Targeting BRCA1- and BRCA2-deficient cells with RAD52 small molecule inhibitors.

RAD52 is a member of the homologous recombination (HR) pathway that is important for maintenance of genome integrity. While single RAD52 mutations show no significant phenotype in mammals, their combination with mutations in genes that cause hereditary breast cancer and ovarian cancer like BRCA1, BRCA2, PALB2 and RAD51C are lethal. Consequently, RAD52 may represent an important target for cancer therapy. In vitro, RAD52 has ssDNA annealing and DNA strand exchange activities. Here, to identify small molecule inhibitors of RAD52 we screened a 372,903-compound library using a fluorescence-quenching assay for ssDNA annealing activity of RAD52. The obtained 70 putative inhibitors were further characterized using biochemical and cell-based assays. As a result, we identified compounds that specifically inhibit the biochemical activities of RAD52, suppress growth of BRCA1- and BRCA2-deficient cells and inhibit RAD52-dependent single-strand annealing (SSA) in human cells. We will use these compounds for development of novel cancer therapy and as a probe to study mechanisms of DNA repair.

Radiotherapy Combined with Intralesional Immunostimulatory Agents for Soft Tissue Sarcomas.

Immunotherapy has shifted the treatment paradigm for many types of cancer. Unfortunately, the most commonly used immunotherapies, such as immune checkpoint inhibitors (ICI), have yielded limited benefit for most types of soft tissue sarcoma (STS). Radiotherapy (RT) is a mainstay of sarcoma therapy and can induce immune modulatory effects. Combining immunotherapy and RT in STS may be a promising strategy to improve sarcoma response to RT and increase the efficacy of immunotherapy. Most combination strategies have employed immunotherapies, such as ICI, that derepress immune suppressive networks. These have yielded only modest results, possibly due to the limited immune stimulatory effects of RT. Combining RT with immune stimulatory agents has yielded promising preclinical and clinical results but can be limited by the toxic nature of systemic administration of immune stimulants. Using intralesional immune stimulants may generate stronger RT immune modulation and less systemic toxicity, which may be a feasible strategy in accessible tumors such as STS. In this review, we summarize the immune modulatory effects of RT, the mechanism of action of various immune stimulants, including toll-like receptor agonists, and data for combinatorial strategies utilizing these agents.

Post-death Vesicles of Senescent Bone Marrow Mesenchymal Stromal Polyploids Promote Macrophage Aging and Breast Cancer.

Potential systemic factors contributing to aging-associated breast cancer (BC) remain elusive. Here, we reveal that the polyploid giant cells (PGCs) that contain more than two sets of genomes prevailing in aging and cancerous tissues constitute 5-10% of healthy female bone marrow mesenchymal stromal cells (fBMSCs). The PGCs can repair DNA damage and stimulate neighboring cells for clonal expansion. However, dying PGCs in advanced-senescent fBMSCs can form "spikings" which are then separated into membraned mtDNA-containing vesicles (Senescent PGC-Spiking Bodies; SPSBs). SPSB-phagocytosed macrophages accelerate aging with diminished clearance on BC cells and protumor M2 polarization. SPSB-carried mitochondrial OXPHOS components are enriched in BC of elder patients and associated with poor prognosis. SPSB-incorporated breast epithelial cells develop aggressive characteristics and PGCs resembling the polyploid giant cancer cells (PGCCs) in clonogenic BC cells and cancer tissues. These findings highlight an aging BMSC-induced BC risk mediated by SPSB-induced macrophage dysfunction and epithelial cell precancerous transition. SIGNIFICANCE: Mechanisms underlying aging-associated cancer risk remain unelucidated. This work demonstrates that polyploid giant cells (PGCs) in bone marrow mesenchymal stromal cells (BMSCs) from healthy female bone marrow donors can boost neighboring cell proliferation for clonal expansion. However, the dying-senescent PGCs in the advanced-senescent fBMSCs can form "spikings" which are separated into mitochondrial DNA (mtDNA)-containing spiking bodies (senescent PGC-spiking bodies; SPSBs). The SPSBs promote macrophage aging and breast epithelial cell protumorigenic transition and form polyploid giant cancer cells. These results demonstrate a new form of ghost message from dying-senescent BMSCs, that may serve as a systemic factor contributing to aging-associated immunosuppression and breast cancer risk.

Advances in Radiotherapy Immune Modulation: From Bench-to-Bedside and Back Again.

Pre-clinical and clinical data clearly demonstrate the immune modulatory effects of radiotherapy (RT) but clinical trials testing RT + immunotherapy have been equivocal. An improved understanding of the immune modulatory effects of RT and how practical parameters of RT delivery (site and number of lesions, dose, fractionation, timing) influence these effects are needed to optimally combine RT with immunotherapy. Additionally, increased exploration of immunotherapy combinations with RT, beyond immune checkpoint inhibitors, are needed. A "bench-to-bedside and back again" approach will improve our understanding of RT immune modulation and allow for the implementation of more effective RT + immunotherapy strategies.

Mass Transport Model of Radiation Response: Calibration and Application to Chemoradiation for Pancreatic Cancer.

PURPOSE: The benefit of radiation therapy for pancreatic ductal adenocarcinoma (PDAC) remains unclear. We hypothesized that a new mechanistic mathematical model of chemotherapy and radiation response could predict clinical outcomes a priori, using a previously described baseline measurement of perfusion from computed tomography scans, normalized area under the enhancement curve (nAUC). METHODS AND MATERIALS: We simplified an existing mass transport model that predicted cancer cell death by replacing previously unknown variables with averaged direct measurements from randomly selected pathologic sections of untreated PDAC. This allowed using nAUC as the sole model input to approximate tumor perfusion. We then compared the predicted cancer cell death to the actual cell death measured from corresponding resected tumors treated with neoadjuvant chemoradiation in a calibration cohort (n = 80) and prospective cohort (n = 25). After calibration, we applied the model to 2 separate cohorts for pathologic and clinical associations: targeted therapy cohort (n = 101), cetuximab/bevacizumab + radiosensitizing chemotherapy, and standard chemoradiation cohort (n = 81), radiosensitizing chemotherapy to 50.4 Gy in 28 fractions. RESULTS: We established the relationship between pretreatment computed v nAUC to pathologically verified blood volume fraction of the tumor (r = 0.65; P = .009) and fractional tumor cell death (r = 0.97-0.99; P < .0001) in the calibration and prospective cohorts. On multivariate analyses, accounting for traditional covariates, nAUC independently associated with overall survival in all cohorts (mean hazard ratios, 0.14-0.31). Receiver operator characteristic analyses revealed discrimination of good and bad prognostic groups in the cohorts with area under the curve values of 0.64 to 0.71. CONCLUSIONS: This work presents a new mathematical modeling approach to predict clinical response from chemotherapy and radiation for PDAC. Our findings indicate that oxygen/drug diffusion strongly influences clinical responses and that nAUC is a potential tool to select patients with PDAC for radiation therapy.

Mathematical prediction of clinical outcomes in advanced cancer patients treated with checkpoint inhibitor immunotherapy.

We present a mechanistic mathematical model of immune checkpoint inhibitor therapy to address the oncological need for early, broadly applicable readouts (biomarkers) of patient response to immunotherapy. The model is built upon the complex biological and physical interactions between the immune system and cancer, and is informed using only standard-of-care CT. We have retrospectively applied the model to 245 patients from multiple clinical trials treated with anti-CTLA-4 or anti-PD-1/PD-L1 antibodies. We found that model parameters distinctly identified patients with common (n = 18) and rare (n = 10) malignancy types who benefited and did not benefit from these monotherapies with accuracy as high as 88% at first restaging (median 53 days). Further, the parameters successfully differentiated pseudo-progression from true progression, providing previously unidentified insights into the unique biophysical characteristics of pseudo-progression. Our mathematical model offers a clinically relevant tool for personalized oncology and for engineering immunotherapy regimens.

The Post-Synaptic Function of Brca2.

Homologous Recombination (HR) is a high-fidelity process with a range of biologic functions from generation of genetic diversity to repair of DNA double-strand breaks (DSBs). In mammalian cells, BRCA2 facilitates the polymerization of RAD51 onto ssDNA to form a presynaptic nucleoprotein filament. This filament can then strand invade a homologous dsDNA to form the displacement loop (D-loop) structure leading to the eventual DSB repair. Here, we have found that RAD51 in stoichiometric excess over ssDNA can cause D-loop disassembly in vitro; furthermore, we show that this RAD51 activity is countered by BRCA2. These results demonstrate that BRCA2 may have a previously unexpected activity: regulation of HR at a post-synaptic stage by modulating RAD51-mediated D-loop dissociation. Our in vitro results suggest a mechanistic underpinning of homeostasis between RAD51 and BRCA2, which is an important factor of HR in mammalian cells.