The transcription corepressor CtBP is often recruited to the target promoter via interaction with a conserved PxDLS motif in the interacting repressor. In this study, we demonstrate that CtBP1 was SUMOylated and that its SUMOylation profoundly affected its subcellular localization. SUMOylation occurred at a single Lys residue, Lys428, of CtBP1. CtBP2, a close homolog of CtBP1, lacked the SUMOylation site and was not modified by SUMO-1. Mutation of Lys428 into Arg (K428R) shifted CtBP1 from the nucleus to the cytoplasm, while it had little effect on its interaction with the PxDLS motif. Consistent with a change in localization, the K428R mutation abolished the ability of CtBP1 to repress the E-cadherin promoter activity. Notably, SUMOylation of CtBP1 was inhibited by the PDZ domain of nNOS, correlating with the known inhibitory effect of nNOS on the nuclear accumulation of CtBP1. This study identifies SUMOylation as a regulatory mechanism underlying CtBP1-dependent transcriptional repression.
The PROTAC (proteolysis-targeting chimera) ARV-825 recruits bromodomain and extraterminal (BET) proteins to the E3 ubiquitin ligase cereblon, leading to degradation of BET proteins, including BRD4. Whereas the BET-protein inhibitor (BETi) OTX015 caused accumulation of BRD4, treatment with equimolar concentrations of ARV-825 caused sustained and profound depletion (>90%) of BRD4 and induced significantly more apoptosis in cultured and patient-derived (PD) CD34+ post-MPN sAML cells, while relatively sparing the CD34+ normal hematopoietic progenitor cells. RNA-Seq, Reversed Phase Protein Array and mass cytometry ‘CyTOF’ analyses demonstrated that ARV-825 caused greater perturbations in mRNA and protein expressions than OTX015 in sAML cells. Specifically, compared to OTX015, ARV-825 treatment caused more robust and sustained depletion of c-Myc, CDK4/6, JAK2, pSTAT3/5, PIM1 and Bcl-xL, while increasing the levels of p21 and p27. Compared to OTX015, PROTAC ARV-771 treatment caused greater reduction in leukemia burden and further improved survival of NSG mice engrafted with luciferase-expressing HEL92.1.7 cells. Co-treatment with ARV-825 and JAK inhibitor ruxolitinib was synergistically lethal against the established and PD-CD34+ sAML cells. Notably, ARV-825 induced high levels of apoptosis in the in vitro generated ruxolitinib-persister or ruxolitinib-resistant sAML cells. These findings strongly support the in vivo testing of the BRD4-PROTAC based combinations against post-MPN sAML.
Bromodomain extraterminal protein (BETP) inhibitors transcriptionally repress oncoproteins and nuclear factor-κB (NF-κB) target genes that undermines the growth and survival of mantle cell lymphoma (MCL) cells. However, BET bromodomain inhibitor (BETi) treatment causes accumulation of BETPs, associated with reversible binding and incomplete inhibition of BRD4 that potentially compromises the activity of BETi in MCL cells. Unlike BETi, BET-PROTACs (proteolysis-targeting chimera) ARV-825 and ARV-771 (Arvinas, Inc.) recruit and utilize an E3-ubiquitin ligase to effectively degrade BETPs in MCL cells. BET-PROTACs induce more apoptosis than BETi of MCL cells, including those resistant to ibrutinib. BET-PROTAC treatment induced more perturbations in the mRNA and protein expressions than BETi, with depletion of c-Myc, CDK4, cyclin D1 and the NF-κB transcriptional targets Bcl-xL, XIAP and BTK, while inducing the levels of HEXIM1, NOXA and CDKN1A/p21. Treatment with ARV-771, which possesses superior pharmacological properties compared with ARV-825, inhibited the in vivo growth and induced greater survival improvement than the BETi OTX015 of immune-depleted mice engrafted with MCL cells. Cotreatment of ARV-771 with ibrutinib or the BCL2 antagonist venetoclax or CDK4/6 inhibitor palbociclib synergistically induced apoptosis of MCL cells. These studies highlight promising and superior preclinical activity of BET-PROTAC than BETi, requiring further in vivo evaluation of BET-PROTAC as a therapy for ibrutinib-sensitive or -resistant MCL.
Several novel protein kinase C (PKC) isozymes have been identified as substrates for caspase-3. We have previously shown that novel PKC⑀ is cleaved during apoptosis in MCF-7 cells that lack any functional caspase-3. In the present study, we show that in vitro-translated PKC⑀ is processed by human recombinant caspase-3, -7, and -9. Tumor necrosis factor-␣ (TNF) triggered processing of PKC⑀ to a 43-kDa carboxyl-terminal fragment, and cell-permeable caspase inhibitors prevented TNF-induced processing of PKC⑀ in MCF-7 cells. PKC⑀ was cleaved primarily at the SSPD2G site to generate two fragments with an approximate molecular mass of 43 kDa. It was also cleaved at the DDVD2C site to generate two fragments with molecular masses of 52 and 35 kDa. Treatment of MCF-7 cells with TNF resulted in the activation of PKC⑀, and mutation at the SSPD2G (D383A) site inhibited proteolytic activation of PKC⑀. Overexpression of wild-type but not dominant-negative PKC⑀ in MCF-7 cells delayed TNF-induced apoptosis, and mutation at the D383A site prevented antiapoptotic activity of PKC⑀. These results suggest that cleavage of PKC⑀ by caspase-7 at the SSPD2G site results in the activation of PKC⑀. Furthermore, activation of PKC⑀ was associated with its antiapoptotic function.
Key Points• BA reduces MYC, CDK4/6, nuclear RelA, and BTK expression and is synergistically lethal with ibrutinib in MCL cells.• Cotreatment with BA and inhibitor of BCL2, CDK4/6, or histone deacetylases is synergistically lethal against ibrutinib-resistant MCL cells.Mantle cell lymphoma (MCL) cells exhibit increased B-cell receptor and nuclear factor (NF)-kB activities. The bromodomain and extra-terminal (BET) protein bromodomain 4 is essential for the transcriptional activity of NF-kB. Here, we demonstrate that treatment with the BET protein bromodomain antagonist (BA) JQ1 attenuates MYC and cyclindependent kinase (CDK)4/6, inhibits the nuclear RelA levels and the expression of NF-kB target genes, including Bruton tyrosine kinase (BTK) in MCL cells. Although lowering the levels of the antiapoptotic B-cell lymphoma (BCL)2 family proteins, BA treatment induces the proapoptotic protein BIM and exerts dose-dependent lethality against cultured and primary MCL cells. Cotreatment with BA and the BTK inhibitor ibrutinib synergistically induces apoptosis of MCL cells. Compared with each agent alone, cotreatment with BA and ibrutinib markedly improved the median survival of mice engrafted with the MCL cells. BA treatment also induced apoptosis of the in vitro isolated, ibrutinib-resistant MCL cells, which overexpress CDK6, BCL2, Bcl-xL, XIAP, and AKT, but lack ibrutinib resistanceconferring BTK mutation. Cotreatment with BA and panobinostat (pan-histone deacetylase inhibitor) or palbociclib (CDK4/6 inhibitor) or synergistically induced apoptosis of the ibrutinib-resistant MCL cells. These findings highlight and support further in vivo evaluation of the efficacy of the BA-based combinations with these agents against MCL, including ibrutinib-resistant MCL. (Blood. 2015;126(13):1565-1574 Introduction Among the genetic alterations described in mantle cell lymphoma (MCL) cells are those that involve p53, cyclin-dependent kinase (CDK)4, CDKN2A, MYC, B-cell lymphoma (BCL)2, B-cell receptor (BCR), and nuclear factor (NF)-kB signaling genes. [1][2][3] These genetic alterations confer a cell autonomous pro-growth and pro-survival advantage on the MCL cells, which is especially dependent on NF-kB, BCL2, and MYC activities. [2][3][4] Next generation sequencing has also disclosed new targets for therapeutic intervention in the deregulated molecular signaling through BCR, toll-like receptor, NOTCH, NFkB, and mitogen-activated protein kinase signaling pathways in the MCL cell lines and patient-derived primary MCL.3-7 Pre-clinical and clinical studies have shown that ibrutinib, a selective, orally bioavailable, irreversible inhibitor of Bruton tyrosine kinase (BTK) in the BCR, also inhibits NF-kB activity and is active against B-cell neoplasms, including chronic lymphocytic leukemia (CLL) and MCL. 6,8 Ibrutinib has demonstrated impressive clinical efficacy and is approved for the treatment of CLL and MCL.9-11 Despite its high level of clinical activity, primary or acquired clinical resistance to ibrutinib therapy is commonly observed....
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