BTN1A1 is a novel immune checkpoint mutually exclusive to PD-L1.

BACKGROUND: While Programmed cell death protein 1 (PD-1)/programmed cell death-ligand 1 (PD-L1) blockade is a potent antitumor treatment strategy, it is effective in only limited subsets of patients with cancer, emphasizing the need for the identification of additional immune checkpoints. Butyrophilin 1A1 (BTN1A1) has been reported to exhibit potential immunoregulatory activity, but its ability to function as an immune checkpoint remains to be systematically assessed, and the mechanisms underlying such activity have yet to be characterized. METHODS: BTN1A1 expression was evaluated in primary tumor tissue samples, and its ability to suppress T-cell activation and T cell-dependent tumor clearance was examined. The relationship between BTN1A1 and PD-L1 expression was further characterized, followed by the development of a BTN1A1-specific antibody that was administered to tumor-bearing mice to test the amenability of this target to immune checkpoint inhibition. RESULTS: BTN1A1 was confirmed to suppress T-cell activation in vitro and in vivo. Robust BTN1A1 expression was detected in a range of solid tumor tissue samples, and BTN1A1 expression was mutually exclusive with that of PD-L1 as a consequence of its inhibition of Janus-activated kinase/signal transducer and activator of transcription signaling-induced PD-L1 upregulation. Antibody-mediated BTN1A1 blockade suppressed tumor growth and enhanced immune cell infiltration in syngeneic tumor-bearing mice. CONCLUSION: Together, these results confirm that the potential of BTN1A1 is a bona fide immune checkpoint and a viable immunotherapeutic target for the treatment of individuals with anti-PD-1/PD-L1 refractory or resistant disease, opening new avenues to improving survival outcomes for patients with a range of cancers.

SMAC Mimetic/IAP Inhibitor Birinapant Enhances Radiosensitivity of Glioblastoma Multiforme.

Birinapant is a novel SMAC peptidomimetic molecule in clinical development. It suppresses the inhibitor of apoptosis proteins (IAPs) and promotes cytochrome-C/Apaf-1/caspase-9 activation to induce effective apoptosis. Because IAP inhibition has been shown to enhance the sensitivity of cancer cells to radiation, we investigated the role of birinapant in radiosensitization of glioblastoma cells in vitro and in vivo. Two glioblastoma cell lines, U-251 and U-87, were used to analyze radiosensitization in vitro with 7-AAD cell death/apoptosis and clonogenic assays. Subcutaneous flank (U-251 and U-87) and intracranial orthotopic (U-251) xenografts in nude mice were used to evaluate radiosensitization in vivo. TNF-α levels in media and serum were measured using electrochemiluminescence. Radiosensitization in vitro was more prominent for U-251 cells than for U-87 cells. In vivo, in both tumor models, significant tumor growth delay was observed with combination treatment compared to radiation alone. There was a survival benefit with combination treatment in the orthotopic U-251 model. TNF-α levels in media correlated directly with radiation dose in vitro. These findings show that birinapant can enhance the radiosensitivity of glioblastoma cell lines in cell-based assays and tumor models via radiation-induced TNF-α. Further study into the use of birinapant with radiation therapy is warranted.

Cadherin 11 Promotes Immunosuppression and Extracellular Matrix Deposition to Support Growth of Pancreatic Tumors and Resistance to Gemcitabine in Mice.

BACKGROUND & AIMS: Pancreatic ductal adenocarcinomas (PDACs) are characterized by fibrosis and an abundance of cancer-associated fibroblasts (CAFs). We investigated strategies to disrupt interactions among CAFs, the immune system, and cancer cells, focusing on adhesion molecule CDH11, which has been associated with other fibrotic disorders and is expressed by activated fibroblasts. METHODS: We compared levels of CDH11 messenger RNA in human pancreatitis and pancreatic cancer tissues and cells with normal pancreas, and measured levels of CDH11 protein in human and mouse pancreatic lesions and normal tissues. We crossed p48-Cre;LSL-KrasG12D/+;LSL-Trp53R172H/+ (KPC) mice with CDH11-knockout mice and measured survival times of offspring. Pancreata were collected and analyzed by histology, immunohistochemistry, and (single-cell) RNA sequencing; RNA and proteins were identified by imaging mass cytometry. Some mice were given injections of PD1 antibody or gemcitabine and survival was monitored. Pancreatic cancer cells from KPC mice were subcutaneously injected into Cdh11+/+ and Cdh11-/- mice and tumor growth was monitored. Pancreatic cancer cells (mT3) from KPC mice (C57BL/6), were subcutaneously injected into Cdh11+/+ (C57BL/6J) mice and mice were given injections of antibody against CDH11, gemcitabine, or small molecule inhibitor of CDH11 (SD133) and tumor growth was monitored. RESULTS: Levels of CDH11 messenger RNA and protein were significantly higher in CAFs than in pancreatic cancer epithelial cells, human or mouse pancreatic cancer cell lines, or immune cells. KPC/Cdh11+/- and KPC/Cdh11-/- mice survived significantly longer than KPC/Cdh11+/+ mice. Markers of stromal activation entirely surrounded pancreatic intraepithelial neoplasias in KPC/Cdh11+/+ mice and incompletely in KPC/Cdh11+/- and KPC/Cdh11-/- mice, whose lesions also contained fewer FOXP3+ cells in the tumor center. Compared with pancreatic tumors in KPC/Cdh11+/+ mice, tumors of KPC/Cdh11+/- mice had increased markers of antigen processing and presentation; more lymphocytes and associated cytokines; decreased extracellular matrix components; and reductions in markers and cytokines associated with immunosuppression. Administration of the PD1 antibody did not prolong survival of KPC mice with 0, 1, or 2 alleles of Cdh11. Gemcitabine extended survival of KPC/Cdh11+/- and KPC/Cdh11-/- mice only or reduced subcutaneous tumor growth in mT3 engrafted Cdh11+/+ mice when given in combination with the CDH11 antibody. A small molecule inhibitor of CDH11 reduced growth of pre-established mT3 subcutaneous tumors only if T and B cells were present in mice. CONCLUSIONS: Knockout or inhibition of CDH11, which is expressed by CAFs in the pancreatic tumor stroma, reduces growth of pancreatic tumors, increases their response to gemcitabine, and significantly extends survival of mice. CDH11 promotes immunosuppression and extracellular matrix deposition, and might be developed as a therapeutic target for pancreatic cancer.

Targeting Glycosylated PD-1 Induces Potent Antitumor Immunity.

Immunotherapies targeting programmed cell death protein 1 (PD-1) and programmed cell death 1 ligand 1 (PD-L1) immune checkpoints represent a major breakthrough in cancer treatment. PD-1 is an inhibitory receptor expressed on the surface of activated T cells that dampens T-cell receptor (TCR)/CD28 signaling by engaging with its ligand PD-L1 expressed on cancer cells. Despite the clinical success of PD-1 blockade using mAbs, most patients do not respond to the treatment, and the underlying regulatory mechanisms of PD-1 remain incompletely defined. Here we show that PD-1 is extensively N-glycosylated in T cells and the intensities of its specific glycoforms are altered upon TCR activation. Glycosylation was critical for maintaining PD-1 protein stability and cell surface localization. Glycosylation of PD-1, especially at the N58 site, was essential for mediating its interaction with PD-L1. The mAb STM418 specifically targeted glycosylated PD-1, exhibiting higher binding affinity to PD-1 than FDA-approved PD-1 antibodies, potently inhibiting PD-L1/PD-1 binding, and enhancing antitumor immunity. Together, these findings provide novel insights into the functional significance of PD-1 glycosylation and offer a rationale for targeting glycosylated PD-1 as a potential strategy for immunotherapy. SIGNIFICANCE: These findings demonstrate that glycosylation of PD-1 is functionally significant and targeting glycosylated PD-1 may serve as a means to improve immunotherapy response.

LHX2 Mediates the FGF-to-SHH Regulatory Loop during Limb Development.

During limb development, fibroblast growth factors (Fgfs) govern proximal⁻distal outgrowth and patterning. FGFs also synchronize developmental patterning between the proximal⁻distal and anterior⁻posterior axes by maintaining Sonic hedgehog (Shh) expression in cells of the zone of polarizing activity (ZPA) in the distal posterior mesoderm. Shh, in turn, maintains Fgfs in the apical ectodermal ridge (AER) that caps the distal tip of the limb bud. Crosstalk between Fgf and Shh signaling is critical for patterned limb development, but the mechanisms underlying this feedback loop are not well-characterized. Implantation of Fgf beads in the proximal posterior limb bud can maintain SHH expression in the former ZPA domain (evident 3 h after application), while prolonged exposure (24 h) can induce SHH outside of this domain. Although temporally and spatially disparate, comparative analysis of transcriptome data from these different populations accentuated genes involved in SHH regulation. Comparative analysis identified 25 candidates common to both treatments, with eight linked to SHH expression or function. Furthermore, we demonstrated that LHX2, a LIM Homeodomain transcription factor, is an intermediate in the FGF-mediated regulation of SHH. Our data suggest that LHX2 acts as a competency factor maintaining distal posterior SHH expression subjacent to the AER.

Eradication of Triple-Negative Breast Cancer Cells by Targeting Glycosylated PD-L1.

Protein glycosylation provides proteomic diversity in regulating protein localization, stability, and activity; it remains largely unknown whether the sugar moiety contributes to immunosuppression. In the study of immune receptor glycosylation, we showed that EGF induces programmed death ligand 1 (PD-L1) and receptor programmed cell death protein 1 (PD-1) interaction, requiring ß-1,3-N-acetylglucosaminyl transferase (B3GNT3) expression in triple-negative breast cancer. Downregulation of B3GNT3 enhances cytotoxic T cell-mediated anti-tumor immunity. A monoclonal antibody targeting glycosylated PD-L1 (gPD-L1) blocks PD-L1/PD-1 interaction and promotes PD-L1 internalization and degradation. In addition to immune reactivation, drug-conjugated gPD-L1 antibody induces a potent cell-killing effect as well as a bystander-killing effect on adjacent cancer cells lacking PD-L1 expression without any detectable toxicity. Our work suggests targeting protein glycosylation as a potential strategy to enhance immune checkpoint therapy.

AAV8-antiVEGFfab Ocular Gene Transfer for Neovascular Age-Related Macular Degeneration.

Sustained suppression of VEGF is needed in many patients with neovascular age-related macular degeneration (NVAMD), and gene transfer of a VEGF-neutralizing protein is a promising approach to achieve it. Initial clinical trials testing this approach have shown encouraging signals, but evidence of robust transgene expression and consistent antiangiogenic and antipermeability activity has been lacking. In this study, we demonstrate expression of an anti-human VEGF antibody fragment (antiVEGFfab) after subretinal injection of AAV8-antiVEGFfab. In transgenic mice expressing human VEGF in retina (rho/VEGF mice), a model of type 3 choroidal neovascularization (NV), eyes injected with ≥1 × 107 gene copies (GC) of AAV8-antiVEGFfab had significantly less mean area of NV than null vector-injected eyes. A dose-dependent response was observed with modest reduction of NV with ≤3 × 107, >50% reduction with ≥1 × 108 GC and almost complete elimination of NV with 3 × 109 or 1 × 1010 GC. In Tet/opsin/VEGF mice, in which doxycycline-induced high expression of VEGF leads to severe vascular leakage and exudative retinal detachment (RD), reduction of total RD by 70%-80% occurred with 3 × 109 or 1 × 1010 GC of AAV8-antiVEGFfab, an effect that was sustained for at least a month. These data strongly support initiating clinical trials testing subretinal injection of AAV8-antiVEGFfab in patients with NVAMD.

Anifrolumab, an Anti-Interferon-α Receptor Monoclonal Antibody, in Moderate-to-Severe Systemic Lupus Erythematosus.

OBJECTIVE: To assess the efficacy and safety of anifrolumab, a type I interferon (IFN) receptor antagonist, in a phase IIb, randomized, double-blind, placebo-controlled study of adults with moderate-to-severe systemic lupus erythematosus (SLE). METHODS: Patients (n = 305) were randomized to receive intravenous anifrolumab (300 mg or 1,000 mg) or placebo, in addition to standard therapy, every 4 weeks for 48 weeks. Randomization was stratified by SLE Disease Activity Index 2000 score (<10 or ≥10), oral corticosteroid dosage (<10 or ≥10 mg/day), and type I IFN gene signature test status (high or low) based on a 4-gene expression assay. The primary end point was the percentage of patients achieving an SLE Responder Index (SRI[4]) response at week 24 with sustained reduction of oral corticosteroids (<10 mg/day and less than or equal to the dose at week 1 from week 12 through 24). Other end points (including SRI[4], British Isles Lupus Assessment Group [BILAG]-based Composite Lupus Assessment [BICLA], modified SRI[6], and major clinical response) were assessed at week 52. The primary end point was analyzed in the modified intent-to-treat (ITT) population and type I IFN-high subpopulation. The study result was considered positive if the primary end point was met in either of the 2 study populations. The Type I error rate was controlled at 0.10 (2-sided), within each of the 2 study populations for the primary end point analysis. RESULTS: The primary end point was met by more patients treated with anifrolumab (34.3% of 99 for 300 mg and 28.8% of 104 for 1,000 mg) than placebo (17.6% of 102) (P = 0.014 for 300 mg and P = 0.063 for 1,000 mg, versus placebo), with greater effect size in patients with a high IFN signature at baseline (13.2% in placebo-treated patients versus 36.0% [P = 0.004] and 28.2% [P = 0.029]) in patients treated with anifrolumab 300 mg and 1,000 mg, respectively. At week 52, patients treated with anifrolumab achieved greater responses in SRI(4) (40.2% versus 62.6% [P < 0.001] and 53.8% [P = 0.043] with placebo, anifrolumab 300 mg, and anifrolumab 1,000 mg, respectively), BICLA (25.7% versus 53.5% [P < 0.001] and 41.2% [P = 0.018], respectively), modified SRI(6) (28.4% versus 49.5% [P = 0.002] and 44.7% [P = 0.015], respectively), major clinical response (BILAG 2004 C or better in all organ domains from week 24 through week 52) (6.9% versus 19.2% [P = 0.012] and 17.3% [P = 0.025], respectively), and several other global and organ-specific end points. Herpes zoster was more frequent in the anifrolumab-treated patients (2.0% with placebo treatment versus 5.1% and 9.5% with anifrolumab 300 mg and 1,000 mg, respectively), as were cases reported as influenza (2.0% versus 6.1% and 7.6%, respectively), in the anifrolumab treatment groups. Incidence of serious adverse events was similar between groups (18.8% versus 16.2% and 17.1%, respectively). CONCLUSION: Anifrolumab substantially reduced disease activity compared with placebo across multiple clinical end points in the patients with moderate-to-severe SLE.

A high content clonogenic survival drug screen identifies mek inhibitors as potent radiation sensitizers for KRAS mutant non-small-cell lung cancer.

INTRODUCTION: Traditional clonogenic survival and high throughput colorimetric assays are inadequate as drug screens to identify novel radiation sensitizers. We developed a method that we call the high content clonogenic survival assay (HCSA) that will allow screening of drug libraries to identify candidate radiation sensitizers. METHODS: Drug screen using HCSA was done in 96 well plates. After drug treatment, irradiation, and incubation, colonies were stained with crystal violet and imaged on the INCell 6000 (GE Health). Colonies achieving 50 or more cells were enumerated using the INCell Developer image analysis software. A proof-of-principle screen was done on the KRAS mutant lung cancer cell line H460 and a Custom Clinical Collection (146 compounds). RESULTS: Multiple drugs of the same class were found to be radiation sensitizers and levels of potency seemed to reflect the clinical relevance of these drugs. For instance, several PARP inhibitors were identified as good radiation sensitizers in the HCSA screen. However, there were also a few PARP inhibitors not found to be sensitizing that have either not made it into clinical development, or in the case of BSI-201, was proven to not even be a PARP inhibitor. We discovered that inhibitors of pathways downstream of activated mutant KRAS (PI3K, AKT, mTOR, and MEK1/2) sensitized H460 cells to radiation. Furthermore, the potent MEK1/2 inhibitor tramenitib selectively enhanced radiation effects in KRAS mutant but not wild-type lung cancer cells. CONCLUSIONS: Drug screening for novel radiation sensitizers is feasible using the HCSA approach. This is an enabling technology that will help accelerate the discovery of novel radiosensitizers for clinical testing.

Mitigating the risk of radiation-induced cancers: limitations and paradigms in drug development.

The United States radiation medical countermeasures (MCM) programme for radiological and nuclear incidents has been focusing on developing mitigators for the acute radiation syndrome (ARS) and delayed effects of acute radiation exposure (DEARE), and biodosimetry technologies to provide radiation dose assessments for guiding treatment. Because a nuclear accident or terrorist incident could potentially expose a large number of people to low to moderate doses of ionising radiation, and thus increase their excess lifetime cancer risk, there is an interest in developing mitigators for this purpose. This article discusses the current status, issues, and challenges regarding development of mitigators against radiation-induced cancers. The challenges of developing mitigators for ARS include: the long latency between exposure and cancer manifestation, limitations of animal models, potential side effects of the mitigator itself, potential need for long-term use, the complexity of human trials to demonstrate effectiveness, and statistical power constraints for measuring health risks (and reduction of health risks after mitigation) following relatively low radiation doses (<0.75 Gy). Nevertheless, progress in the understanding of the molecular mechanisms resulting in radiation injury, along with parallel progress in dose assessment technologies, make this an opportune, if not critical, time to invest in research strategies that result in the development of agents to lower the risk of radiation-induced cancers for populations that survive a significant radiation exposure incident.

Dose-escalation of human anti-interferon-α receptor monoclonal antibody MEDI-546 in subjects with systemic sclerosis: a phase 1, multicenter, open label study.

INTRODUCTION: Type I interferons (IFNs) are implicated in the pathogenesis of systemic sclerosis (SSc). MEDI-546 is an investigational human monoclonal antibody directed against the type I IFN receptor. This Phase 1 study evaluated the safety/tolerability, pharmacokinetics (PK), immunogenicity, and pharmacodynamics (PD) of single and multiple intravenous doses of MEDI-546 in adults with SSc. METHODS: Subjects (≥18 years) with SSc were enrolled in an open-label, dose-escalation study to receive single (0.1, 0.3, 1.0, 3.0, 10.0, or 20.0 mg/kg), or 4 weekly intravenous doses (0.3, 1.0, or 5.0 mg/kg/week) of MEDI-546. Subjects were followed for 12 weeks. Safety assessments included adverse events (AEs), laboratory results, and viral monitoring. Blood samples were collected from all subjects for determination of PK, presence of anti-drug antibodies (ADAs), and expression of type I IFN-inducible genes. RESULTS: Of 34 subjects (mean age 47.4 years), 32 completed treatment and 33 completed the study. Overall, 148 treatment-emergent AEs (TEAEs) were reported (68.9% mild, 27.7% moderate). TEAEs included one grade 1 infusion reaction (5.0 mg/kg/week multiple dose). Of 4 treatment-emergent serious AEs (skin ulcer, osteomyelitis, vertigo, and chronic myelogenous leukemia (CML)), only CML (1.0 mg/kg/week multiple dose) was considered possibly treatment-related. MEDI-546 exhibited non-linear PK at lower doses. ADAs were detected in 5 subjects; no apparent impact on PK was observed. Peak inhibition of the type I IFN signature in whole blood was achieved within 1 day and in skin after 7 days. CONCLUSION: The safety/tolerability, PK, and PD profiles observed in this study support further clinical development of MEDI-546. TRIAL REGISTRATION: ClinicalTrials.gov NCT00930683.

Lessons learned from radiation oncology clinical trials.

A workshop entitled "Lessons Learned from Radiation Oncology Trials" was held on December 7-8, 2011, in Bethesda, MD, to present and discuss some of the recently conducted radiation oncology clinical trials with a focus on those that failed to refute the null hypothesis. The objectives of this workshop were to summarize and examine the questions that these trials provoked, to assess the quality and limitations of the preclinical data that supported the hypotheses underlying these trials, and to consider possible solutions to these challenges for the design of future clinical trials. Several themes emerged from the discussions: (i) opportunities to learn from null-hypothesis trials through tissue and imaging studies; (ii) value of preclinical data supporting the design of combinatorial therapies; (iii) significance of validated biomarkers; (iv) necessity of quality assurance in radiotherapy delivery; (v) conduct of sufficiently powered studies to address the central hypotheses; and (vi) importance of publishing results of the trials regardless of the outcome. The fact that well-designed hypothesis-driven clinical trials produce null or negative results is expected given the limitations of trial design and complexities of cancer biology. It is important to understand the reasons underlying such null results, however, to effectively merge the technologic innovations with the rapidly evolving biology for maximal patient benefit through the design of future clinical trials.

Opportunities and challenges in the era of molecularly targeted agents and radiation therapy.

The first annual workshop for preclinical and clinical development of radiosensitizers took place at the National Cancer Institute on August 8-9, 2012. Radiotherapy is one of the most commonly applied and effective oncologic treatments for solid tumors. It is well recognized that improved clinical efficacy of radiotherapy would make a substantive impact in clinical practice and patient outcomes. Advances in genomic technologies and high-throughput drug discovery platforms have brought a revolution in cancer treatment by providing molecularly targeted agents for various cancers. Development of predictive biomarkers directed toward specific subsets of cancers has ushered in a new era of personalized therapeutics. The field of radiation oncology stands to gain substantial benefit from these advances given the concerted effort to integrate this progress into radiation therapy. This workshop brought together expert clinicians and scientists working in various disease sites to identify the exciting opportunities and expected challenges in the development of molecularly targeted agents in combination with radiation therapy.

Inhibition of nicotinamide phosphoribosyltransferase (NAMPT) activity by small molecule GMX1778 regulates reactive oxygen species (ROS)-mediated cytotoxicity in a p53- and nicotinic acid phosphoribosyltransferase1 (NAPRT1)-dependent manner.

Cancer cells undergo mitosis more frequently than normal cells and thus have increased metabolic needs, which in turn lead to higher than normal reactive oxygen species (ROS) production. Higher ROS production increases cancer cell dependence on ROS scavenging systems to balance the increased ROS. Selectively modulating intracellular ROS in cancers by exploiting cancer dependence on ROS scavenging systems provides a useful therapeutic approach. Essential to developing these therapeutic strategies is to maintain physiologically low ROS levels in normal tissues while inducing ROS in cancer cells. GMX1778 is a specific inhibitor of nicotinamide phosphoribosyltransferase, a rate-limiting enzyme required for the regeneration of NAD(+) from nicotinamide. We show that GMX1778 increases intracellular ROS in cancer cells by elevating the superoxide level while decreasing the intracellular NAD(+) level. Notably, GMX1778 treatment does not induce ROS in normal cells. GMX1778-induced ROS can be diminished by adding nicotinic acid (NA) in a NA phosphoribosyltransferase 1 (NAPRT1)-dependent manner, but NAPRT1 is lost in a high frequency of glioblastomas, neuroblastomas, and sarcomas. In NAPRT1-deficient cancer cells, ROS induced by GMX1778 was not susceptible to treatment with NA. GMX1778-mediated ROS induction is p53-dependent, suggesting that the status of both p53 and NAPRT1 might affect tumor apoptosis, as determined by annexin-V staining. However, as determined by colony formation, GMX1778 long term cytotoxicity in cancer cells was only prevented by the addition of NA to NAPRT1-expressing cells. Exposure to GMX1778 may be a novel way of inducing ROS selectively in NAPRT1-negative tumors without inducing cytotoxic ROS in normal tissue.

GI-Associated Hemangiomas and Vascular Malformations.

Hemangiomas and vascular malformations of the gastrointestinal tract, rare clinical entities, present as overt or occult bleeding. They can be distributed throughout the intestinal digestive system, or present as a singular cavernous hemangioma or malformation, which is often located in the rectosigmoid region. Misdiagnosis is common despite characteristic radiographic features such as radiolucent phleboliths on plain film imaging and a purplish nodule on endoscopy. Adjunctive imaging such as computed tomography and magnetic resonance imaging are suggested as there is potential for local invasion. Endorectal ultrasound with Doppler has also been found to be useful in some instances. Surgical resection is the mainstay of treatment, with an emphasis on sphincter preservation. Nonsurgical endoscopic treatment with banding and sclerotherapy has been reported with success, especially in instances where an extensive resection is not feasible.

Benefits of mock oral examinations in a multi-institutional consortium for board certification in general surgery training.

The Southeast Michigan Center for Medical Education (SEMCME) is a consortium of teaching hospitals in the Greater Detroit metropolitan area. SEMCME pools its resources for several educational means, including mock oral board examinations. The educational and cost benefits to mock oral examinations on a multi-institutional basis in preparation for the American Board of Surgery (ABS) certifying examination were analyzed. Ten-year multi-institution data from the mock oral examinations were correlated with ABS certifying examination pass rates. Mock oral examination scores were available for 107 of 147 graduates, which included 12 candidates who failed their certifying examination on the first attempt (pass rate = 89%). Four of 31 examinees who had a low score (4.9 or less) in their mock oral exams failed their certifying examination in their first attempt. The cost of running the mock examination was low (approximately $35/resident for 50 residents). When graduates from the last 10 years were surveyed, the majority of respondents believed that the mock oral examination helped in their success and with their preparation for the certifying examination. Thus, the many benefits of administering the examination with the resources of a consortium of hospitals result in the accurate reproduction of real-life testing conditions with reasonable overall costs per resident.

Cellular inhibition of checkpoint kinase 2 (Chk2) and potentiation of camptothecins and radiation by the novel Chk2 inhibitor PV1019 [7-nitro-1H-indole-2-carboxylic acid {4-[1-(guanidinohydrazone)-ethyl]-phenyl}-amide].

Chk2 is a checkpoint kinase involved in the ataxia telangiectasia mutated pathway, which is activated by genomic instability and DNA damage, leading to either cell death (apoptosis) or cell cycle arrest. Chk2 provides an unexplored therapeutic target against cancer cells. We recently reported 4,4'-diacetyldiphenylurea-bis(guanylhydrazone) (NSC 109555) as a novel chemotype Chk2 inhibitor. We have now synthesized a derivative of NSC 109555, PV1019 (NSC 744039) [7-nitro-1H-indole-2-carboxylic acid {4-[1-(guanidinohydrazone)-ethyl]-phenyl}-amide], which is a selective submicromolar inhibitor of Chk2 in vitro. The cocrystal structure of PV1019 bound in the ATP binding pocket of Chk2 confirmed enzymatic/biochemical observations that PV1019 acts as a competitive inhibitor of Chk2 with respect to ATP. PV1019 was found to inhibit Chk2 in cells. It inhibits Chk2 autophosphorylation (which represents the cellular kinase activation of Chk2), Cdc25C phosphorylation, and HDMX degradation in response to DNA damage. PV1019 also protects normal mouse thymocytes against ionizing radiation-induced apoptosis, and it shows synergistic antiproliferative activity with topotecan, camptothecin, and radiation in human tumor cell lines. We also show that PV1019 and Chk2 small interfering RNAs can exert antiproliferative activity themselves in the cancer cells with high Chk2 expression in the NCI-60 screen. These data indicate that PV1019 is a potent and selective inhibitor of Chk2 with chemotherapeutic and radiosensitization potential.

Erythrokeratoderma variabilis successfully treated with topical tazarotene.

Erythrokeratoderma variabilis, also known as Mendes da Costa syndrome, is a genodermatosis belonging to the group of diseases known as the erythrokeratodermias. Erythrokeratoderma variabilis is characterized by two distinctive manifestations: well-demarcated, variable, transient, figurate patches of erythema, and localized or generalized hyperkeratotic plaques. Treatments include topical retinoic acid, salicylic acid, and alpha-hydroxy acid in petrolatum, but all have been reported to have limited, variable success rates. We report a child with erythrokeratoderma variabilis with no family history of this entity, successfully treated with topical tazarotene.