Purpose: Glioblastomas are treated by surgical resection followed by radiotherapy X-ray therapy (XRT) and the alkylating chemotherapeutic
agent temozolomide. Recently, inactivating mutations in the ...mismatch repair gene MSH6 were identified in two glioblastomas recurrent post-temozolomide. Because mismatch repair pathway inactivation is a known
mediator of alkylator resistance in vitro , these findings suggested that MSH6 inactivation was causally linked to these two recurrences. However, the extent of involvement of MSH6 in glioblastoma is unknown. We sought to determine the overall frequency and clinical relevance of MSH6 alterations in glioblastomas.
Experimental Design: The MSH6 gene was sequenced in 54 glioblastomas. MSH6 and O 6 -methylguanine methyltransferase (MGMT) immunohistochemistry was systematically scored in a panel of 46 clinically well-characterized
glioblastomas, and the corresponding patient response to treatment evaluated.
Results: MSH6 mutation was not observed in any pretreatment glioblastoma (0 of 40), whereas 3 of 14 recurrent cases had somatic mutations
( P = 0.015). MSH6 protein expression was detected in all pretreatment (17 of 17) cases examined but, notably, expression was
lost in 7 of 17 (41%) recurrences from matched post–XRT + temozolomide cases ( P = 0.016). Loss of MSH6 was not associated with O 6 -methylguanine methyltransferase status. Measurements of in vivo tumor growth using three-dimensional reconstructed magnetic resonance imaging showed that MSH6-negative glioblastomas had
a markedly increased rate of growth while under temozolomide treatment (3.17 versus 0.04 cc/mo for MSH6-positive tumors; P = 0.020).
Conclusions: Loss of MSH6 occurs in a subset of post–XRT + temozolomide glioblastoma recurrences and is associated with tumor progression
during temozolomide treatment, mirroring the alkylator resistance conferred by MSH6 inactivation in vitro . MSH6 deficiency may therefore contribute to the emergence of recurrent glioblastomas during temozolomide treatment.
Protein tyrosine kinase 6 (PTK6, or BRK) is aberrantly expressed in breast cancers, and emerging as an oncogene that promotes tumor cell proliferation, migration and evasion. Both kinase-dependent ...and -independent functions of PTK6 in driving tumor growth have been described, therefore targeting PTK6 kinase activity by small molecule inhibitors as a therapeutic approach to treat cancers remains to be validated. In this study, we identified novel, potent and selective PTK6 kinase inhibitors as a means to investigate the role of PTK6 kinase activity in breast tumorigenesis. We report here the crystal structures of apo-PTK6 and inhibitor-bound PTK6 complexes, providing the structural basis for small molecule interaction with PTK6. The kinase inhibitors moderately suppress tumor cell growth in 2D and 3D cell cultures. However, the tumor cell growth inhibition shows neither correlation with the PTK6 kinase activity inhibition, nor the total or activated PTK6 protein levels in tumor cells, suggesting that the tumor cell growth is independent of PTK6 kinase activity. Furthermore, in engineered breast tumor cells overexpressing PTK6, the inhibition of PTK6 kinase activity does not parallel the inhibition of tumor cell growth with a >500-fold shift in compound potencies (IC50 values). Overall, these findings suggest that the kinase activity of PTK6 does not play a significant role in tumorigenesis, thus providing important evidence against PTK6 kinase as a potential therapeutic target for breast cancer treatment.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Precise regulation of Type I interferon signaling is crucial for combating infection and cancer while avoiding autoimmunity. Type I interferon signaling is negatively regulated by USP18. USP18 ...cleaves ISG15, an interferon-induced ubiquitin-like modification, via its canonical catalytic function, and inhibits Type I interferon receptor activity through its scaffold role. USP18 loss-of-function dramatically impacts immune regulation, pathogen susceptibility, and tumor growth. However, prior studies have reached conflicting conclusions regarding the relative importance of catalytic versus scaffold function. Here, we develop biochemical and cellular methods to systematically define the physiological role of USP18. By comparing a patient-derived mutation impairing scaffold function (I60N) to a mutation disrupting catalytic activity (C64S), we demonstrate that scaffold function is critical for cancer cell vulnerability to Type I interferon. Surprisingly, we discovered that human USP18 exhibits minimal catalytic activity, in stark contrast to mouse USP18. These findings resolve human USP18's mechanism-of-action and enable USP18-targeted therapeutics.
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•USP18 is the primary negative regulator of Type I interferon signaling•In contrast to mouse USP18, human USP18 possesses weak enzymatic activity•USP18 acts chiefly through its scaffold function•Targeting USP18 scaffold function leads to cancer cell killing
Immunity; Immune response; Cell biology; Cancer
BackgroundUSP18 is a key negative regulator of Type I interferon (IFN) signaling. USP18 cleaves ISG15, a ubiquitin-like modification, through its canonical catalytic function, and directly inhibits ...the Type I IFN receptor through its scaffold role. USP18 loss-of-function dramatically impacts autoimmune disease, viral susceptibility, and cancer cell survival. However, published studies have reached different conclusions as to whether catalytic or scaffold function is essential.MethodsThe ability of USP18 to deconjugate ISG15 was determined using purified protein against a reporter substrate or cell lysates. Scaffolding activity was quantified using STAT1 phosphorylation upon IFN stimulation. After characterizing the impact of specific point mutations on USP18 function, mutations that selectively impair catalytic (USP18 C64S) or scaffold function (USP18 I60N) were evaluated for their effects on cancer cell vulnerability. To assess USP18 mechanism-of-action across multiple cancer cell lineages, guide RNAs that introduce the C64S mutation or knock out USP18 were introduced into cancer cells, and cells were passaged for 2 weeks in the presence or absence of IFN. IFN-dependent changes in wild-type, knockout, or C64S USP18 allelic frequency were assessed by DNA sequencing. To determine the effect of USP18 in vivo, mouse Usp18 was knocked out in CT26 colorectal cancer cells, and cells were implanted subcutaneously in wild-type or immunocompromised (NSG) mice.ResultsDeletion of USP18 in a variety of human cancer cell lines of multiple lineages (blood, breast, colon, lung) rendered them vulnerable to IFN. Furthermore, Usp18 knockout in cancer cells prevented tumor formation in wild-type mice, and resulted in partial tumor growth inhibition in immunocompromised mice. Introducing the C64S mutation fully impaired catalytic activity, yet did not result in IFN sensitivity. Furthermore, preventing ISGylation by knocking out the E1 enzyme required for ISGylation, UBA7, did not rescue IFN sensitivity in USP18-deficient cells. By contrast, the I60N mutation led to partial scaffold impairment and partial cancer cell sensitivity. Finally, human USP18 exhibited minimal catalytic activity, in stark contrast to mouse USP18. Therefore, deISGylase activity of USP18 does not mediate IFN sensitivity in human cancer cells.ConclusionsLoss of USP18 creates a key vulnerability of human cancer cells to IFN. This is primarily due to loss of scaffold function, rather than loss of catalytic function (figure 1). Indeed, human USP18 does not appear to function as an enzyme under physiological conditions, unlike mouse USP18. These findings resolve the mechanistic basis for human USP18 function, paving the way to target USP18 for cancer treatment.Ethics ApprovalAll procedures performed on animals were in accordance with regulations and established guidelines and were reviewed and approved by an Institutional Animal Care and Use Committee or through an ethical review process.Abstract 1079 Figure 1The mechanistic basis for human USP18 function and cancer cell vulnerability.
Overexpression of the antiapoptotic members of the Bcl-2 family of proteins is commonly associated with cancer cell survival and resistance to chemotherapeutics. Here, we describe the structure-based ...optimization of a series of N-heteroaryl sulfonamides that demonstrate potent mechanism-based cell death. The role of the acidic nature of the sulfonamide moiety as it relates to potency, solubility, and clearance is examined. This has led to the discovery of novel heterocyclic replacements for the acylsulfonamide core of ABT-737 and ABT-263.
Malignant gliomas have a very poor prognosis. The current standard of care for these cancers consists of extended adjuvant treatment with the alkylating agent temozolomide after surgical resection ...and radiotherapy. Although a statistically significant increase in survival has been reported with this regimen, nearly all gliomas recur and become insensitive to further treatment with this class of agents. We sequenced 500 kb of genomic DNA corresponding to the kinase domains of 518 protein kinases in each of nine gliomas. Large numbers of somatic mutations were observed in two gliomas recurrent after alkylating agent treatment. The pattern of mutations in these cases showed strong similarity to that induced by alkylating agents in experimental systems. Further investigation revealed inactivating somatic mutations of the mismatch repair gene MSH6 in each case. We propose that inactivating somatic mutations of MSH6 confer resistance to alkylating agents in gliomas in vivo and concurrently unleash accelerated mutagenesis in resistant clones as a consequence of continued exposure to alkylating agents in the presence of defective mismatch repair. The evidence therefore suggests that when MSH6 is inactivated in gliomas, alkylating agents convert from induction of tumor cell death to promotion of neoplastic progression. These observations highlight the potential of large scale sequencing for revealing and elucidating mutagenic processes operative in individual human cancers.
Fibroblast growth factor 23 (FGF23) is the causative factor of X-linked hypophosphatemia (XLH), a genetic disorder effecting 1:20,000 that is characterized by excessive phosphate excretion, elevated ...FGF23 levels and a rickets/osteomalacia phenotype. FGF23 inhibits phosphate reabsorption and suppresses 1α,25-dihydroxyvitamin D (1,25D) biosynthesis, analytes that differentially contribute to bone integrity and deleterious soft tissue mineralization. As inhibition of ligand broadly modulates downstream targets, balancing efficacy and unwanted toxicity is difficult when targeting the FGF23 pathway. We demonstrate that a FGF23 c-tail-Fc fusion molecule selectively modulates the phosphate pathway
in vivo
by competitive antagonism of FGF23 binding to the FGFR/ α klotho receptor complex. Repeated injection of FGF23 c-tail Fc in Hyp mice, a pre-clinical model of XLH, increases cell surface abundance of kidney NaPi transporters, normalizes phosphate excretion and significantly improves bone architecture in the absence of soft tissue mineralization. Repeated injection does not modulate either 1,25D or calcium in a physiologically relevant manner in either a wild-type or disease setting. These data suggest that bone integrity can be improved in models of XLH via the exclusive modulation of phosphate. We posit that the selective modulation of the phosphate pathway will increase the window between efficacy and safety risks, allowing increased efficacy to be achieved in the treatment of this chronic disease.
Abstract
Toll-like Receptor-3 (TLR-3), a pattern recognition receptor family member, has been shown to activate immune system pathways; however, it has also been shown to induce apoptosis in certain ...cancer cells. Combination of the synthetic TLR-3 ligand, poly I:C, with the Smac mimetic LBW242, an inhibitor of apoptosis protein (IAP) antagonist, has been shown to potently induce apoptosis in melanoma cells which are insensitive to either single agent. To better elucidate the mechanism responsible for this combination induced cell death, we performed a pooled shRNA screen. We identified receptor interacting protein kinase-1 (RIPK1) and as an important component of this process. Constitutive knockdown of RIPK1 prevents the LBW242 and poly I:C combination induced apoptosis. The IAP proteins cIAP1 and cIAP2 have been shown previously to regulate Caspase-8 activation downstream of Tumor Necrosis Factor Receptor-1 (TNFR1). Interestingly, we found that the combination induced apoptosis was independent of TNF signaling. Utilizing both gain of function and loss of function studies, we found that expression of TLR-3 is necessary for the combination induced apoptosis in melanoma cells. Taken together, our data indicate that the combination of the Smac mimetic LBW242 and TLR-3 agonist poly I:C induces a novel type of TLR-3 dependent apoptosis in melanoma cells that is independent of TNFR1.
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 LB-19. doi:10.1158/1538-7445.AM2011-LB-19
Abstract
Inhibitor of Apoptosis Proteins (IAP) proteins negatively regulate cell death through a variety of mechanisms. The prototypical IAP family member XIAP binds and inhibits the catalytic ...activity of caspases 3/7 and caspase 9 via the BIR2-linker region and BIR3 domains, respectively. CIAP1 and CIAP2 do not directly inhibit caspases but negatively regulate death receptor mediated apoptosis via intrinsic E3 ligase activity towards RIPK and NIK among other client proteins.
IAP inhibitors (IAPi) are low molecular weight compounds that mimic Smac and bind to the IAP binding motif in the BIR3 domain of XIAP, CIAP1 and CIAP2. Smac mimetics induce apoptosis as a single agent in a subset of tumor cell lines in vitro. Cell death is preceded by the rapid proteosome-mediated degradation of CIAP1 followed by activation of both canonical and non-canonical NFKB pathway activation, TNF production and robust activation of caspase 3/7 activity. Multiple nodes in the NFKB signaling pathway were interrogated following IAPi treatment in sensitive and resistant cancer cells to delineate the basis for differential responses. Although canonical and non-canonical NFKB signaling was activated in both sensitive and resistant cells, TNF was induced only in the former.
LCL161 is a second generation orally bioavailable IAPi with nM affinity for XIAP, CIAP1, CIAP2. Consistent with above, tumor cell lines with high baseline TNF levels are predisposed to IAPi sensitivity. Curiously, addition of exogenous TNF can sensitize many otherwise resistant tumor cell lines, but not normal cells, to LCL161. We undertook an unbiased study of the entire TNF super family to determine what other TNF-like cytokines could sensitize tumor cells to LCL161- induced cell death. In addition to TNF, several cytokines synergized with LCL161 and in each case RIPK appeared to play a central role.
LCL161 showed potent single agent activity in the MDA-MB-231 tumor xenograft model. In vivo efficacy was accompanied by a series of tumor pharmacodynamic readouts including CIAP1 elimination, activation of an NFKB transcriptional program and caspase activation. In primary patient derived human tumor xenograft models of triple negative breast cancer and NSCLC, LCL161 had a range of responses from no effect to tumor stasis. Consistent with in vitro mechanistic studies, tumor models which were sensitive had high basal TNF levels. LCL161 lacked single agent activity in the A2058 melanoma model but significantly enhanced the anti-tumor activity of paclitaxel. The LCL161-Taxol combination triggered synergistic activation of caspases and near complete regressions in xenograft tumors.
Clinical trials in man with LCL161 are ongoing in patients with solid tumors. A range of pharmacodynamic readouts have been observed which are consistent with preclinical observations. These findings show promise for IAP inhibitor therapy in humans.
Citation Format: {Authors}. {Abstract title} abstract. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 138.
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