MCL1 is essential for the survival of stem and progenitor cells of multiple lineages, and is unique among pro-survival BCL2 family members in that it is rapidly turned over through the action of ubiquitin ligases. B- and mantle-cell lymphomas, chronic myeloid leukaemia, and multiple myeloma, however, express abnormally high levels of MCL1, contributing to chemoresistance and disease relapse. The mechanism of MCL1 overexpression in cancer is not well understood. Here we show that the deubiquitinase USP9X stabilizes MCL1 and thereby promotes cell survival. USP9X binds MCL1 and removes the Lys 48-linked polyubiquitin chains that normally mark MCL1 for proteasomal degradation. Increased USP9X expression correlates with increased MCL1 protein in human follicular lymphomas and diffuse large B-cell lymphomas. Moreover, patients with multiple myeloma overexpressing USP9X have a poor prognosis. Knockdown of USP9X increases MCL1 polyubiquitination, which enhances MCL1 turnover and cell killing by the BH3 mimetic ABT-737. These results identify USP9X as a prognostic and therapeutic target, and they show that deubiquitinases may stabilize labile oncoproteins in human malignancies.

COP1 (constitutively photomorphogenic 1) is a RING-finger-containing protein that functions to repress plant photomorphogenesis, the light-mediated programme of plant development. Mutants of COP1 are constitutively photomorphogenic, and this has been attributed to their inability to negatively regulate the proteins LAF1 (ref. 1) and HY5 (ref. 2). The role of COP1 in mammalian cells is less well characterized. Here we identify the tumour-suppressor protein p53 as a COP1-interacting protein. COP1 increases p53 turnover by targeting it for degradation by the proteasome in a ubiquitin-dependent fashion, independently of MDM2 or Pirh2, which are known to interact with and negatively regulate p53. Moreover, COP1 serves as an E3 ubiquitin ligase for p53 in vitro and in vivo, and inhibits p53-dependent transcription and apoptosis. Depletion of COP1 by short interfering RNA (siRNA) stabilizes p53 and arrests cells in the G1 phase of the cell cycle. Furthermore, we identify COP1 as a p53-inducible gene, and show that the depletion of COP1 and MDM2 by siRNA cooperatively sensitizes U2-OS cells to ionizing-radiation-induced cell death. Overall, these results indicate that COP1 is a critical negative regulator of p53 and represents a new pathway for maintaining p53 at low levels in unstressed cells.

systems that incorporate features of the tumor microenvironment and model the dynamic response to immune checkpoint blockade (ICB) may facilitate efforts in precision immuno-oncology and the development of effective combination therapies. Here, we demonstrate the ability to interrogate response to ICB using murine- and patient-derived organotypic tumor spheroids (MDOTS/PDOTS). MDOTS/PDOTS isolated from mouse and human tumors retain autologous lymphoid and myeloid cell populations and respond to ICB in short-term three-dimensional microfluidic culture. Response and resistance to ICB was recapitulated using MDOTS derived from established immunocompetent mouse tumor models. MDOTS profiling demonstrated that TBK1/IKKε inhibition enhanced response to PD-1 blockade, which effectively predicted tumor response Systematic profiling of secreted cytokines in PDOTS captured key features associated with response and resistance to PD-1 blockade. Thus, MDOTS/PDOTS profiling represents a novel platform to evaluate ICB using established murine models as well as clinically relevant patient specimens. Resistance to PD-1 blockade remains a challenge for many patients, and biomarkers to guide treatment are lacking. Here, we demonstrate feasibility of profiling of PD-1 blockade to interrogate the tumor immune microenvironment, develop therapeutic combinations, and facilitate precision immuno-oncology efforts..

Plasmids and reagents. Human DET1 cDNA was amplified by PCR from a HEK293 cell library and cloned into pEF6 myc-His (Invitrogen), a modified pFLAG CMV14 vector (Sigma) with a Glu-Glu tag and TEV cleavage site inserted 5' to the C-terminal 3xFLAG sequence, or a modified pFLAG CMV6 vector (Sigma) with a GST tag inserted 3' to the N-terminal FLAG sequence. Human COP1 and hCOP1∆24 cDNA were amplified by PCR from MCF7 and HEK293 libraries, respectively, and cloned into pFLAG CMV6 (Sigma) or pEF6 myc-His (Invitrogen). The hCOP1 RING mutant was generated using a Quikchange Kit as directed by the manufacturer (Stratagene) to mutate C136 to A and C139 to A. The FLAG hCOP1 RING+cc deletion (amino acids 307-731) and the FLAG hCOP1 and hCOP1∆24 C-terminal WD40 deletions (removing the Cterminal 206 amino acids) were generated by standard PCR techniques from the respective pFLAG CMV6 constructs. Human ATF2 cDNA was amplified by PCR from a Jurkat cell library and cloned into pcDNA3

The basal-like subtype of breast cancer has an aggressive clinical behavior compared to that of the luminal subtype. We identified the microRNAs (miRNAs) miR-221 and miR-222 (miR-221/222) as basal-like subtype-specific miRNAs and showed that expression of miR-221/222 decreased expression of epithelial-specific genes and increased expression of mesenchymal-specific genes, and increased cell migration and invasion in a manner characteristic of the epithelial-to-mesenchymal transition (EMT). The transcription factor FOSL1 (also known as Fra-1), which is found in basal-like breast cancers but not in the luminal subtype, stimulated the transcription of miR-221/222, and the abundance of these miRNAs decreased with inhibition of the epidermal growth factor receptor (EGFR) or MEK (mitogen-activated or extracellular signal-regulated protein kinase kinase), placing miR-221/222 downstream of the RAS pathway. Furthermore, miR-221/222-mediated reduction in E-cadherin abundance depended on their targeting the 3' untranslated region of the GATA family transcriptional repressor TRPS1 (tricho-rhino-phalangeal syndrome type 1), which inhibited EMT by decreasing ZEB2 (zinc finger E-box-binding homeobox2) expression. We conclude that by promoting EMT, miR-221/222 may contribute to the more aggressive clinical behavior of basal-like breast cancers.

Overexpression of FGF receptor 3 (FGFR3) is implicated in the development of t(4;14

Here we describe the generation of an antibody-drug conjugate (ADC) consisting of a humanized anti-CD79b antibody that is conjugated to monomethylauristatin E (MMAE) through engineered cysteines (THIOMABs) by a protease cleavable linker. By using flow cytometry, we detected the surface expression of CD79b in almost all non-Hodgkin lymphoma (NHL) and chronic lymphocytic leukemia patients, suggesting that anti-CD79b-vcMMAE could be widely used in these malignancies. By using NHL cell lines to simulate a patient population we discovered that a minimal cell-surface expression level of CD79b was required for in vitro activity. Within the subpopulation of cell lines above this minimal threshold, we found that sensitivity to free MMAE, mutation of cancer genes, and cell doubling time were poorly correlated with in vitro activity; however, the expression level of BCL-XL was correlated with reduced sensitivity to anti-CD79b-vcMMAE.

Reconstitution of the stages in the assembly of the p300⅐p53 transcription complex has identified a novel type of DNA-dependent regulation of p300-catalyzed acetylation. Phosphorylation at the CHK2 site (Ser 20 ) in the N-terminal activation domain of p53 stabilized p300 binding. The phosphopeptide binding activity of p300 was mapped in vitro to two domains: the C-terminal IBiD domain and the N-terminal IHD domain (IBiD homology domain). The IHD or IBiD minidomains can bind to the p53 activation domain in vivo as determined using the mammalian two-hybrid VP16-GAL4 luciferase reporter assay. The IHD and IBiD minidomains of p300 also functioned as dominant negative inhibitors of p53-dependent transcription in vivo. Upon examining the affects of p300 binding on substrate acetylation, we found that the p53 consensus site DNA promotes a striking increase in p53 acetylation in vitro. Co-transfection into cells of the p53 gene and plasmid DNA containing the consensus DNA binding site of p53 activated DNAdependent acetylation of p53 in vivo. The phosphopeptide binding activity of p300 is critical for DNA-dependent acetylation, as p53 acetylation was inhibited by phospho-Ser 20 peptides. Consensus site DNA-dependent acetylation of p53 stabilized the p300⅐p53 protein complex, whereas basal acetylation of p53 by p300 in the presence of nonspecific DNA resulted in p300 dissociation. These data identify at least three distinct stages in the assembly of a p300⅐p53 complex: 1) p300 docking to the activation domain of p53 via the IBiD and/or IHD domains; 2) DNA-dependent acetylation of p53; and 3) stabilization of the p300⅐p53 AC complex after acetylation. The ability of DNA to act as an allosteric ligand to activate substrate acetylation identifies a conformational constraint that can be placed on the p300-acetylation reaction that is likely to be an amplification signal and influence protein-protein contacts at a promoter.