Bcl-xL inhibition radiosensitizes PIK3CA/PTEN wild-type triple negative breast cancers with low Mcl-1 expression.

Patients with radioresistant breast cancers, including a large percentage of women with triple negative breast cancer (TNBC), demonstrate limited response to radiation (RT) and increased locoregional recurrence; thus, strategies to increase the efficacy of RT in TNBC are critically needed. We demonstrate that pan Bcl-2 family inhibition (ABT-263, rER: 1.52-1.56) or Bcl-xL specific inhibition (WEHI-539, A-1331852; rER: 1.31-2.00) radiosensitized wild-type PIK3CA/PTEN TNBC (MDA-MB-231, CAL-120) but failed to radiosensitize mutant PIK3CA/PTEN TNBC (rER: 0.90 - 1.07; MDA-MB-468, CAL-51, SUM-159). Specific inhibition of Bcl-2 or Mcl-1 did not induce radiosensitization, regardless of PIK3CA/PTEN status (rER: 0.95 - 1.07). In wild-type PIK3CA/PTEN TNBC, pan Bcl-2 family inhibition or Bcl-xL specific inhibition with RT led to increased levels of apoptosis (p < 0.001) and an increase in cleaved PARP and cleaved caspase 3. CRISPR-mediated PTEN knockout in wild-type PIK3CA/PTEN MDA-MB-231 and CAL-120 cells induced expression of pAKT/Akt and Mcl-1 and abolished Bcl-xL inhibitor-mediated radiosensitization (rER: 0.94 - 1.07). Similarly, Mcl-1 overexpression abolished radiosensitization in MDA-MB-231 and CAL-120 cells (rER: 1.02 - 1.04) but transient MCL1 knockdown in CAL-51 cells promoted Bcl-xL-inhibitor mediated radiosensitization (rER 2.35 ± 0.05). In vivo, ABT-263 or A-1331852 in combination with RT decreased tumor growth and increased tumor tripling time (p < 0.0001) in PIK3CA/PTEN wild-type TNBC cell line and patient-derived xenografts. Collectively, this study provides the preclinical rationale for early phase clinical trials testing the safety, tolerability, and efficacy of Bcl-xL inhibition and RT in women with wild-type PIK3CA/PTEN wild-type TNBC at high risk for recurrence.

RB expression confers sensitivity to CDK4/6 inhibitor-mediated radiosensitization across breast cancer subtypes.

Standard radiation therapy (RT) does not reliably provide locoregional control for women with multinode-positive breast cancer and triple-negative breast cancer (TNBC). We hypothesized that CDK4/6 inhibition (CDK4/6i) would increase the radiosensitivity not only of estrogen receptor-positive (ER+) cells, but also of TNBC that expresses retinoblastoma (RB) protein. We found that CDK4/6i radiosensitized RB WT TNBC (n = 4, radiation enhancement ratio [rER]: 1.49-2.22) but failed to radiosensitize RB-null TNBC (n = 3, rER: 0.84-1.00). RB expression predicted response to CDK4/6i + RT (R2 = 0.84), and radiosensitization was lost in ER+/TNBC cells (rER: 0.88-1.13) after RB1 knockdown in isogenic and nonisogenic models. CDK4/6i suppressed homologous recombination (HR) in RB WT cells but not in RB-null cells or isogenic models of RB1 loss; HR competency was rescued with RB reexpression. Radiosensitization was independent of nonhomologous end joining and the known effects of CDK4/6i on cell cycle arrest. Mechanistically, RB and RAD51 interact in vitro to promote HR repair. CDK4/6i produced RB-dependent radiosensitization in TNBC xenografts but not in isogenic RB1-null xenografts. Our data provide the preclinical rationale for a clinical trial expanding the use of CDK4/6i + RT to difficult-to-control RB-intact breast cancers (including TNBC) and nominate RB status as a predictive biomarker of therapeutic efficacy.

Short-term CDK4/6 Inhibition Radiosensitizes Estrogen Receptor-Positive Breast Cancers.

PURPOSE: Cyclin-dependent kinase 4/6 (CDK4/6) inhibitors have improved progression-free survival for metastatic, estrogen receptor-positive (ER+) breast cancers, but their role in the nonmetastatic setting remains unclear. We sought to understand the effects of CDK4/6 inhibition (CDK4/6i) and radiotherapy in multiple preclinical breast cancer models. EXPERIMENTAL DESIGN: Transcriptomic and proteomic analyses were used to identify significantly altered pathways after CDK4/6i. Clonogenic assays were used to quantify the radiotherapy enhancement ratio (rER). DNA damage was quantified using γH2AX staining and the neutral comet assay. DNA repair was assessed using RAD51 foci formation and nonhomologous end joining (NHEJ) reporter assays. Orthotopic xenografts were used to assess the efficacy of combination therapy. RESULTS: Palbociclib significantly radiosensitized multiple ER+ cell lines at low nanomolar, sub IC50 concentrations (rER: 1.21-1.52) and led to a decrease in the surviving fraction of cells at 2 Gy (P < 0.001). Similar results were observed in ribociclib-treated (rER: 1.08-1.68) and abemaciclib-treated (rER: 1.19-2.05) cells. Combination treatment decreased RAD51 foci formation (P < 0.001), leading to a suppression of homologous recombination activity, but did not affect NHEJ efficiency (P > 0.05). Immortalized breast epithelial cells and cells with acquired resistance to CDK4/6i did not demonstrate radiosensitization (rER: 0.94-1.11) or changes in RAD51 foci. In xenograft models, concurrent palbociclib and radiotherapy led to a significant decrease in tumor growth. CONCLUSIONS: These studies provide preclinical rationale to test CDK4/6i and radiotherapy in women with locally advanced ER+ breast cancer at high risk for locoregional recurrence.

A novel role for TTK in homologous recombination: implications for breast cancer radiosensitivity.

Basal-like breast cancers have the highest rates of locoregional recurrence after radiation. By correlating gene expression with early locoregional recurrence, we nominate TTK protein kinase as a mediator of radioresistance. TTK inhibition radiosensitizes in vitro and in vivo through a novel mechanism of impaired homologous recombination and represents a promising translational strategy.

BRCA1 Promoter Methylation and Clinical Outcomes in Ovarian Cancer: An Individual Patient Data Meta-Analysis.

BACKGROUND: BRCA1 methylation has been associated with homologous recombination deficiency, a biomarker of platinum sensitivity. Studies evaluating BRCA1-methylated tubal and ovarian cancer (OC) do not consistently support improved survival following platinum chemotherapy. We examine the characteristics of BRCA1-methylated OC in a meta-analysis of individual participant data. METHODS: Data of 2636 participants across 15 studies were analyzed. BRCA1-methylated tumors were defined according to their original study. Associations between BRCA1 methylation and clinicopathological characteristics were evaluated. The effects of methylation on overall survival (OS) and progression-free survival (PFS) were examined using mixed-effects models. All statistical tests were 2-sided. RESULTS: 430 (16.3%) tumors were BRCA1-methylated. BRCA1 methylation was associated with younger age and advanced-stage, high-grade serous OC. There were no survival differences between BRCA1-methylated and non-BRCA1-methylated OC (median PFS = 20.0 vs 18.5 months, hazard ratio [HR] = 1.01, 95% CI = 0.87 to 1.16; P = .98; median OS = 46.6 vs 48.0 months, HR = 1.02, 95% CI = 0.87 to 1.18; P = .96). Where BRCA1/2 mutations were evaluated (n = 1248), BRCA1 methylation displayed no survival advantage over BRCA1/2-intact (BRCA1/2 wild-type non-BRCA1-methylated) OC. Studies used different methods to define BRCA1 methylation. Where BRCA1 methylation was determined using methylation-specific polymerase chain reaction and gel electrophoresis (n = 834), it was associated with improved survival (PFS: HR = 0.80, 95% CI = 0.66 to 0.97; P = .02; OS: HR = 0.80, 95% CI = 0.63 to 1.00; P = .05) on mixed-effects modeling. CONCLUSION: BRCA1-methylated OC displays similar clinicopathological features to BRCA1-mutated OC but is not associated with survival. Heterogeneity within BRCA1 methylation assays influences associations. Refining these assays may better identify cases with silenced BRCA1 function and improved patient outcomes.

Seviteronel, a Novel CYP17 Lyase Inhibitor and Androgen Receptor Antagonist, Radiosensitizes AR-Positive Triple Negative Breast Cancer Cells.

Increased rates of locoregional recurrence (LR) have been observed in triple negative breast cancer (TNBC) despite multimodality therapy, including radiation (RT). Recent data suggest inhibiting the androgen receptor (AR) may be an effective radiosensitizing strategy, and AR is expressed in 15-35% of TNBC tumors. The aim of this study was to determine whether seviteronel (INO-464), a novel CYP17 lyase inhibitor and AR antagonist, is able to radiosensitize AR-positive (AR+) TNBC models. In cell viability assays, seviteronel and enzalutamide exhibited limited effect as a single agent (IC50 > 10 µM). Using clonogenic survival assays, however, AR knockdown and AR inhibition with seviteronel were effective at radiosensitizing cells with radiation enhancement ratios of 1.20-1.89 in models of TNBC with high AR expression. AR-negative (AR-) models, regardless of their estrogen receptor expression, were not radiosensitized with seviteronel treatment at concentrations up to 5 µM. Radiosensitization of AR+ TNBC models was at least partially dependent on impaired dsDNA break repair with significant delays in repair at 6, 16, and 24 h as measured by immunofluorescent staining of γH2AX foci. Similar effects were observed in an in vivo AR+ TNBC xenograft model where there was a significant reduction in tumor volume and a delay to tumor doubling and tripling times in mice treated with seviteronel and radiation. Following combination treatment with seviteronel and radiation, increased binding of AR occurred at DNA damage response genes, including genes involved both in homologous recombination and non-homologous end joining. This trend was not observed with combination treatment of enzalutamide and RT, suggesting that seviteronel may have a different mechanism of radiosensitization compared to other AR inhibitors. Enzalutamide and seviteronel treatment also had different effects on AR and AR target genes as measured by immunoblot and qPCR. These results implicate AR as a mediator of radioresistance in AR+ TNBC models and support the use of seviteronel as a radiosensitizing agent in AR+ TNBC.

TTK inhibition radiosensitizes basal-like breast cancer through impaired homologous recombination.

Increased rates of locoregional recurrence are observed in patients with basal-like breast cancer (BC) despite the use of radiation therapy (RT); therefore, approaches that result in radiosensitization of basal-like BC are critically needed. Using patients' tumor gene expression data from 4 independent data sets, we correlated gene expression with recurrence to find genes significantly correlated with early recurrence after RT. The highest-ranked gene, TTK, was most highly expressed in basal-like BC across multiple data sets. Inhibition of TTK by both genetic and pharmacologic methods enhanced radiosensitivity in multiple basal-like cell lines. Radiosensitivity was mediated, at least in part, through persistent DNA damage after treatment with TTK inhibition and RT. Inhibition of TTK impaired homologous recombination (HR) and repair efficiency, but not nonhomologous end-joining, and decreased the formation of Rad51 foci. Reintroduction of wild-type TTK rescued both radioresistance and HR repair efficiency after TTK knockdown; however, reintroduction of kinase-dead TTK did not. In vivo, TTK inhibition combined with RT led to a significant decrease in tumor growth in both heterotopic and orthotopic, including patient-derived xenograft, BC models. These data support the rationale for clinical development of TTK inhibition as a radiosensitizing strategy for patients with basal-like BC, and efforts toward this end are currently underway.

PARP1 Inhibition Radiosensitizes Models of Inflammatory Breast Cancer to Ionizing Radiation.

Sustained locoregional control of disease is a significant issue in patients with inflammatory breast cancer (IBC), with local control rates of 80% or less at 5 years. Given the unsatisfactory outcomes for these patients, there is a clear need for intensification of local therapy, including radiation. Inhibition of the DNA repair protein PARP1 has had little efficacy as a single agent in breast cancer outside of studies restricted to patients with BRCA mutations; however, PARP1 inhibition (PARPi) may lead to the radiosensitization of aggressive tumor types. Thus, this study investigates inhibition of PARP1 as a novel and promising radiosensitization strategy in IBC. In multiple existing IBC models (SUM-149, SUM-190, MDA-IBC-3), PARPi (AZD2281-olaparib and ABT-888-veliparib) had limited single-agent efficacy (IC50 > 10 µmol/L) in proliferation assays. Despite limited single-agent efficacy, submicromolar concentrations of AZD2281 in combination with RT led to significant radiosensitization (rER 1.12-1.76). This effect was partially dependent on BRCA1 mutational status. Radiosensitization was due, at least in part, to delayed resolution of double strand DNA breaks as measured by multiple assays. Using a SUM-190 xenograft model in vivo, the combination of PARPi and RT significantly delays tumor doubling and tripling times compared with PARPi or RT alone with limited toxicity. This study demonstrates that PARPi improves the effectiveness of radiotherapy in IBC models and provides the preclinical rationale for the opening phase II randomized trial of RT ± PARPi in women with IBC (SWOG 1706, NCT03598257).

Blood-brain barrier-adapted precision medicine therapy for pediatric brain tumors.

Targeted chemotherapeutics provide a promising new treatment option in neuro-oncology. The ability of these compounds to penetrate the blood-brain barrier is crucial for their successful incorporation into patient care. "CNS Targeted Agent Prediction" (CNS-TAP) is a multi-institutional and multidisciplinary translational program established at the University of Michigan for evaluating the central nervous system (CNS) activity of targeted therapies in neuro-oncology. In this report, we present the methodology of CNS-TAP in a series of pediatric and adolescent patients with high-risk brain tumors, for which molecular profiling (academic and commercial) was sought and targeted agents were incorporated. Four of five of the patients had potential clinical benefit (partial response or stable disease greater than 6 months on therapy). We further describe the specific drug properties of each agent chosen and discuss characteristics relevant in their evaluation for therapeutic suitability. Finally, we summarize both tumor and drug characteristics that impact the ability to successfully incorporate targeted therapies into CNS malignancy management.

A cellular automaton based on plasma membrane turnover accurately recapitulates cell mechanics during epithelial scattering.

Epithelial cells can be triggered to actively detach from epithelial tissues and become solitary, migratory and invasive. This process occurs repeatedly in development, where it is termed epithelial-mesenchymal transition (EMT), and can be recapitulated as epithelial scattering in cell culture models. Detachment of cell-cell junctions involves changes in contractile forces, actin cytoskeletal organization, changes in cell-substrate adhesion properties, surface presentation of cell-cell adhesion molecules, and gene expression. That these cellular processes affect each other and share molecular components creates difficulties in generating hypotheses and designing experiments to understand the mechanics of epithelial scattering. Computational modeling is proving a powerful too in such instances. Here we develop a cellular automaton to reveal insights into how cells rupture epithelial cell-cell junctions during scattering. The model is optimized for realistic and stable recapitulation of behavior of single cells, then for realistic simulation of multiple cells forming epithelial colonies. With a workable model of epithelial cell behavior, we then alter model parameters and assess whether we can realistically mimic epithelial scattering. Adjusting model parameters to recapitulate epithelial scattering reveals that induction of cell migration is the major driver of epithelial scattering.

A hybrid approach to appendicitis with right external iliac artery pseudo aneurysm: A case report.

INTRODUCTION: While acute appendicitis is a common surgical problem, the simultaneous occurrence of appendicitis and an infected iliac artery pseudoaneurysm is exceedingly rare. We report the successful treatment of an infected right external iliac artery pseudo aneurysm in the 1setting of acute appendicitis. PRESENTATION OF CASE: The patient is an 83-year-old male who presents with severe sepsis, right lower quadrant and right leg pain. Additional past medical history is significant for rectal cancer status post resection and radiation therapy in 1997. Computed tomography (CT) on admission revealed a right iliopsoas muscle abscess, an inflamed Appendix and a pseudo aneurysm arising from the right external iliac artery. After consultations by multiple specialties, the plan was to proceed with percutaneous drainage of the abscess, antibiotic therapy and subsequent repair of the pseudoaneurysm. CT guided drainage of the iliopsoas abscess was performed with return of hemorrhagic fluid. Due to the concern of contained pseudoaneurysm rupture, the patient was taken for expedited repair. Due to the patient's frailty and hostile abdomen, we performed embolization of the right external iliac artery pseudoaneurysm with Amplatzer I plugs (St. Jude Medical, St. Paul MN) and left common femoral to right superficial femoral bypass with cryopreserved cadaveric femoral vein. Following pseudoaneurysm exclusion, continued percutaneous drainage and antibiotic therapy, the patient has done well with no further evidence of infection. CONCLUSION: Repair of infected pseudo aneurysms can prove challenging. Ongoing infection, a hostile surgical abdomen and patient frailty further complicates the treatment of these patients. This case displays a minimally invasive approach to this rare but morbid condition.

The IncRNAs PCGEM1 and PRNCR1 are not implicated in castration resistant prostate cancer.

Long noncoding RNAs (IncRNAs) are increasingly implicated in cancer biology, contributing to essential cancer cell functions such as proliferation, invasion, and metastasis. In prostate cancer, several lncRNAs have been nominated as critical actors in disease pathogenesis. Among these, expression of PCGEM1 and PRNCR1 has been identified as a possible component in disease progression through the coordination of androgen receptor (AR) signaling (Yang et al., Nature 2013, see ref. [1]). However, concerns regarding the robustness of these findings have been suggested. Here, we sought to evaluate whether PCGEM1 and PRNCR1 are associated with prostate cancer. Through a comprehensive analysis of RNA-sequencing data (RNA-seq), we find evidence that PCGEM1 but not PRNCR1 is associated with prostate cancer. We employ a large cohort of >230 high-risk prostate cancer patients with long-term outcomes data to show that, in contrast to prior reports, neither gene is associated with poor patient outcomes. We further observe no evidence that PCGEM1 nor PRNCR1 interact with AR, and neither gene is a component of AR signaling. Thus, we conclusively demonstrate that PCGEM1 and PRNCR1 are not prognostic lncRNAs in prostate cancer and we refute suggestions that these lncRNAs interact in AR signaling.

Reconstructing targetable pathways in lung cancer by integrating diverse omics data.

Global 'multi-omics' profiling of cancer cells harbours the potential for characterizing the signalling networks associated with specific oncogenes. Here we profile the transcriptome, proteome and phosphoproteome in a panel of non-small cell lung cancer (NSCLC) cell lines in order to reconstruct targetable networks associated with KRAS dependency. We develop a two-step bioinformatics strategy addressing the challenge of integrating these disparate data sets. We first define an 'abundance-score' combining transcript, protein and phospho-protein abundances to nominate differentially abundant proteins and then use the Prize Collecting Steiner Tree algorithm to identify functional sub-networks. We identify three modules centred on KRAS and MET, LCK and PAK1 and ß-Catenin. We validate activation of these proteins in KRAS-dependent (KRAS-Dep) cells and perform functional studies defining LCK as a critical gene for cell proliferation in KRAS-Dep but not KRAS-independent NSCLCs. These results suggest that LCK is a potential druggable target protein in KRAS-Dep lung cancers.

The long noncoding RNA SChLAP1 promotes aggressive prostate cancer and antagonizes the SWI/SNF complex.

Prostate cancers remain indolent in the majority of individuals but behave aggressively in a minority. The molecular basis for this clinical heterogeneity remains incompletely understood. Here we characterize a long noncoding RNA termed SChLAP1 (second chromosome locus associated with prostate-1; also called LINC00913) that is overexpressed in a subset of prostate cancers. SChLAP1 levels independently predict poor outcomes, including metastasis and prostate cancer-specific mortality. In vitro and in vivo gain-of-function and loss-of-function experiments indicate that SChLAP1 is critical for cancer cell invasiveness and metastasis. Mechanistically, SChLAP1 antagonizes the genome-wide localization and regulatory functions of the SWI/SNF chromatin-modifying complex. These results suggest that SChLAP1 contributes to the development of lethal cancer at least in part by antagonizing the tumor-suppressive functions of the SWI/SNF complex.

Parasympathetic stimulation via the vagus nerve prevents systemic organ dysfunction by abrogating gut injury and lymph toxicity in trauma and hemorrhagic shock.

We tested if vagus nerve stimulation (VNS) would prevent gut injury, mesenteric lymph toxicity, and systemic multiple organ dysfunction syndrome following trauma-hemorrhagic shock (T/HS). Four groups of experiments were performed. The first tested whether VNS (5 V for 10 min) would protect against T/HS-induced increases in gut and lung permeability as well as neutrophil priming. In the second experiment, mesenteric lymph was collected from rats subjected to T/HS or trauma-sham shock with or without VNS and then injected into naive mice to assess its biologic activity. Lung permeability, neutrophil priming, and red blood cell deformability were measured. Next, the role of the spleen in VNS-mediated protection was tested by measuring gut and lung injury in splenectomized rats subjected to sham or actual VNS. Lastly, the ability of nicotine to replicate the gut-protective effect of VNS was tested. Vagus nerve stimulation protected against T/HS-induced gut injury, lung injury, and neutrophil priming (P < 0.05). Not only did VNS limit organ injury after T/HS, but in contrast to the mesenteric lymph collected from the sham-VNS T/HS rats, the mesenteric lymph from the VNS T/HS rats did not cause lung injury, neutrophil priming, or loss of red blood cell deformability (P < 0.05) when injected into naive mice. Removal of the spleen did not prevent the protective effects of VNS on gut or lung injury after T/HS. Similar to VNS, the administration of nicotine also protected the gut from injury after T/HS. Vagus nerve stimulation prevents T/HS-induced gut injury, lung injury, neutrophil priming, and the production of biologically active mesenteric lymph. This protective effect of VNS was not dependent on the spleen but appeared to involve a cholinergic nicotinic receptor, because its beneficial effects could be replicated with nicotine.

Trauma hemorrhagic shock-induced lung injury involves a gut-lymph-induced TLR4 pathway in mice.

BACKGROUND: Injurious non-microbial factors released from the stressed gut during shocked states contribute to the development of acute lung injury (ALI) and multiple organ dysfunction syndrome (MODS). Since Toll-like receptors (TLR) act as sensors of tissue injury as well as microbial invasion and TLR4 signaling occurs in both sepsis and noninfectious models of ischemia/reperfusion (I/R) injury, we hypothesized that factors in the intestinal mesenteric lymph after trauma hemorrhagic shock (T/HS) mediate gut-induced lung injury via TLR4 activation. METHODS/PRINCIPAL FINDINGS: The concept that factors in T/HS lymph exiting the gut recreates ALI is evidenced by our findings that the infusion of porcine lymph, collected from animals subjected to global T/HS injury, into naïve wildtype (WT) mice induced lung injury. Using C3H/HeJ mice that harbor a TLR4 mutation, we found that TLR4 activation was necessary for the development of T/HS porcine lymph-induced lung injury as determined by Evan's blue dye (EBD) lung permeability and myeloperoxidase (MPO) levels as well as the induction of the injurious pulmonary iNOS response. TRIF and Myd88 deficiency fully and partially attenuated T/HS lymph-induced increases in lung permeability respectively. Additional studies in TLR2 deficient mice showed that TLR2 activation was not involved in the pathology of T/HS lymph-induced lung injury. Lastly, the lymph samples were devoid of bacteria, endotoxin and bacterial DNA and passage of lymph through an endotoxin removal column did not abrogate the ability of T/HS lymph to cause lung injury in naïve mice. CONCLUSIONS/SIGNIFICANCE: Our findings suggest that non-microbial factors in the intestinal mesenteric lymph after T/HS are capable of recreating T/HS-induced lung injury via TLR4 activation.