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Osteosarcoma is the most common malignancy of the bone, yet the survival for patients with osteosarcoma is virtually unchanged over the past 30 years. This is principally because development of new therapies is hampered by a lack of recurrent mutations that can be targeted in osteosarcoma. Here, we report that epigenetic changes via mRNA methylation holds great promise to better understand the mechanisms of osteosarcoma growth and to develop targeted therapeutics. In patients with osteosarcoma, the RNA demethylase ALKBH5 was amplified and higher expression correlated with copy-number changes. ALKBH5 was critical for promoting osteosarcoma growth and metastasis, yet it was dispensable for normal cell survival. Methyl RNA immunoprecipitation sequencing analysis and functional studies showed that ALKBH5 mediates its protumorigenic function by regulating m6A levels of histone deubiquitinase USP22 and the ubiquitin ligase RNF40. ALKBH5-mediated m6A deficiency in osteosarcoma led to increased expression of USP22 and RNF40 that resulted in inhibition of histone H2A monoubiquitination and induction of key protumorigenic genes, consequently driving unchecked cell-cycle progression, incessant replication, and DNA repair. RNF40, which is historically known to ubiquitinate H2B, inhibited H2A ubiquitination in cancer by interacting with and affecting the stability of DDB1-CUL4–based ubiquitin E3 ligase complex. Taken together, this study directly links increased activity of ALKBH5 with dysregulation of USP22/RNF40 and histone ubiquitination in cancers. More broadly, these results suggest that m6A RNA methylation works in concert with other epigenetic mechanisms to control cancer growth. Significance: RNA demethylase ALKBH5 upregulates USP22 and RNF40 to inhibit histone H2A ubiquitination and induces expression of key replication and DNA repair–associated genes, driving osteosarcoma progression.

Suppl. Fig. 1 confirms that ID8agg is more virulent versus parental ID8 in WT mice. Suppl. Fig. 2 provides relevant details for plasmids used to make recombinant cell lines, and gives Western blot validations for each PD-L1lo clone described. Suppl, Fig. 3 shows that αPD-L1 reduces tumor growth and metastatic spread of B16hi cells in NSG mice and shows color photomicrographs of lungs from treated mice. Suppl. Fig. 4 shows that αPD-L1 immunotherapy fails to treat ID8agg in wild-type mice. Suppl. Fig. 5 shows that tumor PD-L1 is necessary and sufficient for αPD-L1 treatment effects on B16 cells challenged into wild type mice. Suppl. Fig. 6 provides much additional Western blot data on mTORC1 and mTORC2 signals in B16 and ID8agg cells after rapamycin or chloroquine treatments. Suppl. Fig. 7 shows effects of tumor cell PD-L1 expression on B16 and ID8agg cell proliferation sensitivity to TNFα, cis-platinum and paclitaxel in vitro.

Supplementary Figure from M6A RNA Methylation Regulates Histone Ubiquitination to Support Cancer Growth and Progression

Osteosarcoma is the most common malignancy of the bone, yet the survival for patients with osteosarcoma is virtually unchanged over the past 30 years. This is principally because development of new therapies is hampered by a lack of recurrent mutations that can be targeted in osteosarcoma. Here, we report that epigenetic changes via mRNA methylation holds great promise to better understand the mechanisms of osteosarcoma growth and to develop targeted therapeutics. In patients with osteosarcoma, the RNA demethylase ALKBH5 was amplified and higher expression correlated with copy-number changes. ALKBH5 was critical for promoting osteosarcoma growth and metastasis, yet it was dispensable for normal cell survival. Methyl RNA immunoprecipitation sequencing analysis and functional studies showed that ALKBH5 mediates its protumorigenic function by regulating m6A levels of histone deubiquitinase USP22 and the ubiquitin ligase RNF40. ALKBH5-mediated m6A deficiency in osteosarcoma led to increased expression of USP22 and RNF40 that resulted in inhibition of histone H2A monoubiquitination and induction of key protumorigenic genes, consequently driving unchecked cell-cycle progression, incessant replication, and DNA repair. RNF40, which is historically known to ubiquitinate H2B, inhibited H2A ubiquitination in cancer by interacting with and affecting the stability of DDB1-CUL4–based ubiquitin E3 ligase complex. Taken together, this study directly links increased activity of ALKBH5 with dysregulation of USP22/RNF40 and histone ubiquitination in cancers. More broadly, these results suggest that m6A RNA methylation works in concert with other epigenetic mechanisms to control cancer growth.Significance:RNA demethylase ALKBH5 upregulates USP22 and RNF40 to inhibit histone H2A ubiquitination and induces expression of key replication and DNA repair–associated genes, driving osteosarcoma progression.

Supplementary Data from M6A RNA Methylation Regulates Histone Ubiquitination to Support Cancer Growth and Progression

PD-L1 antibodies produce efficacious clinical responses in diverse human cancers, but the basis for their effects remains unclear, leaving a gap in the understanding of how to rationally leverage therapeutic activity. PD-L1 is widely expressed in tumor cells, but its contributions to tumor pathogenicity are incompletely understood. In this study, we evaluated the hypothesis that PD-L1 exerts tumor cell–intrinsic signals that are critical for pathogenesis. Using RNAi methodology, we attenuated PD-L1 in the murine ovarian cell line ID8agg and the melanoma cell line B16 (termed PD-L1lo cells), which express basal PD-L1. We observed that PD-L1lo cells proliferated more weakly than control cells in vitro. As expected, PD-L1lo cells formed tumors in immunocompetent mice relatively more slowly, but unexpectedly, they also formed tumors more slowly in immunodeficient NSG mice. RNA sequencing analysis identified a number of genes involved in autophagy and mTOR signaling that were affected by PD-L1 expression. In support of a functional role, PD-L1 attenuation augmented autophagy and blunted the ability of autophagy inhibitors to limit proliferation in vitro and in vivo in NSG mice. PD-L1 attenuation also reduced mTORC1 activity and augmented the antiproliferative effects of the mTORC1 inhibitor rapamycin. PD-L1lo cells were also relatively deficient in metastasis to the lung, and we found that anti-PD-L1 administration could block tumor cell growth and metastasis in NSG mice. This therapeutic effect was observed with B16 cells but not ID8agg cells, illustrating tumor- or compartmental-specific effects in the therapeutic setting. Overall, our findings extend understanding of PD-L1 functions, illustrate nonimmune effects of anti-PD-L1 immunotherapy, and suggest broader uses for PD-L1 as a biomarker for assessing cancer therapeutic responses. Cancer Res; 76(23); 6964–74. ©2016 AACR.

B cells promote tumor development in murine models of breast cancer (BCa), including - as we have shown - by producing pleiotropic cytokine IL-27, which upregulates expression of the PD-L1 immune checkpoint in BCa cells and tumor-infiltrating B cells (TIL-Bs). Paradoxically, B cell activation has been shown to predict the positive response to immune checkpoint inhibitors (ICIs) by murine BCa tumors and melanoma, sarcoma and kidney cancer in patients. Here, we have addressed the role of B cell-expressed PD-L1 in BCa development and responses to ICIs by generating conditional knockout (KO) mice in which Cd274 (encoding PD-L1) was ablated only in activated B cells (AicdacreCd274fl/fl). Tumor development in AicdacreCd274fl/fl mice from engrafted syngeneic ER+ E0071 BCa cells was significantly delayed, albeit not abolished (as occurring in mice lacking B cells), in association with increased B-cell expression of CD86, a key agonistic ligand for T cell-expressed CD28, a co-stimulatory receptor whose function is inhibited by the PD-L1/PD-1 immune checkpoint in exhausted tumor-infiltrating CD8+ T cells. The heightened B-cell CD86 expression in AicdacreCd274fl/fl mice was recapitulated in AicdacreCd274fl/fl B cells stimulated in vitro by CD154, a T helper cell stimulus critical for B cell activation and differentiation as well as the antibody response. Accordingly, AicdacreCd274fl/fl mice displayed an increased antibody response to a T-dependent antigen. Likewise, treatment with the anti-PD-L1 (αPD-L1) ICI boosted wildtype B cells stimulated with CD154 in vitro to express CD86 and differentiate into potentiated plasma cells. It also effectively regressed the growth of pre-developed E0071 tumor in C57 and AicdacreCd274+/fl mice by rejuvenating tumor-infiltrating CD8+ T cells, likely through activation of previously dampened CD28 signaling. By contrast, residual tumors that had eventually developed in AicdacreCd274fl/fl mice failed to respond to αPD-L1 and continued to grow at a pace comparable to tumors treated with an isotype-matched control antibody, showing that the full anti-tumor efficacy of αPD-L1 was mediated mainly by PD-L1-expressing B cells, but not other immune cell types. Thus, PD-L1+ TIL-Bs use PD-L1 to impose a “brake” on the host anti-tumor activity, but are directly targeted by αPD-L1 to relieve the brake, “jump-start” the CD86:CD28 signaling and potentiate the anti-tumor response, thereby providing a mechanistic explanation for the opposing roles of B cells in murine BCa development and ICI responses. Our findings also provide a strong rationale to investigate the activation of PD-L1-expressing TIL-Bs by Atezolizumab® (αPD-L1) in BCa patients to underpin the patient response to this ICI. Citation Format: Mustafa Tamim Alam Khan, Christian Cervantes, Suryavathi Viswanadhapalli, Yan Hui, Ratna Vadlamudi, Zhenming Xu. Pd-l1-expressing b-cells promote murine breast cancer development and mediate the response to anti-pd-l1 immune checkpoint inhibitor [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P1-04-11.

Our center has developed protocols for nurse-led sedation (NLS) where dedicated cardiac catheterization laboratory nurses directly provide sedation with fentanyl and midazolam. This study compares ...

Background: N6-methyladenosine (m6A) RNA methylation is a dynamic reversible epitranscriptomic modification that includes methyl transferases (writes m6A), demethylases (erases m6A) and reader proteins, which proofreads m6A marks of a specific site of transcripts. Recent studies have suggested that RNA methylation affects several fundamental cellular and molecular functions including mRNA splicing, stability, export, stem cell fate, circadian rhythms, DNA repair and cell survival. The objective of this study is to establish the mechanisms by which RNA demethylase ALKBH5 (AlkB homolog 5) facilitates tumor growth and progression. Methods: To establish the significance of RNA methylation in children’s cancers, we performed siRNA screen targeting m6A writers, erasers, and readers in osteosarcoma (OS). We used several OS cell lines including 143B, MG63, SaOS2, U2OS and multiple patient derived OS cell lines. Mechanistic studies were conducted using ALKBH5 KO and knockdown cells and by measuring the status of Autophagy and UPR associated proteins using Western blot analysis, confocal and electron microscopy. Results: Our results revealed that depletion of ALKBH5, a demethylase that erases the m6A mark from the target gene, altered the autophagy in OS cells. Interestingly, we found that level of LC3, which is the universal marker for autophagy, was significantly increased in OS cells. RNA seq analysis showed that depletion of ALKBH5 significantly altered several autophagy related genes in the OS cells. To better understand the molecular mechanism by which ALKBH5 regulates autophagy, we investigated the Endoplasmic Reticulum (ER) stress-induced Unfolded Protein Response (UPR) pathway, which is a known activator of autophagy. We discovered that ER stress induced UPR signaling pathway is highly activated in ALKBH5 depleted cancer cells. Further, mechanistic studies suggested that ALKBH5 promoted ER homeostasis by controlling the expression of ER lipid raft associated 1 (ERLIN1), which binds to the activated inositol 1, 4, 5,-triphosphate receptor and facilitates its degradation via ERAD to maintain calcium flux between ER and mitochondria. Using functional studies and electron microscopy, we show that ALKBH5-ERLIN1-IP3R-dependent calcium signaling modulates the activity of AMP kinase, and consequently mitochondrial biogenesis. These findings thus reveal that ALKBH5 serves an important role in maintaining ER homeostasis and cellular fitness. Conclusion: These findings provide novel insight into how m6A may promote cancer cell growth by regulating the crosstalk among ER signaling, UPR, autophagy, and mitochondrial function. Our study is the first to show that RNA methylation plays an important role in osteosarcoma by regulating autophagy via UPR. Citation Format: Panneerdoss Subbarayalu, Daisy Medina, Pooja Yadav, Santosh Timilsina, Kunal Baxi, Ratna Vadlamudi, Yidong Chen, Manjeet Rao. ALKBH5 promotes cancer growth by regulating ER homeostasis via UPR, autophagy, and mitochondrial function. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3528.

Introduction: Estrogen receptor gene (ESR1) drives approximately 70-80% of breast cancer cases, which are primarily treated with endocrine therapies. Mutations in the ESR1 exclusively occur in the ligand binding domain (LBD) and are significantly enriched following endocrine therapy. These mutations retain the estrogen receptor (ER) expression thus conferring ligand-independent growth, which is a paramount contributor to endocrine therapy resistant disease. Worse progression-free survival is consistently associated with ESR1 mutation status; therefore, there is an unmet need for therapies against these mutations. To this end, we recently developed TX-245, a bis-benzamide molecule that hampers the function of the two most common ESR1 mutations - Y537S and D538G. Methods: TX-245 was evaluated in vitro using MTT cell proliferation assay and colony formation assay carried out in multiple ER+ breast cancer cells harboring wild type (WT) ESR1 or mutant (MT) ESR1. Approaches such as tumor spheroid cultures were also implemented to study the effect of TX-245 compared to selected SERDs/SERMs via CellTiter-Glo 3D viability assay. Mechanistic insights were obtained performing TR-FRET assay, fluorescence polarization assay, RNA-seq, ChIP-seq, western blot, and qRT-PCR. The therapeutic efficacy in vivo was evaluated using cell xenografts, patient derived xenografts (PDX), patient derived explants (PDE), and metastatic mouse models of MT-ER breast cancer. Results: TX-245 demonstrated greater anti-proliferative dose-dependent activity in models harboring WT and MT ESR1, including those resistant to fulvestrant and tamoxifen, when compared to other SERDs/SERMs. RNAseq data suggests that TX-245 dramatically alters the transcription of ERα-regulated genes, with repression of canonical estradiol-upregulated and induction of estradiol-repressed genes. TX-245 was able to antagonize ER-chromatin interaction and ER-transcriptional signaling in breast cancer cells driven by ESR1 mutation. TX-245 treatment downregulated ER protein expression and decreased expression of ER targeted genes (i.e., GREB and PGR) in multiple ER+ cell lines. TX-245 treatment as a single agent or in combination with palbociclib resulted in tumor regression in xenografts expressing WT, Y537S or D538G ESR1 mutation. PDX and PDE models consistently exhibited substantial tumor growth inhibition as well as decreased proliferation (Ki67 staining). Metastases studies using intracardiac and intratibial injection demonstrated that TX-245 inhibited the progression of established metastatic niches in mice injected with Y537S or D538G ER+ breast cancer cells. Conclusion: Our results indicate that TX-245 inhibits cell proliferation in cell lines that express WT or mutant ESR1, induce ER degradation, suppress ER-mediated signaling and reduce tumor burden in mouse models. These studies highlight the utility of TX-245 in targeting mutations in the ER-LBD. Citation Format: Karla Parra, Suryavathi Viswanadhapalli, Tanner Reese, Xihui Liu, JunHao Liu, Zexuan Liu, Tae-Kyung Lee, Chia Yuan Chen, Kara Kassees, Jung-Mo Ahn, Ratna Vadlamudi, Ganesh Raj, Carlos Roggero. Targeting the mutant estrogen receptor in metastatic breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3440.

BACKGROUND Hypoxia is a major driver of invasiveness and resistance in glioblastoma (GBM). We hypothesize that GBM adapts to chronic hypoxic conditions through utilization of peroxisomal fatty acid oxidation (FAO). We explored the contribution of acyl coenzyme A oxidase 1 (ACOX-1), a rate-limiting enzyme for peroxisomal FAO, in metabolic adaptation to hypoxia and as a potential therapeutic target. METHODS A primary cell line from a bevacizumab-resistant tumor (012015) was cultured under chronic hypoxia (2%O2) or normoxia (21% O2). We used UPLC-MS/MS and GC-MS to determine lipid and polar metabolites. RNA seq was performed on U251 ACOX-1 (KO) cells under normoxic and hypoxic conditions. Differential gene expression was obtained using TopHat2 (genome alignment), HTSeq, and DEseq. Gene set enrichment analysis was used to compare biological pathways between conditions. ACOX-1 was deleted using CRISPR-Cas9 and orthotopically implanted in NCr/SCID nude mice. Animals were treated with 30 mg/kg of pazopanib or vehicle daily through oral gavage. RESULTS In 012015 cells we observed altered carnitine metabolites, increase in specific triglycerides, and S-lactoylglutathione indicating dysfunctional mitochondrial FAO, and increase in oxidative stress between hypoxic and normoxic conditions. Also, decrease in citric acid cycle metabolites, increase in glycolytic metabolites were seen in hypoxia. Further RNA seq on U251 ACOX-1 (KO) cells showed downregulated genes such as PCK1, SCD, SREBF1, PIPOX (p< 1E-8) and significant KEGG pathways such as unsaturated fatty acid (p= 0.009), peroxisome (p= 0.025) and PPAR (p= 0.016) between hypoxia and normoxia suggesting increased dependence of these cells on peroxisomal FAO. ACOX-1 (KO) mice showed increased survival when treated (p= 6.7E-04) with pazopanib. CONCLUSION In ACOX-1 (KO) model, we observed metabolomic reprogramming with downregulated genes, dysregulated metabolites, and pathways in peroxisomal FAO in hypoxia. These identified genes could be potential targets for therapy in combination with anti-angiogenic therapies that increase hypoxia in GBM.

Aberrant activities of various cell cycle and DNA repair proteins promote cancer growth and progression and render them resistant to therapies. Here, we demonstrate that the anti-depressant imipramine blocks growth of triple-negative (TNBC) and estrogen receptor-positive (ER+) breast cancers by inducing cell cycle arrest and by blocking heightened homologous recombination (HR) and non-homologous end joining-mediated (NHEJ) DNA repair activities. Our results reveal that imipramine inhibits the expression of several cell cycle- and DNA repair-associated proteins including E2F1, CDK1, Cyclin D1, and RAD51. In addition, we show that imipramine inhibits the growth of ER + breast cancers by inhibiting the estrogen receptor- α (ER-α) signaling. Our studies in preclinical mouse models and ex vivo explants from breast cancer patients show that imipramine sensitizes TNBC to the PARP inhibitor olaparib and endocrine resistant ER + breast cancer to anti-estrogens. Our studies suggest that repurposing imipramine could enhance routine care for breast cancer patients. Based on these results, we designed an ongoing clinical trial, where we are testing the efficacy of imipramine for treating patients with triple-negative and estrogen receptor-positive breast cancer. Since aberrant DNA repair activity is used by many cancers to survive and become resistant to therapy, imipramine could be used alone and/or with currently used drugs for treating many aggressive cancers.

Catastrophic thrombosis is a rare but frequently fatal event following complex cardiac surgery. It is most often encountered following separation from cardiopulmonary bypass (CPB) and reversal of heparin anticoagulation, and somewhat paradoxically, at the time when bleeding from post-CPB coagulopathy is being treated. We report the case of a 41-year-old female taken to the operating room for repair of an expanding ascending aortic pseudoaneurysm. Following a prolonged operation, she developed intracardiac thrombus during transfusion of hemostatic blood products and procoagulant agents. Potential contributing factors are discussed.

PurposeThe majority of breast cancers are estrogen receptor (ERα) positive making endocrine therapy a mainstay for these patients. Unfortunately, resistance to endocrine therapy is a common occurrence. Fatty acid synthase (FASN) is a key enzyme in lipid biosynthesis and its expression is commensurate with tumor grade and resistance to numerous therapies. MethodsThe effect of the FASN inhibitor TVB-3166 on ERα expression and cell growth was characterized in tamoxifen resistant cell lines, xenografts, and patient explants. Subcellular localization of ERα was assessed using a combination of immunofluorescence and subcellular fractionations. Palmitoylation and ubiquitination of ERα were assessed by immunoprecipitation. ERα and p-eIF2α protein levels were analyzed by western blotting after treatment with TVB-3166 with or without the addition of palmitate or BAPTA. ResultsTVB-3166 treatment leads to a marked inhibition of proliferation in tamoxifen-resistant cells compared to the parental cells. Additionally, TVB-3166 significantly inhibited tamoxifen-resistant breast tumor growth in mice and decreased proliferation of primary tumor explants compared to untreated controls. FASN inhibition significantly reduced ERα levels most prominently in endocrine resistant cells and altered its subcellular localization. Furthermore, we showed that the reduction of ERα expression upon TVB-3166 treatment is mediated through the induction of endoplasmic reticulum stress. ConclusionOur preclinical data provide evidence that FASN inhibition by TVB-3166 presents a promising therapeutic strategy for treatment of endocrine-resistant breast cancer. Further clinical development of FASN inhibitors for endocrine resistant breast cancer should be considered.

The major limitations of DNA-targeting chemotherapy drugs include life-threatening toxicity, acquired resistance and occurrence of secondary cancers. Here, we report a small molecule, Carbazole Blue (CB), that binds to DNA and inhibits cancer growth and metastasis by targeting DNA-related processes that tumor cells use but not the normal cells. We show that CB inhibits the expression of pro-tumorigenic genes that promote unchecked replication and aberrant DNA repair that cancer cells get addicted to survive. In contrast to chemotherapy drugs, systemic delivery of CB suppressed breast cancer growth and metastasis with no toxicity in pre-clinical mouse models. Using PDX and ex vivo explants from estrogen receptor (ER) positive, ER mutant and TNBC patients, we further demonstrated that CB effectively blocks therapy-sensitive and therapy-resistant breast cancer growth without affecting normal breast tissue. Our data provide a strong rationale to develop CB as a viable therapeutic for treating breast cancers.

A 44-year-old man, American Society of Anesthesiologists physical status class IV, presented for fulguration of anal condyloma and diverting colostomy. The patient's medical history includes World Health Organization (WHO) class I pulmonary hypertension (PH), right heart failure, and bilateral lower extremity paralysis due to Pott's disease. The patient was not a candidate for neuraxial anesthesia due to sacral decubitus ulcers, and alternative options to general anesthesia (GA) were considered to avoid the high risk of right ventricular (RV) failure and ensuing complications. The case was successfully performed under sedation with dexmedetomidine infusion and bilateral rectus sheath blocks for surgical anesthesia.

Heart failure (HF) currently affects more than 5 million patients in the United States [1]. Advanced HF is associated with high mortality and poor quality of life. It is estimated that between 5% and 10% of all patients with HF have an advanced form of the disease [1]. Orthotopic heart transplantation (OHT) is an accepted therapy for stage D HF [3] (Fig. 1). Unfortunately, the number of patients with the disease exceeds the number of available organs. This makes appropriate patient selection vital in the field of heart transplantation. Anesthetic evaluation of the patient presenting for OHT or mechanical circulatory support (MCS) implantation is a vital component of the patient's perioperative course. Patients often have had extensive diagnostic testing and assessment prior to being listed for OHT or considered for MCS implantation. Because of the often urgent nature of these procedures, the cardiac anesthesiologist must conduct a focused review of the relevant information and perform a focused patient interview and physical exam.

B cells promote tumor development in murine models of breast cancer (BCa), – as we have shown – by producing pleiotropic cytokine IL-27, which upregulates expression of PD-L1 immune checkpoint in BCa cells and tumor-infiltrating B cells (TIL-Bs). Paradoxically, B cell activation has been shown to predict a positive response to immune checkpoint inhibitors (ICIs) in murine BCa tumors and melanoma, sarcoma and kidney cancer in humans. Here, we have addressed the role of B cell-expressed PD-L1 in BCa development and responses to ICIs by generating conditional knockout mice in which Cd274 (encoding PD-L1) was ablated only in activated B cells (AicdacreCd274fl/fl). Tumor development in AicdacreCd274fl/fl mice from engrafted syngeneic ER+ E0071 BCa cells was significantly delayed, albeit not abolished (as occurring in mice lacking B cells), with a marked increase in CD86 B-cell expression, a key agonistic ligand for T cell-expressed CD28 (a co-stimulatory receptor whose function is inhibited by the PD-L1/PD-1 immune checkpoint). The heightened B-cell CD86 expression in AicdacreCd274fl/fl mice was recapitulated in vitro using CD154, a T helper cell stimulus critical for B cell activation and differentiation. Treatment with anti-PD-L1 (aPD-L1) boosted wildtype B cells, stimulated with CD154 in vitro, to express CD86 and differentiate into potentiated plasma cells. Thus, PD-L1+ TIL-Bs use PD-L1 to impose a “brake” on host anti-tumor activity but are directly targeted by aPD-L1 to relieve the brake, which “jump-starts” CD86:CD28 signaling and potentiates the anti-tumor response. Thus, providing a mechanistic explanation for the opposing roles of B cells in murine BCa development and ICI responses.

Tumor PD-L1 mediates non-canonical, intracellular signals including the DNA damage response (DDR), of unclear significance and mechanisms. We show that genetic PD-L1 depletion (PD-L1KO) destabilized the Chk2 protein, a DNA damage response (DDR) factor, resulting in ATM/Chk2 pathway defects but not ATR/Chk1 in melanoma, bladder, breast, and ovarian cancer. Consistent with ATM/Chk2 defects, PD-L1 KO led to DNA damage (γH2AX) and impaired homologous recombination (HR) (p-RPA32, BRCA1, Rad51 foci). Thus, PD-L1 KO vs control cells were significantly more sensitive to DDR inhibitors (DDRi) against ATR, Chk1, and PARP in vitro and in vivo in NSG mice. Chk2 regulation by PD-L1 was independent of PD-L1 cytoplasmic tail yet required intracellular (vs surface) PD-L1 suggesting physical PD-L1/Chk2 interaction supported by IP and imaging. PD-L1 stabilized Chk2 protein by preventing its lysosomal degradation without altering Chek2 mRNA. αPD-L1 is thought to work by protecting PD-L1 induced anti-tumor T cell suppression via PD-1, but PD-L1 DDR effects were PD-1-independent. Intracellular PD-L1 suppressed DDRi induced cGAS/STING activation by immunoblots and qRT-PCR of type 1 IFN genes. In vivo in WT mice, genetic PD-L1 depletion but not αPD-L1, sensitized highly immunotherapy resistant 4T1 breast cancer to PARPi. Strikingly, PARPi had reduced effect on PD-L1KO tumors in RAG2KO mice despite treating WT mice, indicating a strong immune component to DDRi efficacy. Our work implicates a novel role of intracellular PD-L1 in DDR and tumor immunogenicity and identifies related therapeutic vulnerabilities exposed by intracellular PD-L1 targeting. Surface vs intracellular PD-L1, and specific DDR signals could be treatment response biomarkers.

Triple Negative Breast Cancer (TNBC) is an aggressive subtype of breast cancer that lacks effective targeted therapies with the main therapeutic option being cytotoxic chemotherapies, which have toxic side effects on patient's health. Less toxic alternatives that could target key pathways that are altered in TNBCs could lead to better outcomes for the patients. Important distinguishing traits of TNBCs is the high amounts of genomic instability and high number of alterations in key proteins in DNA repair and cell proliferation that helps the cancer grow, survive and progress. Previous findings from our lab indicate that Imipramine Blue, a derivative of anti-depressant imipramine, may be a potential inhibitor of proliferation and DNA repair pathways in TNBC. Therefore, we tested the therapeutic efficacy of Imipramine on TNBCs using various in vitro assays for viability, migration, invasion as well as in vivo xenograft assays for tumor growth. Our results demonstrate that Imipramine inhibits cancer cell viability and progression in vitro. Similarly, in vivo xenograft assays showed reduced tumor growth in pre-clinical models upon imipramine treatment. Importantly, at the molecular level, Imipramine treatment suppressed key players in both cell proliferation, and DNA repair pathways, including FOXM1; a proto-oncogene that is a master regulator of both cell proliferation and DNA Repair pathways. These findings suggest that Imipramine may be a potential therapeutic option for TNBC patients. A clinical trial is ongoing to test the efficacy of Imipramine in Triple Negative Breast Cancer patients. Citation Format: Santosh Timilsina, Subapriya Rajamanickam, Ismail Jatoi, Yidong Chen, Ratna Vadlamudi, Virginia Kaklamani, Manjeet K. Rao. Imipramine, an FDA approved anti-depressant, suppresses triple negative breast cancer growth and progression through inhibition of FOXM1 and DNA repair pathways [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2975.

Background Consensus statements on lead extraction give consideration to open surgical removal in the setting of large vegetations, to mitigate the risk of massive embolism that may occur with percutaneous lead removal. Vacuum-assisted debulking (VD) of large vegetations as an adjunct to percutaneous lead extraction may provide an opportunity to mitigate these risks. Methods We retrospectively identified all patients undergoing lead extraction at our institution for endovascular infection from 2012 to 2018 and stratified them into two groups based on presence of adjunctive VD (n = 6) or without VD (no-VD, n = 39). VD was performed with the AngioVac system (Angio-Dynamics, Latham, NY, USA). Results Across the cohort, mean age was 62 ± 15 years, ejection fraction was 41 ± 16%, and 39% had end-stage renal disease on dialysis. Defibrillator systems were present in 71%, and 22% had cardiac resynchronization devices. Mean duration of the oldest extracted lead was 6.3 ± 4.9 years. There were no significant differences in baseline covariates between groups. Those in the VD group were significantly less likely to have Staphylococcus aureus as a causative organism (P = .04). In the VD group, vegetations targeted for debulking ranged in size from 1.8 to 6 cm (longest dimension). There were no operative deaths or clinically evident embolic events in either group. The overall nonfatal complication rate in the VD group was higher (33.3% vs 2.3%, P = .043). Conclusion VD can be performed as an adjunct to percutaneous lead extraction with a reasonable safety profile. The relative safety and efficacy of this approach removal requires further study.

Aortic valve disease, particularly aortic stenosis (AS), is a very common form of valvular heart disease in developed countries (Horne et al., Am Heart J 168:414–423, 2014).

Background: We had earlier reported a novel therapeutic agent, ERX-11, that modulates estrogen receptor coregulator interactions. For lead optimization, we designed, synthesized and tested over 500 analogs of ERX-11 in multiple models of BC. We also tested this library of compounds for activity against other cancer cell lines both in vitro and in vivo and against primary tumors ex vivo. Methods: In vitro activity was tested using Cell titer glo, MTT, and apoptosis assays.The utility of the ERX analogs in treating therapy resistant ER-positive BC was evaluated using models with acquired resistance (Tamoxifen, Letrozole), and engineered models that express ER mutations. Xenografts were used for testing the utility of ERX analogs in vivo. Primary patient tumor derived explants were used for ex vivo testing of ERX analogs. Results: Our screening studies identified several ERX analogs with potent activity against BC cells. Subtle changes in the ERX analogs appear to have significant ramifications on both their potency against ER+ BC cell lines and against other tumors types. Some analogs like ERX-41 were more potent than ERX-11 in their ability to block the proliferation of multiple ER-positive BC cell lines (IC50 ranging from 20-200nM). Other analogs like ERX-208 showed similar activity as ERX-11 against ER-positive BC cell lines (IC50 ranging from 100-500nM) but had potent activity against ovarian cancer cell lines. Through iterative changes, we have identified leads compounds with significant activity against other cancers, including in gliomas, ovarian and pancreatic cancers. Although all these compounds were designed to better target the ligand binding pocket of ER, these active compounds do not all target ER and appear to target other proteins, including other nuclear receptors. Some compounds for example have activity in ER-negative breast cancers. In several xenograft models, including pancreatic cancer and ER-negative, the activity of the compounds have been confirmed by oral administration of the ERX analog in vivo. We have also validated the activity of these analogs in patient-derived explants cultured ex vivoin multiple tumor types. Conclusions: From our studies to develop a more potent ERX-11 lead analog, we have identified multiple analogs with distinct activities against multiple cancers. While the intended target of these analogs was ER, our library of analogs has potent activity against both ER-positive and ER-negative tumors. In collaboration, we are pursuing further leads in multiple cancers to further delineate the mechanism of action of these various analogs. Citation Format: Ganesh V. Raj, Xihui Liu, Dede Ekoue, Jung-Mo Ahn, Ratna Vadlamudi. Development of potent lead compounds in multiple tumor types [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 26.

Tumors express intracellular PD-L1 that we showed increases ovarian cancer (OC) and melanoma virulence by increasing mTORC1 and tumor stem cells (TSC). We made PD-L1KO(CRISPR) in ES2 human OC and B16 melanoma cells and re-expressed PD-L1 only on cell surface (PD-L1surf) or in cytoplasm (PD-L1cyto) with specific expression vectors. Subcellular PD-L1 location in all clones was validated by immunoblots, confocal microscopy, and co-IP/mass spec. We selected clones with PD-L1 surf or cyto expression similar to parental cells. PD-L1KO reduced cell diameter 30% which was restored partially in PD-L1surf and fully in PD-L1cyto in ES2 and B16. PD-L1KO significantly decreased viability in all cell lines tested which was fully rescued in PD-L1surf and PD-L1cyto. PD-L1surf ES2 and B16 grew slower in vitro and in vivo than PD-L1cyto and only PD-L1cyto rescued F-actin polymerization phenotype seen in PD-L1KO ES2 and B16. RNA-seq showed that surf and cyto PD-L1 differentially altered canonical signaling pathways, notably mTOR, stemness and gene regulation. PD-L1cyto grew much slower than PD-L1surf B16 in NSG mice but generated more TSC in vitro, and PD-L1surf but not PD-L1cyto B16 responded to αPD-L1 in WT. PD-L1cyto but not PD-L1surf induced mTORC1 activation (p-rpS6, p-P70S6K) through mTORC1 lysosomal docking through LAMTOR1/2 seen by confocal and confirmed by co-IP in ES2 and B16. Imaging CyTOF of human melanomas showed PD-L1cyto association with activated mTOR. Thus, surf versus cyto PD-L1 differentially affects tumor biology, signaling, pathology, TIL and treatment response in OC and melanoma. We define a mechanism for PD-L1-driven mTORC1 activation. Surface vs. cytoplasmic PD-L1 and/or mTORC1 could be response biomarkers for αPD-L1 or αPD-1.

We reported that tumor intrinsic PD-L1 promotes tumor initiating cell (TIC) virulence and mTORC1. Others have focused on PI3K/AKT/mTOR in promoting TIC, but specific downstream mTOR regulators of tumor stemness are unknown. We knocked down Raptor (mTORC1 component, Rptorlo) in ID8agg cells (murine ovarian cancer). Rptorlo cells had reduced mTORC1 with no change in PD-L1 in total cells or CD44+CD24+ TIC. %TIC was reduced in Rptorlo cell cultures, and Rptorlo TIC were functionally defective for self-renewal by tumorosphere formation. Rapamycin (mTORC1 inhibitor) reduced ID8agg Rptor, tumorospheres, and the stemness and virulence genes Oct4 and Nanog. Oct4 knockin to PD-L1lo ID8agg restored TIC numbers and spheres. Together, data confirm PD-L1 drives stemness through Oct4 by increasing mTORC1. Rptorlo TIC formed significantly smaller tumors vs. control TIC in immunodeficient NSG mice, confirming TIC intrinsic Rptor drives immune independent virulence. In wild type mice, Rptorlo vs. control TIC produced less ascites and smaller tumors with lower %TIC in vivo (but similar PD-L1), significantly more recently-activated CD44hiCD62LloCD8+ cells in ascites and draining lymph nodes (DLN). CCR2+CD8+ T cells and CXCR5+TCF1+PD-1+Tim3− stem-like T cells were also increased in Rptorlo DLN. Thus, Rptorlo TIC influence systemic and tumor-associated immunity. In high grade serous ovarian tumors (The Cancer Genome Atlas) high Rptor expression predicted a significantly higher cancer stem cell signature, and lower median survival in late stage patients. Our work establishes tumor intrinsic Rptor driven by PD-L1 plays a key role in regulating TIC and stem like T cells and can be a prognostic marker for late stage ovarian cancer patients.

The host immune response can be corrupted by cancer cells during tumor progression. Here we showed that B lymphocytes played an important role in promoting murine breast cancer (BCa) development, as B cell-deficient mMT mice displayed virtually abrogated tumor growth from transplanted syngeneic ERa+ breast adenocarcinoma cells, concomitant with reduced PD-L1 expression and increased BCa cell death. In murine BCa tumors and tumor-associated lymph nodes, B cells were the major source of IL-27, a pleotropic cytokine with both pro- and anti-inflammatory functions. Deficiency in IL-27 expression specifically in B cells abolished the BCa tumor growth, showing that the effect of B cells in BCa development was mainly through IL-27 production, as likely induced by TLR ligands in the tumor microenvironment together with CD154 and IL-21 expressed by tumor-infiltrating CD4+ T cells. IL-27 treatment of BCa cells resulted in STAT1 and STAT3 phosphorylation, upregulated PD-L1 gene expression and enhanced cell growth. In addition, IL-27 induced activated B cells to express PD-L1 in vitro, consistent with high levels of PD-L1 expression by B cells in tumors. In humans, IL-27 receptor genes were highly expressed in the breast tissue, at levels comparable to those in lymphoid organs, and high IL-27 gene expression was associated with reduced survival in BCa patients, who displayed increased circulating IL-27 levels. Finally, IL-27 supported human BCa cell growth in the presence of tamoxifen, suggesting a role of IL-27 in the drug resistance. Thus, TLR-fueled IL-27 induction in B cells reinforces PD-L1 expression in the tumor environment to drive BCa cell growth and to promote generation of PD-L1hi B regulatory cells, leading to BCa tumor progression.

17β-Estradiol (E2) is a well-known neuroprotective factor in the brain. Recently, our lab demonstrated that the neuroprotective and cognitive effects of E2 require mediation by the estrogen receptor (ER) coregulator protein and proline-, glutamic acid-, and leucine-rich protein 1 (PELP1). In the current study, we examined whether E2, acting via PELP1, can exert anti-inflammatory effects in the ovariectomized rat and mouse hippocampus to regulate NLRP3 inflammasome activation after global cerebral ischemia (GCI). Activation of the NLRP3 inflammasome pathway and expression of its downstream products, cleaved caspase-1 and IL-1β, were robustly increased in the hippocampus after GCI, with peak levels observed at 6-7 days. Expression of P2X7 receptor, an upstream regulator of NLRP3, was also increased after GCI. E2 markedly inhibited NLRP3 inflammasome pathway activation, caspase-1, and proinflammatory cytokine production, as well as P2X7 receptor expression after GCI (at both the mRNA and protein level). Intriguingly, the ability of E2 to exert these anti-inflammatory effects was lost in PELP1 forebrain-specific knockout mice, indicating a key role for PELP1 in E2 anti-inflammatory signaling. Collectively, our study demonstrates that NLRP3 inflammasome activation and proinflammatory cytokine production are markedly increased in the hippocampus after GCI, and that E2 signaling via PELP1 can profoundly inhibit these proinflammatory effects.

PD-L1, expressed on many tumors, inhibits anti-tumor T cells by programmed death (PD)-1 engagement. Tumor PD-L1 predicts anti-PD-L1 treatment outcomes through incompletely understood mechanisms. We used naturally PD-L1+ murine B16 melanoma (ctrl) and made PD-L1KO B16 by CRISPR/Cas9. PD-L1KO B16 grew faster when ctrl B16 was on the opposite flank (trans). By contrast, ctrl B16 grew slower when trans to PD-L1KO B16. Anti-PD-L1 slowed ctrl but not PD-L1KO B16 tumors as expected. However, when PD-L1KO was trans to ctrl αPD-L1 now also slowed PD-L1KO growth. Anti-PD-L1 increased CD3+ T cell infiltration into ctrl and PD-L1KO tumors similarly in vivo, but increased natural killer (NK) cell numbers and functions (e.g., CD107a, IFN-g) in PD-L1KO tumors > ctrl. CD4+ and CD8+ T cells chemotaxed to PD-L1KO slightly > ctrl, but NK cells migrated 2-fold more to PD-L1KO vs ctrl. PD-L1KO tumors produced more CCL2 than ctrl in vitro and in vivo, and this CCL2 induced CCR2+ NK chemotaxis in vitro. Both NK cells and T cells were required for optimal trans responses to anti-PD-L1 treatment in vivo. In wild type mice, anti-CCL2 reduced growth of trans PD-L1KO tumors > ctrl, and reduced anti-PD-L1 efficacy against PD-L1KO> ctrl tumors. In CCR2KO mice, anti-PD-L1 efficacy was retained against trans ctrl B16, but abolished against PD-L1KO trans tumors. PD-L1 also regulated other chemokines recruiting receptor+ cells, with similar effects seen in MB49 bladder cancer and 4T1 breast cancer cells. Thus, tumor PD-L1 alters immune infiltrates and anti-PD-L1 efficacy through novel chemokine effects. Our models help understand anti-PD-L1 (and likely anti-PD-1) responses based on tumor PD-L1 expression and can define ways to improve immunotherapy for PD-L1 null tumors.

We reported that PD-L1 regulates tumor cell-intrinsic mTOR, autophagy and tumor initiating cell (TIC) generation. We found significant cytoplasmic PD-L1 in various mouse and human tumor cell lines by Western, flow cytometry and confocal. To assess subcellular localized PD-L1 effects, we CRISPR knocked out PD-L1 (KO) from mouse B16 melanoma cells and re-expressed PD-L1 only on surface (surfKI; Display vector) or cytoplasm (cytoKI; PcDNA vector). KO cells proliferated slowly vs ctrl in vitro, partially restored by surfKI and cytoKI. KO suppressed mTORC1 (p70S6K), which was reversed by cytoKI but not surfKI. Rapamycin (mTORC1 inhibitor) reduced surfKI and KO proliferation equally, but both > cytoKI (57% vs 88%, p

TIC mediate tumor formation, drug resistance and relapse. We reported that PD-L1 promotes tumor mTORC1, and TIC number and functions. Knocking down PD-L1 reduced expression of stemness genes Oct4 and Nanog and the mTORC1 signaling component Raptor in B16 melanoma and ID8agg ovarian cancer (OC) cells. To understand how PD-L1 controls stemness, we treated control and PD-L1 KO (KO) B16 cells with rapamycin (mTORC1 inhibitor), PI3K inhibitor wortmannin, Notch inhibitor Compound E, and c-Myc inhibitor JQ1. Only rapamycin affected control and KO CD133+CD44+CD24+ TIC differentially (50% reduced TIC in control versus 0% in KO, p=0.0075). We over-expressed Oct4 in B16 but failed to get clones, so over-expressed it in ID8agg PD-L1lo cells (KI) and found that TICs (CD44+CD24+) had improved proliferation and 2-fold more TIC than PD-L1lo cells (p=0.015, p=0.018). We sorted ID8agg, PD-L1lo and KI TIC and cultured them in stem cell medium. KI TICs formed four-fold bigger spheres than PD-L1lo TIC (p

Cardiac arrest-induced global cerebral ischemia (GCI) can lead to death of hippocampal CA1 neurons and cognitive impairment. Previous work has shown that the steroid hormone, 17β-estradiol (E2) is strongly neuroprotective against GCI, an effect that involves, in part, rapid signaling via the membrane estrogen receptor, GPR30. To gain greater insight into E2 actions, the current study examined: 1) whether GPR30 expression in the hippocampus changes after CGI, and 2) the role and potential crosstalk/synergism between local (brain-derived) and exogenous E2 in regulation of GPR30 and rapid prosurvival signaling after GCI. GCI was induced in adult ovariectomized female rats by a 4-vessel occlusion model (12-min occlusion). Vehicle or low diestrous levels of E2 were administered chronically by minipump beginning at the time of ovariectomy. The results showed that GPR30 expression in the hippocampal CA1 was unchanged at 3h, but significantly decreased at 1d and 3d after GCI. In contrast, aromatase expression showed an initial decrease at 3h, a rebound at 1day, and a robust elevation at 3 days after CGI. Administration of exogenous E2 increased both GPR30 and aromatase expression at 3h, 1d and 3d after GCI. Interestingly, central administration of the aromatase inhibitor, letrozole or aromatase antisense (AS) oligonucleotides abolished exogenous E2 enhancement of GPR30 and aromatase at 3d after GCI. Furthermore, letrozole and aromatase-AS treatment attenuated exogenous E2-induced phosphorylation/activation of prosurvival ERK1/2 and CREB following GCI, as well as exogenous E2-induced enhanced expression of BDNF, PSD95 and synaptophsin. As a whole, the results suggest that local-derived E2 may help mediate beneficial protective effects of exogenous E2 after GCI to enhance GPR30 expression, increase activation of ERK1/2-CREB-BDNF pro-survival signaling, and improve synaptic function.

Tumor B7-H1 (PD-L1) promotes tumor immune evasion by interaction with T cell PD-1. B16 melanoma and ID8agg ovarian cancer express B7-H1. We genetically made B7-H1lo or B7-H1hi lines to study additional B7-H1 effects. Control B16 and ID8agg grew more rapidly in vitro versus B7-H1lo cells and B7-H1hi B16 cells grew faster than control. In vivo, B16 and ID8agg grew faster than B7-H1lo lines in immunodeficient NSG mice. B16 challenge in NSG mice with B7-H1hi cells produced more lung metastases versus parental, which produced more metastases than B7-H1lo. αB7-H1 significantly reduced tumor growth and metastases. These data support an immune-independent role for tumor B7-H1 in tumor growth and metastatic spread. Control B16 and ID8agg cells had elevated TORC1 and reduced TORC2 versus B7-H1lo cells. The mTOR inhibitor rapamycin reduced cell growth greater in parental versus B7-H1lo cells in vitro. Also in line with reduced TORC1, B7-H1lo cells had increased autophagy (LC3-II/actin ratio and autophagosome formation) compared to parental cells. Notably, autophagy inhibitors chloroquine and 3-methyladenine had a greater inhibitory effect on in vitro proliferation of control B16 versus B7-H1lo cells. Inhibition of autophagy in vivo with either agent significantly reduced tumor growth of B16 but not B7-H1lo cells, suggesting that B7-H1 replete tumors are more dependent on the stress-modulating function of autophagy versus B7-H1lo tumors. Our studies show novel tumor-intrinsic B7-H1 signal effects that regulate mTOR and autophagy signals directly associated with immune-independent tumor growth, supporting a paradigm shifting concept that tumor B7-H1 augments stress response and metabolism through cell-intrinsic mechanisms.

Cancer stem cells (CSC) resist chemotherapy and radiation, leading to treatment failures and reduced patient survival. Thus, CSC are clinically relevant therapy targets. B7-H1 is an immune checkpoint molecule and a target for αB7-H1 immunotherapy. We generated control (native B7-H1+) and B7-H1lo B16 melanoma lines by shRNA. B16*ctrl cultures were 4.2% CSCs (CD44+CD133+CD24+) vs. 0.19% in B16*B7-H1lo. CSC from B16*B7-H1lo cultures formed fewer and smaller (

m e r l a a a w p r FOR MAJOR BLEEDING related to severe trauma, major surgery, or chronic anticoagulation, a rapid assessment of hemostatic function is crucial so that optimal fluid replacements and blood transfusion can be administered without delays.1-6 Although the safety of blood products with regard to viral transmission risks has improved in recent years,7,8 transfusions of allogeneic erythrocyte and plasma products have been implicated in serious adverse events, including nosocomial infections, acute lung injury, and organ dysfunction.9-12 Obtaining conventional laboratory tests, such as the prothrombin time (PT), activated partial thromboplastin time (aPTT), and fibrinogen level, during acute bleeding is difficult because of a long turn-around time ( 30 min).13,14 Furthermore, laboatory PT/international normalized ratio and aPTT may not be articularly useful in predicting bleeding after trauma or invaive procedures.15,16 The prime example of bleeding management is preemptive transfusions of fresh-frozen plasma (FFP) and platelet concentrates according to the erythrocyte requirement in major trauma cases.17,18 This so-called “damage control resuscitation” (DCR; able 1) originally was advocated for battlefield resuscitation n which laboratory testing and transfusion resources were imited. However, plasma product transfusion according to CR became increasingly popular in US civilian trauma ceners and operating rooms.17,19 The prevention of trauma-induced coagulopathy and subsequent nonsurgical bleeding is a major advantage of DCR,20 but the DCR approach lacks a specific target for replacement and a consideration for interindividual variability in coagulation factor levels and vascular (endothelial) responses. Implementing transfusion algorithms based on point-of-care (POC) coagulation testing can be effective in decreasing transfusion requirements in elective or urgent cardiac surgical settings.2,5,21-23 In this review, the practical use of thromboelastometry is discussed relating to the diagnosis of coagulopathy and optimizing hemostatic interventions.

αB7-H1 (αPD-L1) reportedly treats cancer by blocking PD-1 signals, but few formal data are reported. We showed B16 melanoma is CD80-PD-1-B7-H1+. αB7-H1 slowed subcutaneous (SQ) B16 in wild type (WT), B7-H1 KO and CD80 KO, with little difference between groups. αB7-H1 did not slow B16/B7-H1lo tumors in B7-H1 KO mice, formally showing tumor B7-H1 mediates αB7-H1 effects. αPD-1 slowed B16 in WT or B7-H1 KO, but not B16/B7-H1lo in B7-H1 KO, formally showing B7-H1/PD-1 effects in αB7-H1. Tumor B7-H1 variably predicts αB7-H1 efficacy, possibly due to variable tumor B7-H1 expression. To model variable B7-H1, we gave B16 or B16/B7-H1lo on both flanks, or each on opposite flanks of the same WT mice. αB7-H1 slowed B16, but not B16/B7-H1lo, unless B16 was on the other flank, suggesting spread of anti-tumor immunity, helping explain variable αB7-H1 effects by tumor B7-H1 status. We found EL4 T cell lymphoma is CD80loB7-H1loPD-1hi. αB7-H1 and αPD-1 did not slow SQ EL4. Unexpectedly, EL4/B7-H1hi exhibited significant growth delay after SQ or intravenous challenge in WT. EL4/B7-H1hi grew well in vitro, but in clusters as opposed to EL4. αPD-1 or αB7-H1 in vitro blocked clusters but such pre-treated cells still grew slowly in vivo suggesting negative inter-cell B7-H1/PD-1 signals. Our data formally show B7-H1/PD-1 signals in αB7-H1 treatment effects, help explain variable αB7-H1 effects by tumor B7-H1 status, and suggest novel and specific effects in T cell lymphomas requiring more study.

mTOR signals through 2 complexes: mTORC1 and mTORC2. Approved cancer mTOR inhibitor drugs inhibit mTORC1 and are poorly effective against carcinomas and B cell lymphomas. There is thought that dual mTORC1/2 inhibitors could have better clinical efficacy. As mTOR regulates important T cell functions, we tested mTOR inhibitors in mouse EL4 T cell lymphoma. The mTORC1 inhibitor rapamycin potently suppressed EL4 proliferation in vitro, and significantly reduced EL4 tumor growth in vivo. AZD8055, a dual mTORC1/2 inhibitor, also inhibited EL4 cell proliferation in vitro, decreased in vivo tumor burden and improved survival in EL4 challenged mice, but no better than rapamycin. EL4 tumor growth was slower in females versus males, suggesting hormonal effects. As estrogen receptor (ER)β agonists reportedly can treat lymphomas we tested them. We found that ERβ agonists altered EL4 mTOR signals and a novel ERβ/mTOR physical interaction (mTORC2>>mTORC1) in EL4 by GST pull-down. The ERβ agonist S-equol synergized with AZD8055 to inhibit EL4 cell proliferation in vitro but did not alter EL4 tumor growth in vivo nor boost AZD8055-mediated EL4 growth suppression or survival in vivo. EL4 tumors from AZD8055 treated mice had reduced viability and proliferation (Ki67) without affecting CD3+ T cell Ki67. AZD8055 treated ID8 ovarian cancer in vivo but rapamycin did not, suggesting T cell lymphomas could respond to mTOR inhibitors better than carcinomas. ERβ effects also deserve more study.

Immune co-signaling blockade is effective cancer immunotherapy but is not curative alone. Treg depletion has shown potential human anti-cancer efficacy, but is also not curative alone. As B7-H1 immune co-signaling facilitates Treg generation, we hypothesized that adding αB7-H1 to Treg depletion would improve treatment by hindering Treg regeneration. We show here that αB7-H1 plus denileukin diftitox (DD) to deplete Tregs is a synergistic combo that effects complete and durable (>7 mos) tumor regressions in bulky B16 melanoma, whereas neither agent alone does. αB7-H1 efficacy alone or with DD is lost in IFN-γ KO or T cell KO mice, and is mediated in part by tumor-specific CD8+ (but not CD4+) T cells. αB7-H1 is highly effective in B7-H1 KO mice, demonstrating a major role for tumor B7-H1 in treatment efficacy. Reducing tumor B7-H1-mediated Treg generation appears to be a mechanism as predicted. The αB7-H1/DD combo was also effective in ID8 ovarian cancer showing broad application. We engineered B7-H1hi/lo B16 tumors for further immune studies. Surprisingly, B7-H1hi B16 grew faster than B7-H1lo in RAG KO, suggesting B7-H1 as an adaptive immunity-independent tumor growth/survival factor. IFNγ upregulated tumor B7-H1, but αB7-H1 did not prevent IFN-γ-mediated tumor inhibition in vitro. B7-H1hi/lo tumors in B7-H1 KO are being used to define tumor vs host effects of αB7-H1 ± Treg depletion. As αB7-H1 and DD have been tested clinically, this combo is rapidly clinically translated.