Phase I study of safety and pharmacokinetics of the anti-MUC16 antibody-drug conjugate DMUC5754A in patients with platinum-resistant ovarian cancer or unresectable pancreatic cancer.

BACKGROUND: MUC16 is a tumor-specific antigen overexpressed in ovarian (OC) and pancreatic (PC) cancers. The antibody-drug conjugate (ADC), DMUC5754A, contains the humanized anti-MUC16 monoclonal antibody conjugated to the microtubule-disrupting agent, monomethyl auristatin E (MMAE). PATIENTS AND METHODS: This phase I study evaluated safety, pharmacokinetics (PK), and pharmacodynamics of DMUC5754A given every 3 weeks (Q3W, 0.3-3.2 mg/kg) or weekly (Q1W, 0.8-1.6 mg/kg) to patients with advanced recurrent platinum-resistant OC or unresectable PC. Biomarker studies were also undertaken. RESULTS: Patients (66 OC, 11 PC) were treated with DMUC5754A (54 Q3W, 23 Q1W). Common related adverse events (AEs) in >20% of patients (all grades) over all dose levels were fatigue, peripheral neuropathy, nausea, decreased appetite, vomiting, diarrhea, alopecia, and pyrexia in Q3W patents, and nausea, vomiting, anemia, fatigue, neutropenia, alopecia, decreased appetite, diarrhea, and hypomagnesemia in Q1W patients. Grade ≥3-related AE in ≥5% of patients included neutropenia (9%) and fatigue (7%) in Q3W patients, and neutropenia (17%), diarrhea (9%), and hyponatremia (9%) in Q1W patients. Plasma antibody-conjugated MMAE (acMMAE) and serum total antibody exhibited non-linear PK across tested doses. Minimal accumulation of acMMAE, total antibody, or unconjugated MMAE was observed. Confirmed responses (1 CR, 6 PRs) occurred in OC patients whose tumors were MUC16-positive by IHC (2+ or 3+). Two OC patients had unconfirmed PRs; six OC patients had stable disease lasting >6 months. For CA125, a cut-off of ≥70% reduction was more suitable for monitoring treatment response due to the binding and clearance of serum CA125 by MUC16 ADC. We identified circulating HE4 as a potential novel surrogate biomarker for monitoring treatment response of MUC16 ADC and other anti-MUC16 therapies in OC. CONCLUSIONS: DMUC5754A has an acceptable safety profile and evidence of anti-tumor activity in patients with MUC16-expressing tumors. Objective responses were only observed in MUC16-high patients, although prospective validation is required. CLINICAL TRIAL NUMBER: NCT01335958.

Pseudo-phosphatase Sbf1 contains an N-terminal GEF homology domain that modulates its growth regulatory properties.

Sbf1 (SET binding factor 1) is a pseudo-phosphatase related to the myotubularin family of dual specificity phosphatases, some of which have been implicated in cellular growth and differentiation by virtue of their mutation in human genetic disorders. Sbf1 contains germline-encoded alterations of its myotubularin homology domain that render it non-functional as a phosphatase. We report here the complete structure of Sbfl and further characterization of its growth regulatory properties. In addition to its similarity to myotubularin, the predicted full-length Sbf1 protein contains pleckstrin (PH) and GEF homology domains that are conserved in several proteins implicated in signaling and growth control. Forced expression of wild-type Sbfl in NIH 3T3 cells inhibited their proliferation and altered their morphology. These effects required intact PH, GEF and myotubularin homology domains, implying that growth inhibition may be an intrinsic property of wild-type Sbf1. Conversely, deletion of its conserved N-terminal 44 amino acids alone was sufficient to convert Sbf1 from an inhibitor of cellular growth to a transforming protein in NIH 3T3 cells. Oncogenic forms of Sbf1 partially localized to the nucleus, in contrast to the exclusively cytoplasmic subcellular localization of endogenous Sbf1 in all cell lines and mammalian tissues tested. These data show that the N-terminal GEF homology domain serves to inhibit the transforming effects of Sbf1, possibly sequestering the protein to the cytoplasm, and suggest that this region may be a modulatory domain that relays growth control signals.

Rb targets histone H3 methylation and HP1 to promoters.

In eukaryotic cells the histone methylase SUV39H1 and the methyl-lysine binding protein HP1 functionally interact to repress transcription at heterochromatic sites. Lysine 9 of histone H3 is methylated by SUV39H1 (ref. 2), creating a binding site for the chromo domain of HP1 (refs 3, 4). Here we show that SUV39H1 and HP1 are both involved in the repressive functions of the retinoblastoma (Rb) protein. Rb associates with SUV39H1 and HP1 in vivo by means of its pocket domain. SUV39H1 cooperates with Rb to repress the cyclin E promoter, and in fibroblasts that are disrupted for SUV39, the activity of the cyclin E and cyclin A2 genes are specifically elevated. Chromatin immunoprecipitations show that Rb is necessary to direct methylation of histone H3, and is necessary for binding of HP1 to the cyclin E promoter. These results indicate that the SUV39H1-HP1 complex is not only involved in heterochromatic silencing but also has a role in repression of euchromatic genes by Rb and perhaps other co-repressor proteins.

Set domain-dependent regulation of transcriptional silencing and growth control by SUV39H1, a mammalian ortholog of Drosophila Su(var)3-9.

Mammalian SET domain-containing proteins define a distinctive class of chromatin-associated factors that are targets for growth control signals and oncogenic activation. SUV39H1, a mammalian ortholog of Drosophila Su(var)3-9, contains both SET and chromo domains, signature motifs for proteins that contribute to epigenetic control of gene expression through effects on the regional organization of chromatin structure. In this report we demonstrate that SUV39H1 represses transcription in a transient transcriptional assay when tethered to DNA through the GAL4 DNA binding domain. Under these conditions, SUV39H1 displays features of a long-range repressor capable of acting over several kilobases to silence basal promoters. A possible role in chromatin-mediated gene silencing is supported by the localization of exogenously expressed SUV39H1 to nuclear bodies with morphologic features suggestive of heterochromatin in interphase cells. In addition, we show that SUV39H1 is phosphorylated specifically at the G(1)/S cell cycle transition and when forcibly expressed suppresses cell growth. Growth suppression as well as the ability of SUV39H1 to form nuclear bodies and silence transcription are antagonized by the oncogenic antiphosphatase Sbf1 that when hyperexpressed interacts with the SET domain and stabilizes the phosphorylated form of SUV39H1. These studies suggest a phosphorylation-dependent mechanism for regulating the chromatin organizing activity of a mammalian su(var) protein and implicate the SET domain as a gatekeeper motif that integrates upstream signaling pathways to epigenetic regulation and growth control.

Association of activating transcription factor 2 (ATF2) with the ubiquitin-conjugating enzyme hUBC9. Implication of the ubiquitin/proteasome pathway in regulation of ATF2 in T cells.

Activating transcription factor 2 (ATF2) is regulated by phosphorylation via the Jun N-terminal kinase, and its binding activity is markedly induced at late stages of T and B lymphocyte activation (Feuerstein, N., Firestein, R., Aiyer, N., Xiao, H., Murasko, D., and Cristofalo, V. (1996) J. Immunol. 156, 4582-4593). To identify proteins that interact specifically with ATF2 in lymphocytes, the yeast two-hybrid interaction system was employed using ATF2 cDNA as a "bait." In two separate screenings, a clone was identified that revealed a novel sequence with homology to several members of the ubiquitin-conjugating enzyme family. An identical sequence was recently reported as the human homolog of the yeast UBC9, hUBC9. Northern blot analysis revealed a 1.3-kilobase RNA transcript, which showed differential levels of expression in various human tissues and a moderate induction after a 48-h stimulation of peripheral blood T lymphocytes. An antibody that was generated against the bacterially expressed glutathione S-transferase-hUBC9 detected a approximately 19-kDa protein, which localizes predominantly in the nuclei of T cells. Further quantitative assays using the yeast two-hybrid system confirmed a high and specific level of interaction of hUBC9 with ATF2 and lack of interaction with lamin or control vectors. Two other cyclic AMP-responsive element-binding transcription factors, CREB and ATF1, also showed significant levels of interaction with hUBC9. However, this interaction was severalfold lower as compared with ATF2. Far Western blot analysis confirmed the specific binding of ATF2 and hUBC9 also in vitro. Evidence is presented that indicates a physiological significance for the interaction of hUBC9 with ATF2. (a) We show that ATF2 is ubiquitinated in vivo and in vitro, and (b) ATF2 ubiquitination in vitro is facilitated by addition of purified hUBC9. (c) ATF2 is shown to undergo a proteolytic process, which is rapidly regulated upon T cell activation concomitant with induction of ATF2 phosphorylation. (d) A proteasome inhibitor delays the down-regulation of ATF2 phophorylation after T cell activation. Taken collectively, these results implicate a role for hUBC9 and the ubiquitin/proteasome pathway in regulation of ATF2 in T cells.

Association of SET domain and myotubularin-related proteins modulates growth control.

Several proteins that contribute to epigenetic mechanisms of gene regulation contain a characteristic motif of unknown function called the SET (Suvar3-9, Enhancer-of-zeste, Trithorax) domain. We have demonstrated that SET domains mediate highly conserved interactions with a specific family of proteins that display similarity with dual-specificity phosphatases (dsPTPases). These include myotubularin, the gene of which is mutated in a subset of patients with X-linked myotubular myopathy, and Sbf1, a newly isolated homologue of myotubularin. In contrast with myotubularin, Sbf1 lacks a functional catalytic domain which dephosphorylates phospho-tyrosine and serine-containing peptides in vitro. Competitive interference of endogenous SET domain-dsPTPase interactions by forced expression of Sbf1 induced oncogenic transformation of NIH 3T3 fibroblasts and impaired the in vitro differentiation of C2 myoblast cells. We conclude that myotubularin-type phosphatases link SET-domain containing components of the epigenetic regulatory machinery with signalling pathways involved in growth and differentiation.

Late induction of CREB/ATF binding and a concomitant increase in cAMP levels in T and B lymphocytes stimulated via the antigen receptor.

Transcription factors of the cAMP-responsive element (CRE) binding protein/activating transcription factor (CREB/ATF) family were implicated in the expression of T cell-specific genes and in the expression of oncogenic retroviruses associated with leukemia in T and B lymphocytes. To study the regulation of CREB/ATF transcription factors during lymphocyte activation, studies were pursued in primary cultures of resting murine splenic T and B lymphocytes stimulated via the Ag receptor. Using consensus/CRE and proliferating cell nuclear Ag (PCNA)/CRE as probes in the DNA binding assay, we showed that a marked induction of CRE binding is associated with activation of splenic T lymphocytes with anti-CD3 Ab. CRE binding was markedly induced after 48 h; it gradually declined at 72 h, but remained elevated above control levels after 120 h. Most significant, activation by anti-CD3 was associated with a marked induction of cAMP levels that preceded the onset of DNA synthesis and the induction of IL-2 secretion and reached a peak after 48 h (9.5- to 11-fold), concomitant with the peak in CRE binding. Rapamycin, a potent immunosuppressant, inhibited the induction of cAMP levels by anti-CD3 concomitant with inhibition of CRE binding activity and arrest of DNA synthesis. A marked induction in CRE binding after 48 h was also found in splenic B lymphocytes stimulated by LPS and anti-Ig and was correlated with a 3- to 4-fold increase in the intracellular levels of cAMP. Two inducible CRE complexes were found to bind to consensus/CRE and PCNA/CRE; the major complex contained primarily CREB homodimers and was constitutively expressed in resting lymphocytes. Conversely, stimulation of lymphocytes was associated with formation of a new, slow migrating CRE complex that demonstrated high inducibility in both consensus/CRE and PCNA/CRE. We show that this de novo inducible CRE complex contains CREB and ATF2, but not ATF1. Taken collectively, these results suggest that recruitment of CREB and ATF2 to the promoter of genes is tightly regulated during activation of T and B lymphocytes and implicate a cross-talk of cAMP and non-cAMP pathways in the regulation of transcriptional processes at late stages of activation in T and B lymphocytes stimulated via the Ag receptor.