41.0, while those of the actual offline APT course and our simulated online course is 25.8 and 37.5, respectively. Compared to the offline APT course, our online course improved dose quality for all cases, with the score improvement ranging from 0.4 to 26.9 and an average improvement of 11.7. CONCLUSION: The results of our retrospective study have demonstrated that online APT can better address anatomical changes for HN cancer patients than the current offline replanning practice. The advanced artificial intelligence based automatic replanning technology presents a promising avenue for extending potential benefits of online APT.

SAMHD1 expression contributes to doxorubicin resistance and predicts survival outcomes in diffuse large B-cell lymphoma patients.

Diffuse large B-cell lymphoma (DLBCL) is a commonly diagnosed, aggressive non-Hodgkin's lymphoma. While R-CHOP chemoimmunotherapy is potentially curative, about 40% of DLBCL patients will fail, highlighting the need to identify biomarkers to optimize management. SAMHD1 has a dNTPase-independent role in promoting resection to facilitate DNA double-strand break (DSB) repair by homologous recombination. We evaluated the relationship of SAMHD1 levels with sensitivity to DSB-sensitizing agents in DLBCL cells and the association of SAMHD1 expression with clinical outcomes in 79 DLBCL patients treated with definitive therapy and an independent cohort dataset of 234 DLBCL patients. Low SAMHD1 expression, Vpx-mediated, or siRNA-mediated degradation/depletion in DLBCL cells was associated with greater sensitivity to doxorubicin and PARP inhibitors. On Kaplan-Meier log-rank survival analysis, low SAMHD1 expression was associated with improved overall survival (OS), which on subset analysis remained significant only in patients with advanced stage (III-IV) and moderate to high risk (2-5 International Prognostic Index (IPI)). The association of low SAMHD1 expression with improved OS remained significant on multivariate analysis independent of other adverse factors, including IPI, and was validated in an independent cohort. Our findings suggest that SAMHD1 expression mediates doxorubicin resistance and may be an important prognostic biomarker in advanced, higher-risk DLBCL patients.

Streamlined pin-ridge-filter design for single-energy proton FLASH planning.

BACKGROUND: FLASH radiotherapy (FLASH-RT) with ultra-high dose rate has yielded promising results in reducing normal tissue toxicity while maintaining tumor control. Planning with single-energy proton beams modulated by ridge filters (RFs) has been demonstrated feasible for FLASH-RT. PURPOSE: This study explored the feasibility of a streamlined pin-shaped RF (pin-RF) design, characterized by coarse resolution and sparsely distributed ridge pins, for single-energy proton FLASH planning. METHODS: An inverse planning framework integrated within a treatment planning system was established to design streamlined pin RFs for single-energy FLASH planning. The framework involves generating a multi-energy proton beam plan using intensity-modulated proton therapy (IMPT) planning based on downstream energy modulation strategy (IMPT-DS), followed by a nested pencil-beam-direction-based (PBD-based) spot reduction process to iteratively reduce the total number of PBDs and energy layers along each PBD for the IMPT-DS plan. The IMPT-DS plan is then translated into the pin-RFs and the single-energy beam configurations for IMPT planning with pin-RFs (IMPT-RF). This framework was validated on three lung cases, quantifying the FLASH dose of the IMPT-RF plan using the FLASH effectiveness model. The FLASH dose was then compared to the reference dose of a conventional IMPT plan to measure the clinical benefit of the FLASH planning technique. RESULTS: The IMPT-RF plans closely matched the corresponding IMPT-DS plans in high dose conformity (conformity index of <1.2), with minimal changes in V7Gy and V7.4 Gy for the lung (<3%) and small increases in maximum doses (Dmax) for other normal structures (<3.4 Gy). Comparing the FLASH doses to the doses of corresponding IMPT-RF plans, drastic reductions of up to nearly 33% were observed in Dmax for the normal structures situated in the high-to-moderate-dose regions, while negligible changes were found in Dmax for normal structures in low-dose regions. Positive clinical benefits were seen in comparing the FLASH doses to the reference doses, with notable reductions of 21.4%-33.0% in Dmax for healthy tissues in the high-dose regions. However, in the moderate-to-low-dose regions, only marginal positive or even negative clinical benefit for normal tissues were observed, such as increased lung V7Gy and V7.4 Gy (up to 17.6%). CONCLUSIONS: A streamlined pin-RF design was developed and its effectiveness for single-energy proton FLASH planning was validated, revealing positive clinical benefits for the normal tissues in the high dose regions. The coarsened design of the pin-RF demonstrates potential advantages, including cost efficiency and ease of adjustability, making it a promising option for efficient production.

The putative RNA helicase DDX1 associates with the nuclear RNA exosome and modulates RNA/DNA hybrids (R-loops).

The RNA exosome is a ribonuclease complex that mediates both RNA processing and degradation. This complex is evolutionarily conserved, ubiquitously expressed, and required for fundamental cellular functions, including rRNA processing. The RNA exosome plays roles in regulating gene expression and protecting the genome, including modulating the accumulation of RNA-DNA hybrids (R-loops). The function of the RNA exosome is facilitated by cofactors, such as the RNA helicase MTR4, which binds/remodels RNAs. Recently, missense mutations in RNA exosome subunit genes have been linked to neurological diseases. One possibility to explain why missense mutations in genes encoding RNA exosome subunits lead to neurological diseases is that the complex may interact with cell- or tissue-specific cofactors that are impacted by these changes. To begin addressing this question, we performed immunoprecipitation of the RNA exosome subunit, EXOSC3, in a neuronal cell line (N2A), followed by proteomic analyses to identify novel interactors. We identified the putative RNA helicase, DDX1, as an interactor. DDX1 plays roles in double-strand break repair, rRNA processing, and R-loop modulation. To explore the functional connections between EXOSC3 and DDX1, we examined the interaction following double-strand breaks and analyzed changes in R-loops in N2A cells depleted for EXOSC3 or DDX1 by DNA/RNA immunoprecipitation followed by sequencing. We find that EXOSC3 interaction with DDX1 is decreased in the presence of DNA damage and that loss of EXOSC3 or DDX1 alters R-loops. These results suggest EXOSC3 and DDX1 interact during events of cellular homeostasis and potentially suppress unscrupulous expression of genes promoting neuronal projection.

CBCT-Based synthetic CT image generation using conditional denoising diffusion probabilistic model.

BACKGROUND: Daily or weekly cone-beam computed tomography (CBCT) scans are commonly used for accurate patient positioning during the image-guided radiotherapy (IGRT) process, making it an ideal option for adaptive radiotherapy (ART) replanning. However, the presence of severe artifacts and inaccurate Hounsfield unit (HU) values prevent its use for quantitative applications such as organ segmentation and dose calculation. To enable the clinical practice of online ART, it is crucial to obtain CBCT scans with a quality comparable to that of a CT scan. PURPOSE: This work aims to develop a conditional diffusion model to perform image translation from the CBCT to the CT distribution for the image quality improvement of CBCT. METHODS: The proposed method is a conditional denoising diffusion probabilistic model (DDPM) that utilizes a time-embedded U-net architecture with residual and attention blocks to gradually transform the white Gaussian noise sample to the target CT distribution conditioned on the CBCT. The model was trained on deformed planning CT (dpCT) and CBCT image pairs, and its feasibility was verified in brain patient study and head-and-neck (H&N) patient study. The performance of the proposed algorithm was evaluated using mean absolute error (MAE), peak signal-to-noise ratio (PSNR) and normalized cross-correlation (NCC) metrics on generated synthetic CT (sCT) samples. The proposed method was also compared to four other diffusion model-based sCT generation methods. RESULTS: In the brain patient study, the MAE, PSNR, and NCC of the generated sCT were 25.99 HU, 30.49 dB, and 0.99, respectively, compared to 40.63 HU, 27.87 dB, and 0.98 of the CBCT images. In the H&N patient study, the metrics were 32.56 HU, 27.65 dB, 0.98 and 38.99 HU, 27.00, 0.98 for sCT and CBCT, respectively. Compared to the other four diffusion models and one Cycle generative adversarial network (Cycle GAN), the proposed method showed superior results in both visual quality and quantitative analysis. CONCLUSIONS: The proposed conditional DDPM method can generate sCT from CBCT with accurate HU numbers and reduced artifacts, enabling accurate CBCT-based organ segmentation and dose calculation for online ART.

Synthetic CT generation from MRI using 3D transformer-based denoising diffusion model.

BACKGROUND AND PURPOSE: Magnetic resonance imaging (MRI)-based synthetic computed tomography (sCT) simplifies radiation therapy treatment planning by eliminating the need for CT simulation and error-prone image registration, ultimately reducing patient radiation dose and setup uncertainty. In this work, we propose a MRI-to-CT transformer-based improved denoising diffusion probabilistic model (MC-IDDPM) to translate MRI into high-quality sCT to facilitate radiation treatment planning. METHODS: MC-IDDPM implements diffusion processes with a shifted-window transformer network to generate sCT from MRI. The proposed model consists of two processes: a forward process, which involves adding Gaussian noise to real CT scans to create noisy images, and a reverse process, in which a shifted-window transformer V-net (Swin-Vnet) denoises the noisy CT scans conditioned on the MRI from the same patient to produce noise-free CT scans. With an optimally trained Swin-Vnet, the reverse diffusion process was used to generate noise-free sCT scans matching MRI anatomy. We evaluated the proposed method by generating sCT from MRI on an institutional brain dataset and an institutional prostate dataset. Quantitative evaluations were conducted using several metrics, including Mean Absolute Error (MAE), Peak Signal-to-Noise Ratio (PSNR), Multi-scale Structure Similarity Index (SSIM), and Normalized Cross Correlation (NCC). Dosimetry analyses were also performed, including comparisons of mean dose and target dose coverages for 95% and 99%. RESULTS: MC-IDDPM generated brain sCTs with state-of-the-art quantitative results with MAE 48.825 ± 21.491 HU, PSNR 26.491 ± 2.814 dB, SSIM 0.947 ± 0.032, and NCC 0.976 ± 0.019. For the prostate dataset: MAE 55.124 ± 9.414 HU, PSNR 28.708 ± 2.112 dB, SSIM 0.878 ± 0.040, and NCC 0.940 ± 0.039. MC-IDDPM demonstrates a statistically significant improvement (with p < 0.05) in most metrics when compared to competing networks, for both brain and prostate synthetic CT. Dosimetry analyses indicated that the target dose coverage differences by using CT and sCT were within ± 0.34%. CONCLUSIONS: We have developed and validated a novel approach for generating CT images from routine MRIs using a transformer-based improved DDPM. This model effectively captures the complex relationship between CT and MRI images, allowing for robust and high-quality synthetic CT images to be generated in a matter of minutes. This approach has the potential to greatly simplify the treatment planning process for radiation therapy by eliminating the need for additional CT scans, reducing the amount of time patients spend in treatment planning, and enhancing the accuracy of treatment delivery.

Streamlined Pin-Ridge-Filter Design for Single-energy Proton FLASH Planning.

PURPOSE: This study explored the feasibility of a streamlined pin-shaped ridge filter (pin-RF) design for single-energy proton FLASH planning. METHODS: An inverse planning framework integrated within a TPS was established for FLASH planning. The framework involves generating a IMPT plan based on downstream energy modulation strategy (IMPT-DS), followed by a nested spot reduction process to iteratively reduce the total number of pencil beam directions (PBDs) and energy layers along each PBD for the IMPT-DS plan. The IMPT-DS plan is then translated into the pin-RFs for a single-energy IMPT plan (IMPT-RF). The framework was validated on three lung cases, quantifying the FLASH dose of the IMPT-RF plan using the FLASH effectiveness model and comparing it with the reference dose of a conventional IMPT plan to assess the clinical benefit of the FLASH planning technique. RESULTS: The IMPT-RF plans closely matched the corresponding IMPT-DS plans in high dose conformity, with minimal changes in V7Gy and V7.4Gy for the lung (< 5%) and small increases in Dmax for other OARs (< 3.2 Gy). Comparing the FLASH doses to the doses of corresponding IMPT-RF plans, drastic reductions of up to ~33% were observed in Dmax for OARs in the high-to-moderate-dose regions with negligible changes in Dmax for OARs in low-dose regions. Positive clinical benefits were observed with notable reductions of 18.4-33.0% in Dmax for OARs in the high-dose regions. However, in the moderate-to-low-dose regions, only marginal positive or even negative clinical benefit for OARs were observed, such as increased lung V7Gy and V7.4Gy (16.4-38.9%). CONCLUSIONS: A streamlined pin-RF design for single-energy proton FLASH planning was validated, revealing positive clinical benefits for OARs in the high dose regions. The coarsened design of the pin-RF demonstrates potential cost efficiency and efficient production.

DNA-PK is activated by SIRT2 deacetylation to promote DNA double-strand break repair by non-homologous end joining.

DNA-dependent protein kinase (DNA-PK) plays a critical role in non-homologous end joining (NHEJ), the predominant pathway that repairs DNA double-strand breaks (DSB) in response to ionizing radiation (IR) to govern genome integrity. The interaction of the catalytic subunit of DNA-PK (DNA-PKcs) with the Ku70/Ku80 heterodimer on DSBs leads to DNA-PK activation; however, it is not known if upstream signaling events govern this activation. Here, we reveal a regulatory step governing DNA-PK activation by SIRT2 deacetylation, which facilitates DNA-PKcs localization to DSBs and interaction with Ku, thereby promoting DSB repair by NHEJ. SIRT2 deacetylase activity governs cellular resistance to DSB-inducing agents and promotes NHEJ. SIRT2 furthermore interacts with and deacetylates DNA-PKcs in response to IR. SIRT2 deacetylase activity facilitates DNA-PKcs interaction with Ku and localization to DSBs and promotes DNA-PK activation and phosphorylation of downstream NHEJ substrates. Moreover, targeting SIRT2 with AGK2, a SIRT2-specific inhibitor, augments the efficacy of IR in cancer cells and tumors. Our findings define a regulatory step for DNA-PK activation by SIRT2-mediated deacetylation, elucidating a critical upstream signaling event initiating the repair of DSBs by NHEJ. Furthermore, our data suggest that SIRT2 inhibition may be a promising rationale-driven therapeutic strategy for increasing the effectiveness of radiation therapy.

The RNA helicase DDX1 associates with the nuclear RNA exosome and modulates R-loops.

The RNA exosome is a ribonuclease complex that mediates both RNA processing and degradation. This complex is evolutionarily conserved, ubiquitously expressed, and required for fundamental cellular functions, including rRNA processing. The RNA exosome plays roles in regulating gene expression and protecting the genome, including modulating the accumulation of RNA-DNA hybrids (R-loops). The function of the RNA exosome is facilitated by cofactors, such as the RNA helicase MTR4, which binds/remodels RNAs. Recently, missense mutations in RNA exosome subunit genes have been linked to neurological diseases. One possibility to explain why missense mutations in genes encoding RNA exosome subunits lead to neurological diseases is that the complex may interact with cell- or tissue-specific cofactors that are impacted by these changes. To begin addressing this question, we performed immunoprecipitation of the RNA exosome subunit, EXOSC3, in a neuronal cell line (N2A) followed by proteomic analyses to identify novel interactors. We identified the putative RNA helicase, DDX1, as an interactor. DDX1 plays roles in double-strand break repair, rRNA processing, and R-loop modulation. To explore the functional connections between EXOSC3 and DDX1, we examined the interaction following double-strand breaks, and analyzed changes in R-loops in N2A cells depleted for EXOSC3 or DDX1 by DNA/RNA immunoprecipitation followed by sequencing (DRIP-Seq). We find that EXOSC3 interaction with DDX1 is decreased in the presence of DNA damage and that loss of EXOSC3 or DDX1 alters R-loops. These results suggest EXOSC3 and DDX1 interact during events of cellular homeostasis and potentially suppress unscrupulous expression of genes promoting neuronal projection.

YB1 modulates the DNA damage response in medulloblastoma.

Y-box binding protein 1 (YBX1 or YB1) is a therapeutically relevant oncoprotein capable of RNA and DNA binding and mediating protein-protein interactions that drive proliferation, stemness, and resistance to platinum-based therapies. Given our previously published findings, the potential for YB1-driven cisplatin resistance in medulloblastoma (MB), and the limited studies exploring YB1-DNA repair protein interactions, we chose to investigate the role of YB1 in mediating radiation resistance in MB. MB, the most common pediatric malignant brain tumor, is treated with surgical resection, cranio-spinal radiation, and platinum-based chemotherapy, and could potentially benefit from YB1 inhibition. The role of YB1 in the response of MB to ionizing radiation (IR) has not yet been studied but remains relevant for determining potential anti-tumor synergy of YB1 inhibition with standard radiation therapy. We have previously shown that YB1 drives proliferation of cerebellar granular neural precursor cells (CGNPs) and murine Sonic Hedgehog (SHH) group MB cells. While others have demonstrated a link between YB1 and homologous recombination protein binding, functional and therapeutic implications remain unclear, particularly following IR-induced damage. Here we show that depleting YB1 in both SHH and Group 3 MB results not only in reduced proliferation but also synergizes with radiation due to differential response dynamics. YB1 silencing through shRNA followed by IR drives a predominantly NHEJ-dependent repair mechanism, leading to faster γH2AX resolution, premature cell cycle re-entry, checkpoint bypass, reduced proliferation, and increased senescence. These findings show that depleting YB1 in combination with radiation sensitizes SHH and Group 3 MB cells to radiation.

HELZ promotes R loop resolution to facilitate DNA double-strand break repair by homologous recombination.

R loops are RNA-DNA hybrid containing structures involved in diverse cellular processes, including DNA double-strand break (DSB) repair. R loop homeostasis involving the formation and resolution of R loops is critical for DSB repair, and its dysregulation leads to genome instability. Here we show that the HELZ helicase promotes R loop resolution to facilitate DSB repair by homologous recombination (HR). HELZ depletion causes hypersensitivity to DSB-inducing agents, and HELZ localizes and binds to DSBs. HELZ depletion further leads to genomic instability in a R loop dependent manner and the accumulation of R loops globally and at DSBs. HELZ binds to R loops in response to DSBs and promotes their resolution, thereby facilitating HR to promote genome integrity. Our findings thus define a role for HELZ in promoting the resolution of R loops critical for DSB repair by HR.

SAMHD1 deacetylation by SIRT1 promotes DNA end resection by facilitating DNA binding at double-strand breaks.

Sterile alpha motif and HD domain-containing protein 1 (SAMHD1) has a dNTPase-independent function in promoting DNA end resection to facilitate DNA double-strand break (DSB) repair by homologous recombination (HR); however, it is not known if upstream signaling events govern this activity. Here, we show that SAMHD1 is deacetylated by the SIRT1 sirtuin deacetylase, facilitating its binding with ssDNA at DSBs, to promote DNA end resection and HR. SIRT1 complexes with and deacetylates SAMHD1 at conserved lysine 354 (K354) specifically in response to DSBs. K354 deacetylation by SIRT1 promotes DNA end resection and HR but not SAMHD1 tetramerization or dNTPase activity. Mechanistically, K354 deacetylation by SIRT1 promotes SAMHD1 recruitment to DSBs and binding to ssDNA at DSBs, which in turn facilitates CtIP ssDNA binding, leading to promotion of genome integrity. These findings define a mechanism governing the dNTPase-independent resection function of SAMHD1 by SIRT1 deacetylation in promoting HR and genome stability.

Deformable CT image registration via a dual feasible neural network.

PURPOSE: A quality assurance (QA) CT scans are usually acquired during cancer radiotherapy to assess for any anatomical changes, which may cause an unacceptable dose deviation and therefore warrant a replan. Accurate and rapid deformable image registration (DIR) is needed to support contour propagation from the planning CT (pCT) to the QA CT to facilitate dose volume histogram (DVH) review. Further, the generated deformation maps are used to track the anatomical variations throughout the treatment course and calculate the corresponding accumulated dose from one or more treatment plans. METHODS: In this study, we aim to develop a deep learning (DL)-based method for automatic deformable registration to align the pCT and the QA CT. Our proposed method, named dual-feasible framework, was implemented by a mutual network that functions as both a forward module and a backward module. The mutual network was trained to predict two deformation vector fields (DVFs) simultaneously, which were then used to register the pCT and QA CT in both directions. A novel dual feasible loss was proposed to train the mutual network. The dual-feasible framework was able to provide additional DVF regularization during network training, which preserves the topology and reduces folding problems. We conducted experiments on 65 head-and-neck cancer patients (228 CTs in total), each with 1 pCT and 2-6 QA CTs. For evaluations, we calculated the mean absolute error (MAE), peak-signal-to-noise ratio (PSNR), structural similarity index (SSIM), target registration error (TRE) between the deformed and target images and the Jacobian determinant of the predicted DVFs. RESULTS: Within the body contour, the mean MAE, PSNR, SSIM, and TRE are 122.7 HU, 21.8 dB, 0.62 and 4.1 mm before registration and are 40.6 HU, 30.8 dB, 0.94, and 2.0 mm after registration using the proposed method. These results demonstrate the feasibility and efficacy of our proposed method for pCT and QA CT DIR. CONCLUSION: In summary, we proposed a DL-based method for automatic DIR to match the pCT to the QA CT. Such DIR method would not only benefit current workflow of evaluating DVHs on QA CTs but may also facilitate studies of treatment response assessment and radiomics that depend heavily on the accurate localization of tissues across longitudinal images.

Protocol for in vitro lysine deacetylation to test putative substrates of class III deacetylases.

Lysine acetylation is an important post-translational modification that is used in multiple cellular pathways, such as the regulation of gene expression at the histone level. The purpose of this assay is to test for putative substrates of class III deacetylases using an in vitro method. The in vitro analysis helps circumvent confounding variables when assessing for a direct relationship between deacetylase and substrate, such as the effects of other cellular deacetylases or acetyltransferases that modify the substrate in vivo. For complete details on the use and execution of this protocol, please refer to Minten et al. (2021).

Quantitative proteomic analysis of the lysine acetylome reveals diverse SIRT2 substrates.

Sirtuin 2 (SIRT2) is a NAD+-dependent deacetylase, which regulates multiple biological processes, including genome maintenance, aging, tumor suppression, and metabolism. While a number of substrates involved in these processes have been identified, the global landscape of the SIRT2 acetylome remains unclear. Using a label-free quantitative proteomic approach following enrichment for acetylated peptides from SIRT2-depleted and SIRT2-overexpressing HCT116 human colorectal cancer cells, we identified a total of 2,846 unique acetylation sites from 1414 proteins. 896 sites from 610 proteins showed a > 1.5-fold increase in acetylation with SIRT2 knockdown, and 509 sites from 361 proteins showed a > 1.5-fold decrease in acetylation with SIRT2 overexpression, with 184 proteins meeting both criteria. Sequence motif analyses identified several site-specific consensus sequence motifs preferentially recognized by SIRT2, most commonly KxxxxK(ac). Gene Ontology, KEGG, and MetaCore pathway analyses identified SIRT2 substrates involved in diverse pathways, including carbon metabolism, glycolysis, spliceosome, RNA transport, RNA binding, transcription, DNA damage response, the cell cycle, and colorectal cancer. Collectively, our findings expand on the number of known acetylation sites, substrates, and cellular pathways targeted by SIRT2, providing support for SIRT2 in regulating networks of proteins in diverse pathways and opening new avenues of investigation into SIRT2 function.

Structural and functional characterization explains loss of dNTPase activity of the cancer-specific R366C/H mutant SAMHD1 proteins.

Elevated intracellular levels of dNTPs have been shown to be a biochemical marker of cancer cells. Recently, a series of mutations in the multifunctional dNTP triphosphohydrolase (dNTPase), sterile alpha motif and histidine-aspartate domain-containing protein 1 (SAMHD1), have been reported in various cancers. Here, we investigated the structure and functions of SAMHD1 R366C/H mutants, found in colon cancer and leukemia. Unlike many other cancer-specific mutations, the SAMHD1 R366 mutations do not alter cellular protein levels of the enzyme. However, R366C/H mutant proteins exhibit a loss of dNTPase activity, and their X-ray structures demonstrate the absence of dGTP substrate in their active site, likely because of a loss of interaction with the γ-phosphate of the substrate. The R366C/H mutants failed to reduce intracellular dNTP levels and restrict HIV-1 replication, functions of SAMHD1 that are dependent on the ability of the enzyme to hydrolyze dNTPs. However, these mutants retain dNTPase-independent functions, including mediating dsDNA break repair, interacting with CtIP and cyclin A2, and suppressing innate immune responses. Finally, SAMHD1 degradation in human primary-activated/dividing CD4+ T cells further elevates cellular dNTP levels. This study suggests that the loss of SAMHD1 dNTPase activity induced by R366 mutations can mechanistically contribute to the elevated dNTP levels commonly found in cancer cells.

SIRT2 promotes BRCA1-BARD1 heterodimerization through deacetylation.

The breast cancer type I susceptibility protein (BRCA1) and BRCA1-associated RING domain protein I (BARD1) heterodimer promote genome integrity through pleiotropic functions, including DNA double-strand break (DSB) repair by homologous recombination (HR). BRCA1-BARD1 heterodimerization is required for their mutual stability, HR function, and role in tumor suppression; however, the upstream signaling events governing BRCA1-BARD1 heterodimerization are unclear. Here, we show that SIRT2, a sirtuin deacetylase and breast tumor suppressor, promotes BRCA1-BARD1 heterodimerization through deacetylation. SIRT2 complexes with BRCA1-BARD1 and deacetylates conserved lysines in the BARD1 RING domain, interfacing BRCA1, which promotes BRCA1-BARD1 heterodimerization and consequently BRCA1-BARD1 stability, nuclear retention, and localization to DNA damage sites, thus contributing to efficient HR. Our findings define a mechanism for regulation of BRCA1-BARD1 heterodimerization through SIRT2 deacetylation, elucidating a critical upstream signaling event directing BRCA1-BARD1 heterodimerization, which facilitates HR and tumor suppression, and delineating a role for SIRT2 in directing DSB repair by HR.