Search

PDF file - 87K, Survivin protein levels normalized to tubulin.

PDF file - 84K

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.

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 is an anti-apoptotic protein that is highly expressed in most malignant tumors but minimally expressed in normal tissues. Here, we investigate how induced oxidative stress in the form of hydrogen peroxide (H2O2) affects survivin expression levels in the MCF-7 breast cancer cell line. Numerous studies have demonstrated that intracellular H2O2 is a key regulator of signal transduction however its putative role in oncogenesis remains controversial. Conflicting reports have suggested that altering intracellular H2O2 levels can both inhibit and promote apoptosis in cancer cells. Recent studies have shown that compounds that raise cancer cells' intracellular H2O2 levels may make them more susceptible to chemotherapy, while studies at the molecular level show that an increase in intracellular H2O2 activates key oncogenes that block regulated apoptosis and promote an environment optimal for malignant transformation. Based on these reports, we investigated the effects of H2O2 treatment on survivin expression in MCF-7 cells under two different conditions 1) normal proliferation in complete media, 2) cell-cycle arrest by 16-hour serum starvation. Our results show that H2O2 treatment in quiescent cells induced survivin expression in a dose-dependent manner. By contrast, H2O2 treatment in proliferating cells led to a dose-dependent decrease in survivin expression. This result was unexpected as reports indicate that survivin expression is highly induced by growth factors present in serum-complemented media. Further investigation into potential upstream signaling mediators revealed that activation of extracellular signal related kinases 1 and 2 (ERK1/2) may play an important role in H2O2-induced survivin expression. We observed an increase in ERK1/2 phosphorylation that directly corresponded to an increase in survivin expression in serum-deprived cells. However, phospho-ERK1/2 levels remained relatively constant in proliferating MCF-7 cells despite a dose-dependent decrease in survivin expression. One possible explanation for this difference is a dramatic reduction in endogenous H2O2 levels following serum-deprivation. Compared to normal cells, cancer cells have a significantly higher level of intracellular H2O2 potentially making them more susceptible to apoptosis following increased oxidative stress. This mechanism may account for the dose-dependent decrease in survivin expression observed in MCF-7 cells treated with H2O2 in the presence of complete media. However, growth factor withdrawal decreases intracellular H2O2 levels. Therefore, under conditions of serum deprivation, H2O2 may activate mitogen-activated protein (MAP) kinase signal transduction cascades that promote growth and proliferation via oxidative stress signals. Together, our results support the hypothesis that endogenous levels of H2O2 may play a critical role in how cancer cells respond to treatment regulated by the MAP kinase pathway. Based on this model, agents that increase H2O2 levels in rapidly dividing cancer cells should be explored as an adjunct to chemotherapy, representing a novel approach to induce selective apoptosis by exploiting the oxidative environment tumor cells manifest in order to sustain their excessive metabolic demands. Citation Information: Cancer Res 2009;69(23 Suppl):A55.