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PDF file - 53K, Serum levels of KPT-330 in mice.

PDF file - 51K, Knockdown of XIAP has no effect on survivin degradation.

PDF file - 66K, Molecular structures of the KPT-SINES.

Inhibition of XPO1 (CRM1)-mediated nuclear export of multiple tumor suppressor proteins has been proposed as a novel cancer therapeutic strategy to turn off oncogenic signals and enhance tumor suppression. Survivin is a multifunctional protein with oncogenic properties when expressed in the cytoplasm that requires the XPO1–RanGTP complex for its nuclear export. We investigated the antitumor mechanisms of the drug-like selective inhibitors of nuclear export (SINE) XPO1 antagonists KPT-185, KPT-251 KPT-276, and KPT-330 in estrogen receptor–positive and triple-negative breast cancer (TNBC) cell lines and xenograft models of human breast tumors. KPT compounds significantly inhibited breast cancer cell growth and induced tumor cell death, both in vitro and in vivo. These drugs initially promoted survivin accumulation within tumor cell nuclei. However, their major in vitro effect was to decrease survivin cytoplasmic protein levels, correlating with the onset of apoptosis. XPO1 inhibition repressed Survivin transcription by inhibiting CREB-binding protein-mediated STAT3 acetylation, and blocking STAT3 binding to the Survivin promoter. In addition, caspase-3 was activated to cleave survivin, rendering it unavailable to bind X-linked inhibitor of apoptosis protein and block the caspase cascade. Collectively, these data demonstrate that XPO1 inhibition by SINE compounds represses STAT3 transactivation to block the selective oncogenic properties of survivin and supports their clinical use in TNBC. Mol Cancer Ther; 13(3); 675–86. ©2014 AACR.

Supplementary Data from First-in-Class Anti-immunoglobulin–like Transcript 4 Myeloid-Specific Antibody MK-4830 Abrogates a PD-1 Resistance Mechanism in Patients with Advanced Solid Tumors

Purpose:In this first-in-human study (NCT03564691) in advanced solid tumors, we investigated a novel first-in-class human IgG4 monoclonal antibody targeting the immunoglobulin-like transcript 4 (ILT4) receptor, MK-4830, as monotherapy and in combination with pembrolizumab.Patients and Methods:Patients with histologically/cytologically confirmed advanced solid tumors, measurable disease by RECIST v1.1, and evaluable baseline tumor sample received escalating doses of intravenous MK-4830 every 3 weeks as monotherapy (parts A and B) and in combination with pembrolizumab (part C). Safety and tolerability were the primary objectives. Pharmacokinetics, objective response rate per RECIST v1.1, and molecular biomarkers were also evaluated.Results:Of 84 patients, 50 received monotherapy and 34 received combination therapy. No dose-limiting toxicities were observed; maximum tolerated dose was not reached. MK-4830 showed dose-related target engagement. Eleven of 34 patients in the dose-escalation phase who received combination therapy achieved objective responses; 5 previously had progressive disease on anti–PD-1/PD-L1 therapies. Exploratory evaluation of the association between response and pretreatment gene expression related to interferon-gamma signaling in tumors suggested higher sensitivity to T-cell inflammation with combination therapy than historically expected with pembrolizumab monotherapy, with greater response at more moderate levels of inflammation.Conclusions:This first-in-class MK-4830 antibody dosed as monotherapy and in combination with pembrolizumab was well tolerated with no unexpected toxicities, and demonstrated dose-related evidence of target engagement and antitumor activity. Inflammation intrinsic to the ILT4 mechanism may be facilitated by alleviating the myeloid-suppressive components of the tumor microenvironment, supporting the target of ILT4 as a potential novel immunotherapy in combination with an anti–PD-1/PD-L1 agent.

TPS484 Background: Advanced G/GEJ cancer is a major cause of cancer-related mortality requiring novel therapies. DKN-01 is a humanized mAb that binds and neutralizes DKK1. DKK1 plays a major role in WNT-pathway regulation and is overexpressed in multiple cancers. DKK1 expression is associated with poor prognosis, an immunosuppressive tumor microenvironment, and resistance to chemotherapy in G/GEJ cancer. The non-randomized Part A of DisTinGuish demonstrated durable clinical benefit in patients with G/GEJ adenocarcinoma. Higher response rates were observed in patients with high tumoral DKK1 expression, when DKN-01 was administered in combination with an anti-PD1 mAb, tislelizumab, and chemotherapy1,2. Furthermore, in Part B, durable responses were seen in 2L patients with DKK1-high G/GEJ cancers treated with a chemotherapy-free doublet regimen, DKN-01 plus tislelizumab1. Part C will evaluate the efficacy of this chemoimmunotherapy combination in a randomized fashion in treatment-naïve G/GEJ adenocarcinoma patients. Methods: Part C is an ongoing, global, randomized phase 2 open-label study evaluating DKN-01 in combination with tislelizumab + chemotherapy (CAPOX or mFOLFOX6) vs tislelizumab + chemotherapy in patients with advanced G/GEJ adenocarcinoma. Patients are randomized 1:1 and stratified by DKK1 and PD-L1 expression. Approximately 160 patients will be enrolled from 5 different countries. Key inclusion criteria include patients with HER2 negative, G/GEJ adenocarcinoma who have received no prior systemic treatment in the unresectable locally advanced/metastatic setting and can provide tumor tissue to document PD-L1 CPS and DKK1 expression, as performed at a central lab. Key exclusion criteria include prior treatment with anti-PD1/L1/L2. The primary endpoint is PFS according to RECIST v1.1 as assessed by the investigator in patients with DKK1-high tumors. Key secondary and exploratory end points include PFS, OS, ORR, DoR in patients with tumors with any level of DKK1 expression, as assessed by both investigator and by blinded, central review. Recruitment is ongoing. Clinical trial information: NCT04363801 . References SJ Klempner, et al. DKN-01 and tislelizumab ± chemotherapy as a first-line (1L) and second-line (2L) investigational therapy in advanced gastroesophageal adenocarcinoma (GEA): DisTinGuish Trial. JCO 40, no. 4_suppl, 2022, 292-292. SJ Klempner, et al. DKN-01 and Tislelizumab + Chemotherapy as First-line (1L) Investigational Therapy in Advanced Gastroesophageal Adenocarcinoma (GEA): DisTinGuish Trial. ESMO Annual Meeting September 2022. Clinical trial information: NCT04363801 .

Summary In mammalian cells, histone deacetylase (HDAC) and Sirtuin (SIRT) are two families responsible for removing acetyl groups from acetylated proteins. Here, we describe protein deacetylation coupled with deacetylimination as a function of lysyl oxidase (LOX) family members. LOX-like 3 (Loxl3) associates with Stat3 in the nucleus to deacetylate and deacetyliminate Stat3 on multiple acetyl-lysine sites. Surprisingly, Loxl3 N-terminal scavenger receptor cysteine-rich (SRCR) repeats, rather than the C-terminal oxidase catalytic domain, represent the major deacetylase/deacetyliminase activity. Loxl3-mediated deacetylation/deacetylimination disrupts Stat3 dimerization, abolishes Stat3 transcription activity, and restricts cell proliferation. In Loxl3 −/− mice, Stat3 is constitutively acetylated and naive CD4 + T cells are potentiated in Th17/Treg cell differentiation. When overexpressed, the SRCR repeats from other LOX family members can catalyze protein deacetylation/deacetylimination. Thus, our findings delineate a hitherto-unknown mechanism of protein deacetylation and deacetylimination catalyzed by lysyl oxidases.

B-lymphoblastic leukemia is a clonal hematopoietic disorder of precursors B-lymphoblasts being most frequently encountered in children. Expression of CD19, a pan B-cell marker is noted in the majority of the cases with lack of CD19 expression being extremely rarely reported in the medical literature. We report the very rare case of a B-lymphoblastic leukemia with a triple negative immunophenotype: CD19 negative, CD45 negative and CD10 negative with vacuolated lymphoblasts and an aleukemic presentation in a 20-year-old man.

Mutations in PIEZO1 are the primary cause of hereditary xerocytosis, a clinically heterogeneous, dominantly inherited disorder of erythrocyte dehydration. We used next-generation sequencing-based techniques to identify PIEZO1 mutations in individuals from 9 kindreds referred with suspected hereditary xerocytosis (HX) and/or undiagnosed congenital hemolytic anemia. Mutations were primarily found in the highly conserved, COOH-terminal pore-region domain. Several mutations were novel and demonstrated ethnic specificity. We characterized these mutations using genomic-, bioinformatic-, cell biology-, and physiology-based functional assays. For these studies, we created a novel, cell-based in vivo system for study of wild-type and variant PIEZO1 membrane protein expression, trafficking, and electrophysiology in a rigorous manner. Previous reports have indicated HX-associated PIEZO1 variants exhibit a partial gain-of-function phenotype with generation of mechanically activated currents that inactivate more slowly than wild type, indicating that increased cation permeability may lead to dehydration of PIEZO1-mutant HX erythrocytes. In addition to delayed channel inactivation, we found additional alterations in mutant PIEZO1 channel kinetics, differences in response to osmotic stress, and altered membrane protein trafficking, predicting variant alleles that worsen or ameliorate erythrocyte hydration. These results extend the genetic heterogeneity observed in HX and indicate that various pathophysiologic mechanisms contribute to the HX phenotype.

Targeted next-generation sequencing (NGS) identified a novel loss of function mutation in GARS, a gene linked to Charcot–Marie–Tooth disease (CMT), in a paediatric acute lymphoblastic leukaemia patient with severe chemotherapy-induced peripheral neuropathy (CIPN) due to vincristine. The patient was clinically asymptomatic, and lacked a family history of neuropathy. The effect of the mutation was modelled in a zebrafish knockdown system that recapitulated the symptoms of the patient both prior to and after treatment with vincristine. Confocal microscopy of pre- and post-synaptic markers revealed that the GARS knockdown results in changes to peripheral motor neurons, acetylcholine receptors and their co-localisation in neuromuscular junctions (NMJs), whereas a sensitive and reproducible stimulus–response assay demonstrated that the changes correlating with the GARS mutation in themselves fail to produce peripheral neuropathy symptoms. However, with vincristine treatment the GARS knockdown exacerbates decreased stimulus response and NMJ lesions. We propose that there is substantial benefit in the use of a targeted NGS screen of cancer patients who are to be treated with microtubule targeting agents for deleterious mutations in CMT linked genes, and for the screening in zebrafish of reagents that might inhibit CIPN. Studying a young cancer patient’s DNA in a zebrafish model helped reveal why she experienced a severe complication of chemotherapy. The 12-year-old girl developed back pain, muscle weakness and other symptoms of nerve damage after receiving two doses of a chemotherapy drug called vincristine to treat her acute lymphoblastic leukemia. To determine the cause of the problem, a team led by Michael Holloway from the Warren Alpert Medical School at Brown University, USA, sequenced 15 genes linked to Charcot-Marie-Tooth disease, the most common hereditary explanation for this kind of nerve damage. They found a novel mutation in a gene called GARS. The researchers knocked down this gene’s function in zebrafish, and saw no obvious signs of disease until they added vincristine and observed disruption of neuromuscular junctions.

Global gene expression profiling studies led to the recent classification of breast cancer into 4 distinct molecular subtypes including luminal, human epidermal growth factor receptor 2 enriched, basal like, and unclassified. Here, we used immunohistochemistry to evaluate expression of the antiapoptotic protein Survivin and its recently described acetylated form, Survivin acetyl129, in normal breast tissue and in 226 primary breast tumors of different molecular subtypes. Correlation of Survivin expression with molecular markers and its impact on patient outcomes were analyzed. Eighty-four percent of basal-like tumors expressed high levels of total Survivin, whereas 52% of luminal tumors expressed high levels of acetylated Survivin (P < .001). Overall survival (91%) for tumors expressing low levels of total Survivin was better than that for tumors expressing high levels of total Survivin (72%, P = .02), whereas the reverse was true for tumors expressing acetylated Survivin. In hierarchical cluster analysis, total Survivin clustered with basal marker expression, whereas acetylated Survivin clustered with luminal marker expression. In multivariate analysis, high total Survivin expression was an independent predictor of worse overall survival in patients with breast cancer (relative risk, 11; P < .01). These data indicate that high levels of total Survivin predict poor outcome in patients with grade 3 invasive ductal carcinoma and correlate directly with a basal-like phenotype. In contrast, high expression of the acetylated form of the protein associates with a favorable outcome and preferentially correlates with luminal-type tumors. Survivin likely has different functions in distinct breast cancer subtypes, and diagnostic strategies that incorporate immunohistochemical markers that detect both Survivin forms may help better strategize patient risk and direct therapy.

Accumulating evidence suggests that adult hippocampal neurogenesis relies on the controlled and continued proliferation of neural progenitor cells (NPCs). With age, neurogenesis decreases through mechanisms that remain unclear but are believed to involve changes in the NPC microenvironment. Here, we provide evidence that NPC proliferation in the adult brain is in part regulated by astrocytes via Wnt signaling and that this cellular cross-talk is modified in the aging brain, leading to decreased proliferation of NPCs. Furthermore, we show that astrocytes regulate the NPC cell cycle by acting on the expression levels of survivin, a known mitotic regulator. Among cell cycle genes found down-regulated in aged NPCs, survivin was the only one that restored NPC proliferation in the aged brain. Our results provide a mechanism for the gradual loss of neurogenesis in the brain associated with aging and suggest that targeted modulation of survivin expression directly or through Wnt signaling could be used to stimulate adult neurogenesis.

Survivin is an oncogenic protein that is highly expressed in breast cancer and has a dual function that is dependent on its subcellular localization. In the cytosol, survivin blocks programmed cell death by inactivating caspase proteins; however, in the nucleus it facilitates cell division by regulating chromosomal movement and cytokinesis. In prior work, we showed that survivin is acetylated by CREB-binding protein (CBP), which restricts its localization to the nuclear compartment and thereby inhibits its anti-apoptotic function. Here, we identify histone deacetylase 6 (HDAC6) as responsible for abrogating CBP-mediated survivin acetylation in the estrogen receptor (ER)-positive breast cancer cell line, MCF-7. HDAC6 directly binds survivin, an interaction that is enhanced by CBP. In quiescent breast cancer cells in culture and in malignant tissue sections from ER+ breast tumors, HDAC6 localizes to a perinuclear region of the cell, undergoing transport to the nucleus following CBP activation where it then deacetylates survivin. Genetically modified mouse embryonic fibroblasts that lack mhdac6 localize survivin predominantly to the nuclear compartment, whereas wild-type mouse embryonic fibroblasts localize survivin to distinct cytoplasmic structures. Together, these data imply that HDAC6 deacetylates survivin to regulate its nuclear export, a feature that may provide a novel target for patients with ER+ breast cancer.

The multiple functions of the oncofetal protein survivin are dependent on its selective expression patterns within immunochemically distinct subcellular pools. The mechanism by which survivin localizes to these compartments, however, is only partly understood. Here we show that nuclear accumulation of survivin is promoted by CREB-binding protein (CBP)-dependent acetylation on lysine 129 (129K, Lys-129). We demonstrate a mechanism by which survivin acetylation at this position results in its homodimerization, while deacetylation promotes the formation of survivin monomers that heterodimerize with CRM1 and facilitate its nuclear export. Using proteomic analysis, we identified the oncogenic transcription factor STAT3 as a binding partner of nuclear survivin. We show that acetylated survivin binds to the N-terminal transcriptional activation domain of the STAT3 dimer and represses STAT3 transactivation of target gene promoters. Using multiplex PCR and DNA sequencing, we identified a single-nucleotide polymorphism (A → G) at Lys-129 that exists as a homozygous mutation in a neuroblastoma cell line and corresponds with a defect in survivin nuclear localization. Our results demonstrate that the dynamic equilibrium between survivin acetylation and deacetylation at amino acid 129 determines its interaction with CRM1, its subsequent subcellular localization, and its ability to inhibit STAT3 transactivation, providing a potential route for therapeutic intervention in STAT3-dependent tumors.

Mevalonate kinase deficiency (MKD) is a rare inborn error of metabolism caused by mutations in the mevalonate kinase (MVK) gene. The clinical phenotype is variable, ranging from the hyperimmunoglobulinemia D and periodic fever syndrome (HIDS) to mevalonic aciduria (MA), a severe metabolic disease. We report here for the first time (to our knowledge) the case of a patient with MKD and congenital dyserythropoietic anemia. Clinical and laboratory characteristics of inflammatory attacks were compatible with HIDS, but mild dysmorphic features and elevated urinary mevalonic acid levels in the absence of an inflammatory attack suggested an intermediate phenotype between HIDS and MA. Genomic sequencing of the MVK gene revealed compound heterozygosity for a missense mutation previously described in MA (V310M) and a novel missense mutation (Y116H). By contrast, sequencing of the novel CDAII (SEC23B) gene revealed no mutations, suggesting that the bone marrow abnormalities were causally related to the MKD. Treatment with corticosteroids and colchicine directed at controlling the autoinflammatory disease resulted in improvement of the anemia.

RUNX1T1/RUNX1 (formerly ETO/AML1) is a molecular marker that is usually associated with a favorable outcome in both pediatric and adult patients with acute myeloid leukemia (AML). We describe a 10-year-old girl with AML associated with an RUNX1T1/RUNX1 fusion. The patient's karyotype at the time of diagnosis was 46,X,-X,t(4;21;8)(q25;q22;q22),+6. She had an early relapse while being treated on a standard protocol and had significant difficulty in attaining a second remission. She subsequently underwent a matched related donor bone marrow transplant, but a second bone marrow relapse with extensive extramedullary disease followed on day +199. Cytogenetic analysis at second relapse showed evidence of clonal evolution in the form of a highly complex karyotype with numeric and structural abnormalities in addition to the t(4;21;8) and trisomy 6 detected in the diagnostic sample. Trisomy 6 is an uncommon cytogenetic abnormality in myeloid diseases. As a sole abnormality, it has been associated mainly with myelodysplastic syndrome and AML. The presence of this novel variant of t(8;21)(q22;q22) associated with trisomy 6 may have abrogated the usual favorable prognosis associated with RUNX1T1/RUNX1 in AML.

OBJECTIVE—Diabetes results from a deficiency of functional β-cells due to both an increase in β-cell death and an inhibition of β-cell replication. The molecular mechanisms responsible for these effects in susceptible individuals are mostly unknown. The objective of this study was to determine whether a gene critical for cell division and cell survival in cancer cells, survivin, might also be important for β-cells. RESEARCH DESIGN AND METHODS—We generated mice harboring a conditional deletion of survivin in pancreatic endocrine cells using mice with a Pax-6-Cre transgene promoter construct driving tissue-specific expression of Cre-recombinase in these cells. We performed metabolic studies and immunohistochemical analyses to determine the effects of a mono- and biallelic deletion of survivin. RESULTS—Selective deletion of survivin in pancreatic endocrine cells in the mouse had no discernible effects during embryogenesis but was associated with striking decreases in β-cell number after birth, leading to hyperglycemia and early-onset diabetes by 4 weeks of age. Serum insulin levels were significantly decreased in animals lacking endocrine cell survivin, with relative stability of other hormones. Exogenous expression of survivin in mature β-cells lacking endogenous survivin completely rescued the hyperglycemic phenotype and the decrease in β-cell mass, confirming the specificity of the survivin effect in these cells. CONCLUSIONS—Our findings implicate survivin in the maintenance of β-cell mass through both replication and antiapoptotic mechanisms. Given the widespread involvement of survivin in cancer, a novel role for survivin may well be exploited in β-cell regulation in diseased states, such as diabetes.

We explored the physiologic role of endothelial cell apoptosis during development by generating mouse embryos lacking the inhibitor of apoptosis protein (IAP) survivin in endothelium. This was accomplished by intercrossing survivinlox/lox mice with mice expressing cre recombinase under the control of the endothelial cell specific tie1 promoter (tie1-cre mice). Lack of endothelial cell survivin resulted in embryonic lethality. Mutant embryos had prominent and diffuse hemorrhages from embryonic day 9.5 (E9.5) and died before E13.5. Heart development was strikingly abnormal. Survivin-null endocardial lineage cells could not support normal epithelial-mesenchymal transformation (EMT), resulting in hypoplastic endocardial cushions and in utero heart failure. In addition, 30% of mutant embryos had neural tube closure defects (NTDs) that were not caused by bleeding or growth retardation, but were likely due to alterations in the release of soluble factors from endothelial cells that otherwise support neural stem cell proliferation and neurulation. Thus, regulation of endothelial cell survival, and maintenance of vascular integrity by survivin are crucial for normal embryonic angiogenesis, cardiogenesis, and neurogenesis.

Survivin is an inhibitor of apoptosis protein (IAP) expressed in a large number of adult malignancies. Its expression levels correlate with more aggressive disease and poor clinical outcome in many of these tumors. As its expression is restricted in normal adult differentiated tissues, it has become of great interest as both a tumor prognostic marker and as a potential biologic target for future anti-cancer therapies. Survivin expression and Survivin-based therapies have been examined in many of the more common pediatric malignancies. We present an overview of Survivin function and current research exploring its biologic and therapeutic roles in pediatric tumors. Pediatr Blood Cancer © 2006 Wiley-Liss, Inc.

Background: Survivin is proposed to play a central role in the progression and resistance to therapy of diverse tumour types. High levels of this molecule in tumour cells also correlate with loss of the TP53 tumour suppressor gene, suggesting a molecular connection between TP53 loss and transcriptional induction of Survivin. Patients with TP53 germline mutations, such as those with Li-Fraumeni syndrome, are particularly susceptible to sarcomas, including rhabdomyosarcomas. Our study aimed to identify rhabdomyosarcoma tumours that express Survivin, in order to test novel Survivin-targeted therapies in these tumours. Methods: Tumour microarray slides composed of 63 primary rhabdomyosarcoma tumours were stained with a polyclonal antibody to Survivin to identify tumours expressing Survivin. Subcutaneous tumours were then established in NOD/SCID mice using RH30 red cells, a red fluorescent clone of the RH30 human alveolar rhabdomyosarcoma cell line. Tumours were treated by hydrodynamic injection with a cocktail of Survivin-shRNA-encoding plasmids for a period of 2 weeks. Results: Over 80% of primary rhabdomyosarcoma tumours expressed Survivin. Treatment of rhabdomyosarcoma xenografts showed greater than 70% reduction in growth when compared with control injected tumours at study completion (average tumour sizes: 1683 v 304 mm 3 , p Conclusions: Our findings support a role for Survivin in rhabdomyosarcoma biology and provide preliminary evidence for the therapeutic use of Survivin-targeted RNA interference for human tumours that express high levels of this molecule.

Survivin is an apoptotic inhibitor that is expressed at high levels in a variety of malignancies. Survivin has four known alternative splice forms (Survivin, Survivin-2B, Survivin-deltaEx3, and Survivin-3B), and the recent literature suggests that these splice variants have unique functions and subcellular localisation patterns. We evaluated 19 fresh-frozen paediatric medulloblastomas for the expression of three Survivin isoforms by quantitative PCR. Survivin was most highly expressed when compared with normal cerebellar tissue. We also investigated Survivin protein expression in 40 paraffin-embedded paediatric medulloblastoma tumours by immunohistochemistry. We found a statistically significant association between the percentage of Survivin-positive cells and histologic subtype, with the large-cell-anaplastic variant expressing Survivin at higher levels than the classic subtype. We also found a statistically significant relationship between the percent of Survivin-positive cells in the tumours and clinical outcome, with higher levels of Survivin correlating with a worse prognosis. In summary, our study demonstrates a role for Survivin as a marker of tumour morphology and clinical outcome in medulloblastoma. Survivin may be a promising future prognostic tool and potential biologic target in this malignancy.

Low levels of total magnesium in sickle cell erythrocytes have been linked to increased sickling due to cell dehydration. We tested the null hypothesis that adult sickle cell anemia (SCA) patients have the same serum level of ionized Mg (Mg(2+)) and Ca(2+)/Mg(2+) ratio as healthy African Americans (AA) and healthy Caucasians (CAUC). We measured serum Mg(2+) and ionized calcium (Ca(2+)) with ion-selective electrodes and calculated the serum Ca(2+)/Mg(2+) ratios in patients with SCA and control groups (AA and CAUC). Seventy-four SCA patients and 61 controls were compared. SCA patients had significantly (P < 0.001) lower levels of serum Mg(2+) (0.52 +/- 0.05) compared to healthy AA (0.57 +/- 0.04) and CAUC (0.62 +/- 0.03). Eighty-six percent of the adult SCA patients had serum Mg(2+) levels below the mean for the AA group, and 96% of SCA patients were above the AA group's mean serum Ca(2+)/Mg(2+). Of the SCA patients studied, 25.6% (95% CI, 16.2-37.2%) had serum Mg(2+) levels below the racially adjusted lower limit of normal and 50% (95% CI, 38.1-61.9%) were above the upper limit of serum Ca(2+)/Mg(2+) for AA controls. By measuring serum Mg(2+) and Ca(2+), we were able to define a subset of SCA patients with hypomagnesemia and elevated Ca(2+)/Mg(2+) ratios, who may benefit from magnesium supplementation.

Inflammatory myofibroblastic tumors are benign neoplasms histologically composed of lymphocytes, histiocytes, macrophages, foam cells, and plasma cells among a spindle-shaped stroma. Their etiology and potential for metastatic spread is controversial. Numerous predisposing factors have been suggested, including preceding infections, radiotherapy, and local trauma. We present two cases of pseudotumors that developed in children following hematopoietic stem cell transplantation. These are the first cases after hematopoietic transplant reported in the literature. As these neoplasms are difficult to diagnose and are often confused with highly aggressive tumors, our cases demonstrate that a high index of suspicion for such lesions must be maintained when evaluating masses in post transplant patients.

Inhibition of XPO1 (CRM1)-mediated nuclear export of multiple tumor suppressor proteins has been proposed as a novel cancer therapeutic strategy to turn off oncogenic signals and enhance tumor suppression. Survivin is a multifunctional protein with oncogenic properties when expressed in the cytoplasm that requires the XPO1–RanGTP complex for its nuclear export. We investigated the antitumor mechanisms of the drug-like selective inhibitors of nuclear export (SINE) XPO1 antagonists KPT-185, KPT-251 KPT-276, and KPT-330 in estrogen receptor–positive and triple-negative breast cancer (TNBC) cell lines and xenograft models of human breast tumors. KPT compounds significantly inhibited breast cancer cell growth and induced tumor cell death, both in vitro and in vivo. These drugs initially promoted survivin accumulation within tumor cell nuclei. However, their major in vitro effect was to decrease survivin cytoplasmic protein levels, correlating with the onset of apoptosis. XPO1 inhibition repressed Survivin transcription by inhibiting CREB-binding protein-mediated STAT3 acetylation, and blocking STAT3 binding to the Survivin promoter. In addition, caspase-3 was activated to cleave survivin, rendering it unavailable to bind X-linked inhibitor of apoptosis protein and block the caspase cascade. Collectively, these data demonstrate that XPO1 inhibition by SINE compounds represses STAT3 transactivation to block the selective oncogenic properties of survivin and supports their clinical use in TNBC. Mol Cancer Ther; 13(3); 675–86. ©2014 AACR.

Idiopathic myelofibrosis can develop in children as well as adults. However, the disease appears to be much more aggressive in adults, being characterized by poor survival rates and a high frequency of malignant transformation. Here, we describe three cases of idiopathic myelofibrosis in infants, two of whom were followed for 16 and 22 years after diagnosis. Neither of these patients required more than minimal supportive care, and both have had spontaneous erythropoietic recovery as early as 2–3 years after diagnosis. There have been no indications of malignant transformation or clinical deterioration. Thus, idiopathic myelofibrosis may have a different pathogenesis and clinical course in infants from adults, requiring a more conservative approach to management.

Undifferentiated embryonal sarcoma of the liver is a rare hepatic malignancy of childhood with a historically poor prognosis. Recent improvements in outcomes have been reported in small numbers of cases with the use of combination therapy involving aggressive surgical resection and chemotherapy. Complete surgical resection is frequently difficult to achieve when the location of the tumor is along the margins of the major hepatic vessels (portal vein, hepatic vein, and hepatic artery). Here we report a case of undifferentiated embryonal sarcoma of the liver that recurred along surgical hepatic vein margins in a 9-year-old boy who subsequently underwent orthotopic liver transplantation from a cadaveric donor. The patient has been in continuous clinical remission for the last 5 years.

Leukemic lymphoblasts expressing the E2A-HLF oncoprotein possess wild-type p53 genes, but do not undergo apoptosis in response to DNA damage. Experimentally, E2A-HLF prevents apoptosis due to growth factor deprivation or γ-irradiation in interleukin-3 (IL-3)–dependent murine pro-B cells. To directly test the chimeric protein’s ability to abrogate p53-mediated cell death, we used mouse myeloid leukemia cells (M1p53tsval) that constitutively express a temperature-sensitive (ts) mutant p53 gene and undergo apoptosis when p53 assumes an active wild-type configuration. This effect is blocked by treatment with IL-6, which allows the cells to survive in culture despite wild-type p53 activation. We introduced E2A-HLF into M1p53tsval cells and found that they were resistant to p53-mediated apoptosis and that E2A-HLF effectively substituted for the survival functions of IL-6. The expression of p53-responsive genes such as p21 and Bax was upregulated normally, suggesting that E2A-HLF acts downstream of p53 to block execution of the p53-induced apoptotic program. NFIL3, a growth factor-regulated bZIP protein that binds to the same DNA-consensus site as E2A-HLF, delays apoptosis in IL-3–dependent pro-B cells deprived of growth factor. By contrast, in the present study, enforced expression of NFIL3 failed to protect M1p53tsval cells from p53-dependent apoptosis and actively antagonized the ability of IL-6 to rescue cells from that fate, consistent with its role as either a transcriptional repressor or activator, depending on the cell type in which it is expressed. We conclude that the E2A-HLF chimera abrogates p53-induced apoptosis in leukemic cells, possibly through the transcriptional modulation of cell death pathways that are activated by p53 in response to DNA damage. © 1998 by The American Society of Hematology.

Leukemic lymphoblasts expressing the E2A-HLF oncoprotein possess wild-type p53 genes, but do not undergo apoptosis in response to DNA damage. Experimentally, E2A-HLF prevents apoptosis due to growth factor deprivation or γ-irradiation in interleukin-3 (IL-3)–dependent murine pro-B cells. To directly test the chimeric protein’s ability to abrogate p53-mediated cell death, we used mouse myeloid leukemia cells (M1p53tsval) that constitutively express a temperature-sensitive (ts) mutant p53 gene and undergo apoptosis when p53 assumes an active wild-type configuration. This effect is blocked by treatment with IL-6, which allows the cells to survive in culture despite wild-type p53 activation. We introduced E2A-HLF into M1p53tsval cells and found that they were resistant to p53-mediated apoptosis and that E2A-HLF effectively substituted for the survival functions of IL-6. The expression of p53-responsive genes such as p21 and Bax was upregulated normally, suggesting that E2A-HLF acts downstream of p53 to block execution of the p53-induced apoptotic program. NFIL3, a growth factor-regulated bZIP protein that binds to the same DNA-consensus site as E2A-HLF, delays apoptosis in IL-3–dependent pro-B cells deprived of growth factor. By contrast, in the present study, enforced expression of NFIL3 failed to protect M1p53tsval cells from p53-dependent apoptosis and actively antagonized the ability of IL-6 to rescue cells from that fate, consistent with its role as either a transcriptional repressor or activator, depending on the cell type in which it is expressed. We conclude that the E2A-HLF chimera abrogates p53-induced apoptosis in leukemic cells, possibly through the transcriptional modulation of cell death pathways that are activated by p53 in response to DNA damage. © 1998 by The American Society of Hematology.

Childhood neuroblastoma, an embryonal neoplasm of sympathetic nervous system progenitors, occurs in a familial form with an autosomal dominant mode of inheritance. Genetic susceptibility to this disorder is thought to arise via a germline mutation affecting a tumor suppressor gene, in accord with the two-hit model established for familial and sporadic retinoblastoma. Surprisingly, the familial neuroblastoma predisposition locus does not map to chromosome band 1p36, a genomic region likely to contain one or more neuroblastoma suppressor genes. We reasoned that inherited point mutations affecting one allele would be unmasked in many cases by somatically acquired deletions of the second allele that included the target gene in the tumor cells from these patients. Thus, to identify chromosomal regions that might contain suppressor genes important in hereditary neuroblastoma, we analyzed six familial tumors by comparative genomic hybridization. Recurrent losses of genetic material were detected on chromosome arms 3p (consensus region, 3p24-pter), 10p (consensus, 10p12-p13), 10q (consensus, 10q25-qter), 16q (consensus, 16q12-q22), and 20q (consensus, 20q13.3-qter), in addition to the regions commonly deleted in sporadic neuroblastomas (1p36 and 11q). These chromosomal sites may harbor novel tumor suppressor genes that could aid in our understanding of the predisposition to and pathogenesis of familial neuroblastoma and potentially sporadic tumors as well.

CRM1 (Chromosomal Maintenance 1, also known as Exportin 1) is the major mammalian export protein that facilitates the transport of large macromolecules including RNA and protein across the nuclear membrane to the cytoplasm. The gene encoding CRM1 was originally identified in yeast as required to maintain higher order chromosome structure. In mammalian cells, CRM1 was found to bind several nuclear pore proteins hence its role in nuclear-cytosolic transport was discovered. In addition to nuclear-cytosolic transport, CRM1 also plays a role in centrosome duplication and spindle assembly, especially in response to DNA damage. The crystal structure of CRM1 suggests a complex protein that binds the Ran protein bound to GTP, allowing for a conformational change that facilitates binding to different cargo proteins through a nuclear export signal (NES). Included in the cadre of cargo are multiple tumor suppressor and oncoproteins as p53, BRCA1, Survivin, NPM, and APC, which function in the nucleus to regulate transcription or aid in chromosomal assembly and movement. An imbalance in the cytosolic level of these proteins has been observed in cancer cells, resulting in either inactivation (tumor suppressor) or an excess of anti-apoptotic activity (oncoprotein). Thus, the concept of inhibiting CRM1 has been explored as a potential therapeutic intervention. Indeed, inhibition of CRM1 by a variety of small molecules that interfere with cargo-NES binding results in cancer cell death. Whether all of these proteins together are responsible for this phenotype or whether specific proteins are required for this effect is unclear at this time.

Background Postnatal expansion of the pancreatic β-cell mass is required to maintain glucose homeostasis immediately after birth. This β-cell expansion is regulated by multiple growth factors, including glucose, insulin, insulin-like growth factor (IGF-1) and epidermal growth factor (EGF). These mitogens signal through several downstream pathways (AKT, ERK, STAT3, and JNK) to regulate the survival and proliferation of β-cells. Survivin, an oncofetal protein with both pro-proliferative and anti-apoptotic properties, is a known transcriptional target of both IGF-1 and EGF in cancer cells. Here, we analyzed the effects of the β-cell mitogens IGF-1 and EGF on survivin regulation in the established pancreatic β-cell model cell lines, MIN6 and INS-1 and in primary mouse islets. Results In pancreatic β-cells, treatment with glucose, insulin, or EGF increased survivin protein levels at early time points. By contrast, no significant effects on survivin were observed following IGF-1 treatment. EGF-stimulated increases in survivin protein were abrogated in the presence of downstream inhibitors of the Raf-1/MEK/ERK pathway. EGF had no significant effect on survivin transcription however it prolonged the half-life of the survivin protein and stabilized survivin protein levels by inhibiting surviving ubiquitination. Conclusions This study defines a novel mechanism of survivin regulation by EGF through the Raf-1/MEK/ERK pathway in pancreatic β-cells, via prolongation of survivin protein half-life and inhibition of the ubiquitin-mediated proteasomal degradation pathway. This mechanism may be important for regulating β-cell expansion after birth.

The identification of alternative splice variants of Survivin that possess distinct functions from those originally identified for the main Survivin isoform has greatly increased the complexity of our understanding of the role of Survivin in different cells. Previous functional studies of the Survivin splice variants have been performed almost exclusively in cancer cells. However, Survivin has increasingly been implicated in other normal physiologic and pathophysiologic processes, including angiogenesis. In this study, we dissect the involvement of Survivin ΔEx3 in angiogenesis. We show by confocal microscopy that a pool of endothelial Survivin ΔEx3 is localized to membrane ruffles. We also demonstrate that Survivin ΔEx3 is the Survivin splice variant responsible for modulating angiogenesis in vitro, in tube formation assays, and in vivo, in an in vivo angiogenesis assay. Our data indicate that Survivin ΔEx3 may regulate angiogenesis via several mechanisms including cell invasion, migration, and Rac1 activation. Our findings identify a novel pathway regulating angiogenesis through Survivin ΔEx3 and a novel mechanism for Rac1 activation during angiogenesis. In conclusion, our results provide new insights into the regulation of endothelial cell homeostasis and angiogenesis by the Survivin proteins.

Survivin is an antiapoptotic protein highly expressed in malignant cells that confers resistance to cytotoxic therapy. Granzyme B is a potent cytotoxic protein that is released from mammalian natural killer cells and CTLs following noxious stimuli, including foreign invaders. Here, we took advantage of the properties of these two functionally divergent molecules to create a molecular agent that specifically activates Granzyme B within tumor cells. We designed Survivin and Granzyme B–induced apoptosis (SAGA), which consists of a fusion of the Survivin gene promoter to the coding sequence of active Granzyme B. In cultured human tumor cells transfected with SAGA DNA, Granzyme B is rapidly expressed and results in significant tumor cell death. In vivo, mice harboring human ovarian tumors had statistically significant clinical responses to SAGA treatment that were magnified following combination therapy with SAGA and paclitaxel. At the completion of a 3-week therapeutic trial, 3 of 15 animals were free of disease in the SAGA-treated group, and an additional eight animals had tumors that were nonpalpable and only detected on surgical resection. In contrast, 15 of 15 animals in the control and paclitaxel-only–treated groups had tumors at end of therapy. Treatment with SAGA with or without paclitaxel also prevented disease dissemination in 19 of 20 animals. These results strongly suggest that SAGA has the potential to be a potent agent for the treatment of primary and recurrent human ovarian carcinoma. Moreover, we predict that SAGA will be useful therapeutically in any human cancer that expresses Survivin. [Mol Cancer Ther 2006;5(3):693–703]

Apoptosis is an essential process during normal neuronal development. Approximately one-half of the neurons produced during neurogenesis die before completion of CNS maturation. To characterize the role of the inhibitor of apoptosis gene,survivin, during neurogenesis, we used the Cre-loxP-system to generate mice lackingsurvivinin neuronal precursor cells. Conditional deletion ofsurvivinstarting at embryonic day 10.5 leads to massive apoptosis of neuronal precursor cells in the CNS. Conditional mutants were born at the expected Mendelian ratios; however, these died shortly after birth from respiratory insufficiency, without primary cardiopulmonary pathology. Newborn conditional mutants showed a marked reduction in the size of the brain associated with severe, mutifocal apoptosis in the cerebrum, cerebellum, brainstem, spinal cord, and retina. Caspase-3 and caspase-9 activities in the mutant brains were significantly elevated, whereas bax expression was unchanged from controls. These results show that survivin is critically required for the survival of developing CNS neurons, and may impact on our understanding of neural repair, neural development, and neurodegenerative diseases. Our study is the first to solidify a role for survivin as an antiapoptotic protein during normal neuronal developmentin vivo.

Background Survivin and its alternative splice forms are involved in critical cellular processes, including cell division and programmed cell death. Survivin is expressed in the majority of human cancers, but minimally in differentiated normal tissues. Expression levels correlate with tumor aggressiveness and resistance to therapy. Results In the present study, we identify and characterize a novel survivin isoform that we designate survivin 2α. Structurally, the transcript consists of 2 exons: exon 1 and exon 2, as well as a 3' 197 bp region of intron 2. Acquisition of a new in-frame stop codon within intron 2 results in an open reading frame of 225 nucleotides, predicting a truncated 74 amino acid protein. Survivin 2α is expressed at high levels in several malignant cell lines and primary tumors. Functional assays show that survivin 2α attenuates the anti-apoptotic activity of survivin. Subcellular localization and immunoprecipitation of survivin 2α suggests a physical interaction with survivin. Conclusion We characterized a novel survivin splice variant that we designated survivin 2α. We hypothesize that survivin 2α can alter the anti-apoptotic functions of survivin in malignant cells. Thus survivin 2α may be useful as a therapeutic tool in sensitizing chemoresistant tumor cells to chemotherapy.

Survivin is an inhibitor of apoptosis protein that also plays critical roles in regulating the cell cycle and mitosis. Its prominent expression in essentially all human malignancies, and low or absent expression in most normal tissues, suggests that it would be an ideal target for cancer-directed therapy. Impeding development of safe and effective survivin antagonists for clinical use is a lack of understanding of the molecular mechanisms by which survivin differentially affects apoptosis and cell division, in normal and malignant cells. We show that the diverse functional roles of survivin can be explained, in part, by its heterodimerization with survivin splice variants in tumor cells. Survivin and survivin-DeltaEx3 interact within the mitochondria where they may inhibit mitochondrial-dependent apoptosis. If the expression of all survivin forms is eliminated by siRNA transfections, cells undergo both apoptosis and defective cell division. Overall, we provide new insights suggesting that targeting specific survivin isoforms, rather than survivin alone, may selectively and effectively destroy tumor cells. These findings are likely to have a significant impact in the design of biologic agents for clinical therapy.

Survivin is a putative oncogene that is aberrantly expressed in cancer cells. It has been hypothesized to play a central role in cancer progression and resistance to therapy in diverse tumor types. Although some of the transcriptional processes regulating its expression have been established, the diversity of genes that may be controlling the levels of its expression in both normal cells as well as in cancer cells has not been fully explored. The most common genetically mutated pathways in human malignancies are the p53 tumor suppressor pathway and the RB/E2F pathway. Both of these pathways, when intact, provide essential checkpoints in the maintenance of normal cell growth and protect the cell from DNA damage. Using non-transformed embryonic fibroblasts, we provide evidence of a molecular link between the regulation of survivin transcription and the RB/E2F family of proteins. We demonstrate that both pRB and p130 can interact with the survivin promoter and can repress survivin transcription. We also show that the E2F activators (E2F1, E2F2, and E2F3) can bind to the survivin promoter and induce survivin transcription. Genetically modified cells that harbor deletions in various members of the RB/E2F family confirm our data from the wild-type cells. Our findings implicate several members of the RB/E2F pathway in an intricate mechanism of survivin gene regulation that, when genetically altered during the process of tumorigenesis, may function within cancer cells to aberrantly alter survivin levels and enhance tumor progression.

Objective: To obtain quantitative serum levels of total and ionized magnesium (Mg 2+ ) in children with homozygous sickle cell anemia (SCA) undergoing therapy with hydroxyurea. Study design: Five children, ages 11 to 14 years with homozygous SCA, were enrolled in a dose-escalating trial of hydroxyurea over an 18-month period. Serum levels of total and ionized magnesium together with ionized K + , Na + , and Ca 2+ were measured before hydroxyurea and every 6 months during hydroxyurea therapy. Results: Before treatment, 4 of the 5 patients had low or below-normal serum concentrations of Mg 2+ (normal range, 0.51-0.67 mmol/L). All 5 became Mg 2+ -deficient during hydroxyurea therapy, with no indication of recovery until after 12 to 18 months of drug administration ( P + , Na + , and Ca 2+ remained consistently within normal ranges. Conclusions: These findings warrant a controlled study of the effects of magnesium supplementation in patients with SCA receiving hydroxyurea. Potentially, such therapy could alleviate or prevent vaso-occlusive crises. (J Pediatr 2002;140:565-9)

Survivin is a member of the inhibitor of apoptosis protein (IAP) family based on its N-terminal baculovirus IAP repeat (BIR) domain. The protein is largely undetectable in differentiated tissues, but is highly expressed in most human tumors. Its cytoplasmic expression in these tumors correlates with reduced tumor cell death, increased resistance to cancer therapy, and decreased patient survival. Survivin is actively exported into the cytoplasm exclusively by the chromosomal region maintenance 1 (CRM1/Exportin1) protein, one of seven known nuclear export proteins. CRM1 binds to the canonical nuclear export signal (NES) domain within the survivin protein. Inhibition of CRM1-mediated nuclear export has been suggested as a novel cancer therapeutic strategy that restores the tumor suppressor function of multiple nuclear proteins. Here, we investigated the anti-tumor mechanisms of two novel, drug-like CRM1 inhibitors, KPT-185 and KPT-276, in estrogen-receptor positive and triple negative breast cancer cell lines. Tumor and control cells were treated with varying doses of the KPT compounds over time. Cell proliferation and apoptosis were measured using standard assays. Survivin localization and expression in the presence and absence of the KPT drugs was assessed by immunofluorescence microscopy and cell fractionation. Breast cancer cell lines were engineered to express higher or lower levels of Survivin to determine the contribution of Survivin inhibition to the anti-tumor effects of the CRM1 inhibitors. Results showed that the KPT compounds have potent anti-proliferative properties and that they induce time- and dose-dependent apoptosis. KPT-185 and KPT-276 initially enhanced nuclear Survivin expression but produced a decrease in total cellular Survivin levels at later time points. Survivin protein degraded at a faster rate following KPT treatment, which was blocked by treatment with proteasome inhibitors. Knockdown of Survivin increased KPT-mediated cell apoptosis while exogenous expression of Survivin rescued cells from KPT-mediated apoptosis. In summary, our data demonstrate that inhibition of nuclear export by CRM1 inhibition suppresses breast tumor cell growth and enhances tumor cell death, in part through specific targeting of the Survivin-CRM1 complex. Citation Format: Yan Cheng, Michael Holloway, Kevin Nguyen, Michael Kauffman, Sharon Shacham, Rachel A. Altura. Inhibition of the nuclear transport protein CRM1 induces human breast cancer cell death by regulating survivin degradation. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 853. doi:10.1158/1538-7445.AM2013-853

Survivin is an oncogenic protein that participates in cell division and inhibits apoptosis in cancer cells. These functions are dependent on its interaction with partner proteins within distinct subcellular compartments. Survivin is highly expressed in malignant tumors yet minimally expressed in normal tissues, making it an attractive target for molecular therapy. Inhibition of Survivin function enhances tumor cell sensitivity to radiation, the mechanism of which has been reported to result from a disruption of DNA-double-strand break repair processes. Recently, we demonstrated that Survivin is post-translationally modified by acetylation at numerous lysine residues, one of which is responsible for its subcellular transport. Acetylation at Lys-129 facilitates Survivin homodimerization which prevents its binding to the CRM1 nuclear export protein, inhibiting its translocation from the nucleus to the cytoplasm. The object of this study was to determine the potential relationship between Survivin acetylation and its function in nuclear repair processes. To elucidate the association between Survivin acetylation, nuclear localization and DNA double-strand break repair we subjected HeLa cells to 4Gy γXRT and examined Survivin acetylation at serial time points (20, 40, 60 min) after irradiation. An immediate deacetylation of Survivin was observed at 20 minutes, followed by an increase in both global acetylation and acetylation at Lys-129 at 40 minutes. The increase in Lys-129 acetylation correlated with its nuclear accumulation, as demonstrated by indirect immunofluorescence staining and immunoblotting after subcellular fractionation. Acetylated Survivin was found in endogenous immunoprecipitants with the DNA damage repair protein, Ku70, following γ-irradiation. Indirect immunoflourescence revealed co-localization of Ku70 and acetylated Survivin within the nucleus at similar time points after irradiation suggesting that Survivin acetylation may be required for Ku70 complex formation within the nucleus. These results implicate acetylated Survivin as a regulator of DNA damage repair through the Ku70 DNA repair pathway and further our understanding of Survivin function within the nuclear compartment of cancer cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2476. doi:10.1158/1538-7445.AM2011-2476

Background: Basal-like breast cancer is a highly aggressive breast tumor subtype associated with an adverse prognosis for which biologic therapies are not available. These tumors have a distinct molecular profile characterized by an absence of estrogen receptor expression, absence of HER2 amplification, and positive expression of EGFR and/or cytokeratin (CK) 5/6. The object of this study was to evaluate the expression of the anti-apoptotic protein Survivin in basal-like breast cancer and compare it with other molecular subtypes. Our laboratory recently identified that post-translational modification of Survivin by acetylation (Ac) at Lys-129 regulates its export from the nucleus and therefore its anti-apoptotic function. In an effort to understand the expression profile of Survivin and Ac-Survivin in breast tumors, we developed an antibody to the Ac Lys-129 residue. Methods: 4 μM sections of 238 consecutive grade 3 invasive ductal carcinoma cases, arranged on 15 tissue microarrays were stratified into 67 luminal (ER+), 66 HER2 positive (HER2+), 93 basal-like (ER-, HER2-, CK5/6+ and/or EGFR+), and 12 ER-/HER2-/CK5/6-/EGFR- carcinomas. TMAs were immunostained with a rabbit polyclonal Survivin antibody generated to the Lys-129 acetylated residue, a full-length (FL) rabbit polyclonal Survivin antibody (NB-500-201), or normal rabbit serum control. Tumors were scored semiquantitatively and compared with recurrence-free (RF) and overall survival (OS) and with expression of ER, PR, HER2, CK 5/6, and EGFR. Results: In all breast carcinomas examined, FL-Survivin and Ac-Survivin had a predominantly nuclear localization pattern. Ac-Survivin was also expressed in the nucleus of normal breast epithelium. RF (70%) and OS (90%) for FL-Survivin low tumors was significantly better than for tumors with strong FL-Survivin expression (40% and 70%, respectively) at 10 yrs, p = 0.02. By contrast, strong expression of Ac-Survivin was associated with improved RF (70% vs. 40%) and OS (85% vs. 75%), p = 0.03. Molecular subset analyses showed that FL-Survivin expression was associated with a basal-like expression profile, while Ac-Survivin expression was associated with a luminal-type profile, p Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2272. doi:10.1158/1538-7445.AM2011-2272

Survivin, a member of the inhibitor of apoptosis (IAP) family of proteins, is widely expressed in transformed cell lines and in many different primary cancer cells, including both hematopoietic and non-hematopoietic malignancies. It is not expressed in many non-malignant adult tissues, but is essential for fetal development, as demonstrated by conventional gene-targeting experiments in mice that show embryonic lethality at day 4–6 of development. In adult cancers, including lymphoma and many epithelial carcinomas (colon, breast, gastric) the expression level of survivin, as assayed by immunohistochemical analysis and RT-PCR, correlates with overall survival. We have designed a novel gene therapy approach that takes advantage of the high expression levels of survivin in malignant cells, in which the survivin promoter is used to drive the expression of a suicide gene to kill cancer cells by programmed cell death. Our system is based on perforin-independent granzyme B cytotoxicity and therefore does not require pro-drug activation. We designated it SAGA, for survivin and granzyme apoptosis. We used Jurkat cells as an in vitro model for T-cell leukemia, and 697Bcl2 cells as a model for pre-B Bcl2-expressing leukemia, to show this approach is more efficient in killing leukemic cells than conventional chemotherapy. Jurkat cells responded to both vincristine therapy and SAGA whereas 697Bcl2 were unaffected by vincristine, but responded to SAGA. Cell growth curves of Jurkat cells and 697Bcl2 cells are shown in Figure 1. Our approach not only inhibits cell growth, but also induces apoptosis. We detected apoptotic events by Annexin V staining and by changes in mitochondrial potential, as early as 12 hours post-treatment. Rates of early apoptotic events are shown in Table 1. In addition to these events, we also documented DNA fragmentation and caspase-3 activation in treated cells. Cytotoxicity was clearly visible by microscopic analysis 24 hours post-treatment (Figure 2). Our results strongly suggest that survivin-driven suicide gene therapy effectively enhances cell death of leukemic blasts derived from two common sub-types of ALL, one of which expresses the potent anti-apoptotic inhibitor, Bcl-2, known to be clinically more resistant to standard therapy. Experiments evaluating the in vivo effects of SAGA in mouse leukemia models are currently underway. Cell death at 24 hours Early Apoptosis Necrosis control vincristine SAGA control vincristine SAGA T-ALL 2% 46% 41% 2% 18% 19% B-ALL 1% 2% 29% 0.4% 1% 30% Figure Figure Figure Figure

The E2F family of transcription factors is critical for the control of cell cycle progression. We now show that the specific inactivation of E2F3 in mouse embryo fibroblasts (MEFs) results in a disruption of the centrosome duplication cycle. Loss of E2F3, but not E2F1, E2F2, E2F4, or E2F5 results in unregulated cyclin E-dependent kinase activity, defects in nucleophosmin B association with centrosomes, and premature centriole separation and duplication. Consequently, this defect leads to centrosome amplification, mitotic spindle defects, and aneuploidy. Our findings implicate the E2F3 transcription factor as an important link that orchestrates DNA and centrosome duplication cycles, ensuring the faithful transmission of genetic material to daughter cells.