The multi-specific VH-based Humabody CB213 co-targets PD1 and LAG3 on T cells to promote anti-tumour activity.

BACKGROUND: Improving cancer immunotherapy long-term clinical benefit is a major priority. It has become apparent that multiple axes of immune suppression restrain the capacity of T cells to provide anti-tumour activity including signalling through PD1/PD-L1 and LAG3/MHC-II. METHODS: CB213 has been developed as a fully human PD1/LAG3 co-targeting multi-specific Humabody composed of linked VH domains that avidly bind and block PD1 and LAG3 on dual-positive T cells. We present the preclinical primary pharmacology of CB213: biochemistry, cell-based function vs. immune-suppressive targets, induction of T cell proliferation ex vivo using blood obtained from NSCLC patients, and syngeneic mouse model anti-tumour activity. CB213 pharmacokinetics was assessed in cynomolgus macaques. RESULTS: CB213 shows picomolar avidity when simultaneously engaging PD1 and LAG3. Assessing LAG3/MHC-II or PD1/PD-L1 suppression individually, CB213 preferentially counters the LAG3 axis. CB213 showed superior activity vs. αPD1 antibody to induce ex vivo NSCLC patient T cell proliferation and to suppress tumour growth in a syngeneic mouse tumour model, for which both experimental systems possess PD1 and LAG3 suppressive components. Non-human primate PK of CB213 suggests weekly clinical administration. CONCLUSIONS: CB213 is poised to enter clinical development and, through intercepting both PD1 and LAG3 resistance mechanisms, may benefit patients with tumours escaping front-line immunological control.

Neoadjuvant Chemoradiotherapy and Liver Transplantation for Unresectable Hilar Cholangiocarcinoma: The Irish Experience of the Mayo Protocol.

BACKGROUND: Pioneered by the Mayo Clinic, multimodal therapy with neoadjuvant chemoradiotherapy and orthotopic liver transplant has emerged as a promising option for unresectable hilar cholangiocarcinoma (hCCA). This study reports the experience of the Irish National Liver Transplant Programme with the Mayo Protocol. METHODS: All patients diagnosed with unresectable hCCA between 2004 and 2016, who were eligible for the treatment protocol, were prospectively studied. RESULTS: Thirty-seven patients commenced chemoradiotherapy. Of those, 11 were excluded due to disease progression and 26 proceeded to liver transplantation. There were 24 males, the median age was 49, and 88% had underlying primary sclerosing cholangitis. R0 and pathologic complete response rates were 96% and 62%, respectively. Overall median survival was 53 months and 1-, 3-, and 5-year survival was 81%, 69%, and 55%, respectively. The median survival of patients achieving a pathologic complete response was 83.8 months compared with 20.9 months in the group with residual disease (P = 0.036). Six patients (23%) developed disease recurrence. Among the patients who developed metastatic disease during neoadjuvant treatment, median survival was 10.5 months compared with 53 months in patients who proceeded to transplant (P < 0.001). CONCLUSIONS: Neoadjuvant chemoradiotherapy followed by liver transplantation substantially increases the survival of patients with unresectable hCCA. Achieving a pathologic complete response confers a significant survival benefit.

Growing Teratoma Syndrome of the Liver in a 22-Year-Old Female.

Following a presentation with abdominal pain, a 22-year-old female was diagnosed with a massive primary liver immature teratoma with evidence of omental and pelvic metastases. Despite chemotherapy, the teratoma continued to rapidly increase in size. Significant treatment-associated myelosuppression was challenging as the patient did not want to receive any blood products (religious objections). The only feasible approach was surgical resection. Surgical resection of the primary tumor and abdominal metastases was undertaken despite unappealing perioperative risk with histological specimens demonstrating only mature teratoma. We report the first case of a liver teratoma suggestive of growing teratoma syndrome treated with myelosuppressive chemotherapy and major hepatectomy without the use of any blood products.

Pain and quality of life after inguinal hernia surgery: a multicenter randomized controlled trial comparing lightweight vs heavyweight mesh (Supermesh Study).

Mesh repair has significantly reduced recurrence rate after groin hernia surgery. Recently, attention has shifted to issues such as chronic pain and discomfort, leading to development of lightweight and partially re-absorbable meshes. The aim of the study was to evaluate the effect of lightweight mesh vs heavyweight mesh on post-operative pain, discomfort and quality of life in short and medium term after inguinal hernia surgery. Eight hundred and eight patients with primary inguinal hernia were allocated to anterior repair (Lichtenstein technique) using a lightweight mesh (Ultrapro®) or a heavyweight mesh (Prolene®). Primary outcomes were incidence of chronic pain and discomfort at 6-month follow-up. Secondary endpoints were quality of life (QoL), pain and complication at 1 week, 1 and 6 months. At 6 months, 25% of patients reported pain of some intensity; severe pain was reported by 1% of patients in both groups. A statistically significant difference in favour of lightweight mesh was found at multivariable analysis for pain (1 week and 6 months after surgery: p = 0.02 and p = 0.04, respectively) and QoL at 1 month and 6 months (p = 0.05 and p = 0.02, respectively). There was no difference in complication rate and no hernia recurrences were detected. The use of lightweight mesh in anterior Lichtenstein inguinal hernia repair significantly reduced the incidence of pain and favourably affected the perceived quality of life at 6 months after surgery compared to heavyweight mesh.

The Mitotic Checkpoint Complex Requires an Evolutionary Conserved Cassette to Bind and Inhibit Active APC/C.

The Spindle Assembly Checkpoint (SAC) ensures genomic stability by preventing sister chromatid separation until all chromosomes are attached to the spindle. It catalyzes the production of the Mitotic Checkpoint Complex (MCC), which inhibits Cdc20 to inactivate the Anaphase Promoting Complex/Cyclosome (APC/C). Here we show that two Cdc20-binding motifs in BubR1 of the recently identified ABBA motif class are crucial for the MCC to recognize active APC/C-Cdc20. Mutating these motifs eliminates MCC binding to the APC/C, thereby abolishing the SAC and preventing cells from arresting in response to microtubule poisons. These ABBA motifs flank a KEN box to form a cassette that is highly conserved through evolution, both in the arrangement and spacing of the ABBA-KEN-ABBA motifs, and association with the amino-terminal KEN box required to form the MCC. We propose that the ABBA-KEN-ABBA cassette holds the MCC onto the APC/C by binding the two Cdc20 molecules in the MCC-APC/C complex.

The ABBA motif binds APC/C activators and is shared by APC/C substrates and regulators.

The anaphase-promoting complex or cyclosome (APC/C) is the ubiquitin ligase that regulates mitosis by targeting specific proteins for degradation at specific times under the control of the spindle assembly checkpoint (SAC). How the APC/C recognizes its different substrates is a key problem in the control of cell division. Here, we have identified the ABBA motif in cyclin A, BUBR1, BUB1, and Acm1, and we show that it binds to the APC/C coactivator CDC20. The ABBA motif in cyclin A is required for its proper degradation in prometaphase through competing with BUBR1 for the same site on CDC20. Moreover, the ABBA motifs in BUBR1 and BUB1 are necessary for the SAC to work at full strength and to recruit CDC20 to kinetochores. Thus, we have identified a conserved motif integral to the proper control of mitosis that connects APC/C substrate recognition with the SAC.

Mechanisms controlling the temporal degradation of Nek2A and Kif18A by the APC/C-Cdc20 complex.

The Anaphase Promoting Complex/Cyclosome (APC/C) in complex with its co-activator Cdc20 is responsible for targeting proteins for ubiquitin-mediated degradation during mitosis. The activity of APC/C-Cdc20 is inhibited during prometaphase by the Spindle Assembly Checkpoint (SAC) yet certain substrates escape this inhibition. Nek2A degradation during prometaphase depends on direct binding of Nek2A to the APC/C via a C-terminal MR dipeptide but whether this motif alone is sufficient is not clear. Here, we identify Kif18A as a novel APC/C-Cdc20 substrate and show that Kif18A degradation depends on a C-terminal LR motif. However in contrast to Nek2A, Kif18A is not degraded until anaphase showing that additional mechanisms contribute to Nek2A degradation. We find that dimerization via the leucine zipper, in combination with the MR motif, is required for stable Nek2A binding to and ubiquitination by the APC/C. Nek2A and the mitotic checkpoint complex (MCC) have an overlap in APC/C subunit requirements for binding and we propose that Nek2A binds with high affinity to apo-APC/C and is degraded by the pool of Cdc20 that avoids inhibition by the SAC.

How cyclin A destruction escapes the spindle assembly checkpoint.

The anaphase-promoting complex/cyclosome (APC/C) is the ubiquitin ligase essential to mitosis, which ensures that specific proteins are degraded at specific times to control the order of mitotic events. The APC/C coactivator, Cdc20, is targeted by the spindle assembly checkpoint (SAC) to restrict APC/C activity until metaphase, yet early substrates, such as cyclin A, are degraded in the presence of the active checkpoint. Cdc20 and the cyclin-dependent kinase cofactor, Cks, are required for cyclin A destruction, but how they enable checkpoint-resistant destruction has not been elucidated. In this study, we answer this problem: we show that the N terminus of cyclin A binds directly to Cdc20 and with sufficient affinity that it can outcompete the SAC proteins. Subsequently, the Cks protein is necessary and sufficient to promote cyclin A degradation in the presence of an active checkpoint by binding cyclin A-Cdc20 to the APC/C.

Nuclear reformation after mitosis requires downregulation of the Ran GTPase effector RanBP1 in mammalian cells.

The GTPase Ran regulates nucleocytoplasmic transport in interphase and spindle organisation in mitosis via effectors of the importin beta superfamily. Ran-binding protein 1 (RanBP1) regulates guanine nucleotide turnover on Ran, as well as its interactions with effectors. Unlike other Ran network members that are steadily expressed, RanBP1 abundance is modulated during the mammalian cell cycle, peaking in mitosis and declining at mitotic exit. Here, we show that RanBP1 downregulation takes place in mid to late telophase, concomitant with the reformation of nuclei. Mild RanBP1 overexpression in murine cells causes RanBP1 to persist in late mitosis and hinders a set of events underlying the telophase to interphase transition, including chromatin decondensation, nuclear expansion and nuclear lamina reorganisation. Moreover, the reorganisation of nuclear pores fails associated with defective nuclear relocalisation of NLS cargoes. Co-expression of importin beta, together with RanBP1, however mitigates these defects. Thus, RanBP1 downregulation is required for nuclear reorganisation pathways operated by importin beta after mitosis.

Measuring proteolysis in mitosis.

The targeted destruction of key regulators helps to drive the cell cycle. Here we describe a quantitative assay to measure destruction of different regulators in mitotic cells. This assay uses GFP-tagged substrates and time-lapse fluorescence microscopy of single cells to pinpoint the timing of destruction of different substrates at different stages in mitosis.

Defining the role of Emi1 in the DNA replication-segregation cycle.

Ordered progression through the cell cycle is essential to maintain genomic stability, and fundamental to this is ubiquitin-mediated proteolysis. In particular, the anaphase-promoting complex/cyclosome (APC/C) ubiquitin ligase destabilises specific regulators at defined times in the cycle to ensure that each round of DNA replication is followed by cell division. Thus, the proper regulation of the APC/C is crucial in each cell cycle. There are several APC/C regulators that restrict its activity to specific cell cycle phases, and amongst these the early mitotic inhibitor 1 (Emi1) protein has recently come to prominence. Emi1 has been proposed to control APC/C in early mitosis; however, recent evidence questions this role. In this review we discuss new evidence that indicates that Emi1 is essential to restrict APC/C activity in interphase and, by doing so, ensure the proper coordination between DNA replication and mitosis.

Emi1 is needed to couple DNA replication with mitosis but does not regulate activation of the mitotic APC/C.

Ubiquitin-mediated proteolysis is critical for the alternation between DNA replication and mitosis and for the key regulatory events in mitosis. The anaphase-promoting complex/cyclosome (APC/C) is a conserved ubiquitin ligase that has a fundamental role in regulating mitosis and the cell cycle in all eukaryotes. In vertebrate cells, early mitotic inhibitor 1 (Emi1) has been proposed as an important APC/C inhibitor whose destruction may trigger activation of the APC/C at mitosis. However, in this study, we show that the degradation of Emi1 is not required to activate the APC/C in mitosis. Instead, we uncover a key role for Emi1 in inhibiting the APC/C in interphase to stabilize the mitotic cyclins and geminin to promote mitosis and prevent rereplication. Thus, Emi1 plays a crucial role in the cell cycle to couple DNA replication with mitosis, and our results also question the current view that the APC/C has to be inactivated to allow DNA replication.

The small-molecule inhibitor BI 2536 reveals novel insights into mitotic roles of polo-like kinase 1.

BACKGROUND: The mitotic kinases, Cdk1, Aurora A/B, and Polo-like kinase 1 (Plk1) have been characterized extensively to further understanding of mitotic mechanisms and as potential targets for cancer therapy. Cdk1 and Aurora kinase studies have been facilitated by small-molecule inhibitors, but few if any potent Plk1 inhibitors have been identified. RESULTS: We describe the cellular effects of a novel compound, BI 2536, a potent and selective inhibitor of Plk1. The fact that BI 2536 blocks Plk1 activity fully and instantaneously enabled us to study controversial and unknown functions of Plk1. Cells treated with BI 2536 are delayed in prophase but eventually import Cdk1-cyclin B into the nucleus, enter prometaphase, and degrade cyclin A, although BI 2536 prevents degradation of the APC/C inhibitor Emi1. BI 2536-treated cells lack prophase microtubule asters and thus polymerize mitotic microtubules only after nuclear-envelope breakdown and form monopolar spindles that do not stably attach to kinetochores. Mad2 accumulates at kinetochores, and cells arrest with an activated spindle-assembly checkpoint. BI 2536 prevents Plk1's enrichment at kinetochores and centrosomes, and when added to metaphase cells, it induces detachment of microtubules from kinetochores and leads to spindle collapse. CONCLUSIONS: Our results suggest that Plk1's accumulation at centrosomes and kinetochores depends on its own activity and that this activity is required for maintaining centrosome and kinetochore function. Our data also show that Plk1 is not required for prophase entry, but delays transition to prometaphase, and that Emi1 destruction in prometaphase is not essential for APC/C-mediated cyclin A degradation.

A novel mutation of the DHCR7 gene in a sicilian compound heterozygote with Smith-Lemli-Opitz Syndrome.

INTRODUCTION: Smith-Lemli-Opitz syndrome (SLOS) is an autosomal recessive disorder of cholesterol biosynthesis, resulting from deficient 7-dehydrocholesterol reductase (3beta-hydroxysterol Delta7-reductase) activity, the enzyme responsible for conversion of 7-dehydrocholesterol to cholesterol. SLOS is most common among people of European descent, with a reported incidence of 1 per 20,000-60,000 newborns, depending on the diagnostic criteria and the reference population. More than 80 different mutations have been identified in several hundred patients. In Italy, SLOS appears to be a rare condition, probably because of underdiagnosis. METHOD: We analyzed by direct sequencing the 7-dehydrocholesterol reductase gene (DHCR7) in a Sicilian patient with Smith-Lemli-Opitz syndrome and his parents in order to characterize the molecular defect. RESULTS: The molecular analysis of the coding exons and the intron-exon boundaries of the DHCR7 gene demonstrated the presence of two missense mutations: a novel mutation (I251N) and a known mutation (E288K) responsible in a compound heterozygous state for a severe form of SLOS. CONCLUSION: The present study describes a Sicilian patient, a carrier of a novel mutation of the DHCR7 gene (I251N), which is responsible in a compound heterozygous state for a severe form of SLOS.

Importin beta is transported to spindle poles during mitosis and regulates Ran-dependent spindle assembly factors in mammalian cells.

Spatial control is a key issue in cell division. The Ran GTPase regulates several fundamental processes for cell life, largely acting through importin molecules. The best understood of these is protein import through the nuclear envelope in interphase, but roles in mitotic spindle assembly are also established. In mammalian cells, in which centrosomes are major spindle organizers, a link is emerging between the Ran network, centrosomes and spindle poles. Here, we show that, after nuclear envelope breakdown, importin beta is transported to the spindle poles in mammalian cells. This localization is temporally regulated from prometaphase until anaphase, when importin beta dissociates from poles and is recruited back around reforming nuclei. Importin beta sediments with mitotic microtubules in vitro and its accumulation at poles requires microtubule integrity and dynamics in vivo. Furthermore, RNA interference-dependent inactivation of TPX2, the major Ran-dependent spindle organizer, abolishes importin beta accumulation at poles. Importin beta has a functional role in spindle pole organization, because overexpression yields mitotic spindles with abnormal, fragmented poles. Coexpression of TPX2 with importin beta mitigates these abnormalities. Together, these results indicate that the balance between importins and spindle regulators of the TPX2 type is crucial for spindle formation. Targeting of TPX2/importin-beta complexes to poles is a key aspect in Ran-dependent control of the mitotic apparatus in mammalian cells.

Mitotic functions of the Ran GTPase network: the importance of being in the right place at the right time.

The Ran GTPase has important roles in nucleocytoplasmic transport, cell cycle progression, nuclear organization and nuclear envelope (NE) assembly. In this review, we discuss emerging evidence that implicate the Ran GTPase system in mitotic control in mammalian cells. Recent work indicates that members of the Ran network control two fundamental aspects of the mammalian mitotic apparatus: (i) centrosome and spindle pole function, and (ii) kinetochore function. It is also emerging that, after NE breakdown, specific Ran network components assemble in local combinations at crucial sites of the mitotic apparatus. In the light of these findings, the original notion that nucleotide-bound forms of the Ran GTPase are distributed along a unique "gradient" in mitotic cells should be re-examined. Available data also suggest that the Ran system is deregulated in certain cellular contexts: this may represent a favoring condition for the onset and propagation of mitotic errors that can predispose cells to become genetically unstable and facilitate neoplastic growth.

Part of Ran is associated with AKAP450 at the centrosome: involvement in microtubule-organizing activity.

The small Ran GTPase, a key regulator of nucleocytoplasmic transport, is also involved in microtubule assembly and nuclear membrane formation. Herein, we show by immunofluorescence, immunoelectron microscopy, and biochemical analysis that a fraction of Ran is tightly associated with the centrosome throughout the cell cycle. Ran interaction with the centrosome is mediated by the centrosomal matrix A kinase anchoring protein (AKAP450). Accordingly, when AKAP450 is delocalized from the centrosome, Ran is also delocalized, and as a consequence, microtubule regrowth or anchoring is altered, despite the persisting association of gamma-tubulin with the centrosome. Moreover, Ran is recruited to Xenopus sperm centrosome during its activation for microtubule nucleation. We also demonstrate that centrosomal proteins such as centrin and pericentrin, but not gamma-tubulin, AKAP450, or ninein, undertake a nucleocytoplasmic exchange as they concentrate in the nucleus upon export inhibition by leptomycin B. Together, these results suggest a challenging possibility, namely, that centrosome activity could depend upon nucleocytoplasmic exchange of centrosomal proteins and local Ran-dependent concentration at the centrosome.

Mammalian RanBP1 regulates centrosome cohesion during mitosis.

The Ran GTPase plays a central function in control of nucleo-cytoplasmic transport in interphase. Mitotic roles of Ran have also been firmly established in Xenopus oocyte extracts. In this system, Ran-GTP, or the RCC1 exchange factor for Ran, drive spindle assembly by regulating the availability of 'aster-promoting activities'. In previous studies to assess whether the Ran network also influences mitosis in mammalian cells, we found that overexpression of Ran-binding protein 1 (RanBP1), a major effector of Ran, induces multipolar spindles. We now show that these abnormal spindles are generated through loss of cohesion in mitotic centrosomes. Specifically, RanBP1 excess induces splitting of mother and daughter centrioles at spindle poles; the resulting split centrioles can individually organize functional microtubule arrays, giving rise to functional spindle poles. RanBP1-dependent centrosome splitting is specifically induced in mitosis and requires microtubule integrity and Eg5 activity. In addition, we have identified a fraction of RanBP1 at the centrosome. These data indicate that overexpressed RanBP1 interferes with crucial factor(s) that control structural and dynamic features of centrosomes during mitosis and contribute to uncover novel mitotic functions downstream of the Ran network.

All trans retinoic acid sensitizes colon cancer cells to hyperthermia cytotoxic effects.

The effects of all trans retinoic acid and hyperthermia were studied in the human colon adenocarcinoma cell line HT29. Cell cytotoxicity after exposure to ATRA or heat-shock, alone or in association, was evaluated by the MTT assay while cell surface and ultrastructure modifications and actin fibre assembly changes were investigated by electron microscopy and by the FITC-phalloidin method. Apoptosis was evaluated by flow cytofluorimetry and electron microscopy. Reverse transcriptase-polymerase chain reaction was employed to study mRNA expression of genes involved in apoptosis, differentiation and growth arrest. Joint treatments were more effective in reducing the vital cell yield, being this effect only partially due to apoptosis. A marked up-regulation of the cyclin-dependent kinase inhibitor p21WAF1/Cip1 expression, not followed by any differentiation process, was responsible for growth arrest. Modulation of Hsp-70 expression, involved in cell response to treatments, was considered. Our results demonstrate that cell treatment with ATRA followed by heat-shock may elicit useful effects to treat tumours, which are responsive to retinoids, as well as those malignant cells which may be constitutively thermotolerant.