Inhibiting PRMT5 induces DNA damage and increases anti-proliferative activity of Niraparib, a PARP inhibitor, in models of breast and ovarian cancer.

BACKGROUND: Inhibitors of Poly (ADP-Ribose) Polymerases (PARP) provide clinical benefit to patients with breast and ovarian cancers, by compromising the DNA repair activity of cancer cells. Although these agents extend progression-free survival in many patients, responses can be short lived with many patients ultimately progressing. Identification of combination partners that increase dependence of cancer cells to the DNA repair activity of PARPs may represent a strategy to increase the utility of PARP inhibitors. Protein arginine methyltransferase 5 (PRMT5) regulates DNA damage response pathways through splicing and protein modification, and inhibitors of PRMT5 have recently entered clinical trials. METHODS: The effect of PRMT5 inhibition on the levels of DNA damage and repair markers including γH2AX, RAD51, and 53BP1 was determined using high content immunofluorescent imaging. The anti-proliferative activity of the combination of PRMT5 and PARP inhibitors was evaluated using in vitro models of breast and ovarian cancers using both cell lines and ex vivo patient derived xenografts. Finally, the combinations of PRMT5 and PARP inhibitors were evaluated in cell line xenograft models in vivo. RESULTS: Inhibition of PRMT5 by GSK3326595 led to increased levels of markers of DNA damage. The addition of GSK3326595 to the PARP inhibitor, niraparib, resulted in increased growth inhibition of breast and ovarian cancer cell lines and patient derived spheroids. In vivo, the combination improved the partial effects on tumor growth inhibition achieved by either single agent, producing complete tumor stasis and regression. CONCLUSION: These data demonstrate that inhibition of PRMT5 induced signatures of DNA damage in models of breast and ovarian cancer. Furthermore, combination with the PARP inhibitor, Niraparib, resulted in increased anti-tumor activity in vitro and in vivo. Overall, these data suggest inhibition of PRMT5 as a mechanism to broaden and enhance the clinical application of PARP inhibitors.

GSK137, a potent small-molecule BCL6 inhibitor with in vivo activity, suppresses antibody responses in mice.

B-cell lymphoma 6 (BCL6) is a zinc finger transcriptional repressor possessing a BTB-POZ (BR-C, ttk, and bab for BTB; pox virus and zinc finger for POZ) domain, which is required for homodimerization and association with corepressors. BCL6 has multiple roles in normal immunity, autoimmunity, and some types of lymphoma. Mice bearing disrupted BCL6 loci demonstrate suppressed high-affinity antibody responses to T-dependent antigens. The corepressor binding groove in the BTB-POZ domain is a potential target for small compound-mediated therapy. Several inhibitors targeting this binding groove have been described, but these compounds have limited or absent in vivo activity. Biophysical studies of a novel compound, GSK137, showed an in vitro pIC50 of 8 and a cellular pIC50 of 7.3 for blocking binding of a peptide derived from the corepressor silencing mediator for retinoid or thyroid hormone receptors to the BCL6 BTB-POZ domain. The compound has good solubility (128 µg/ml) and permeability (86 nM/s). GSK137 caused little change in cell viability or proliferation in four BCL6-expressing B-cell lymphoma lines, although there was modest dose-dependent accumulation of G1 phase cells. Pharmacokinetic studies in mice showed a profile compatible with achieving good levels of target engagement. GSK137, administered orally, suppressed immunoglobulin G responses and reduced numbers of germinal centers and germinal center B cells following immunization of mice with the hapten trinitrophenol. Overall, we report a novel small-molecule BCL6 inhibitor with in vivo activity that inhibits the T-dependent antigen immune response.

Safety, pharmacokinetic, pharmacodynamic and clinical activity of molibresib for the treatment of nuclear protein in testis carcinoma and other cancers: Results of a Phase I/II open-label, dose escalation study.

Molibresib is an orally bioavailable, selective, small molecule BET protein inhibitor. Results from a first time in human study in solid tumors resulted in the selection of a 75 mg once daily dose of the besylate formulation of molibresib as the recommended Phase 2 dose (RP2D). Here we present the results of Part 2 of our study, investigating safety, pharmacokinetics, pharmacodynamics and clinical activity of molibresib at the RP2D for nuclear protein in testis carcinoma (NC), small cell lung cancer, castration-resistant prostate cancer (CRPC), triple-negative breast cancer, estrogen receptor-positive breast cancer and gastrointestinal stromal tumor. The primary safety endpoints were incidence of adverse events (AEs) and serious AEs; the primary efficacy endpoint was overall response rate. Secondary endpoints included plasma concentrations and gene set enrichment analysis (GSEA). Molibresib 75 mg once daily demonstrated no unexpected toxicities. The most common treatment-related AEs (any grade) were thrombocytopenia (64%), nausea (43%) and decreased appetite (37%); 83% of patients required dose interruptions and 29% required dose reductions due to AEs. Antitumor activity was observed in NC and CRPC (one confirmed partial response each, with observed reductions in tumor size), although predefined clinically meaningful response rates were not met for any tumor type. Total active moiety median plasma concentrations after single and repeated administration were similar across tumor cohorts. GSEA revealed that gene expression changes with molibresib varied by patient, response status and tumor type. Investigations into combinatorial approaches that use BET inhibition to eliminate resistance to other targeted therapies are warranted.

Limited antitumor activity of combined BET and MEK inhibition in neuroblastoma.

BACKGROUND: The treatment of high-risk neuroblastoma continues to present a formidable challenge to pediatric oncology. Previous studies have shown that Bromodomain and extraterminal (BET) inhibitors can inhibit MYCN expression and suppress MYCN-amplified neuroblastoma in vivo. Furthermore, alterations within RAS-MAPK (mitogen-activated protein kinase) signaling play significant roles in neuroblastoma initiation, maintenance, and relapse, and mitogen-activated extracellular signal-regulated kinase (MEK) inhibitors demonstrate efficacy in subsets of neuroblastoma preclinical models. Finally, hyperactivation of RAS-MAPK signaling has been shown to promote resistance to BET inhibitors. Therefore, we examined the antitumor efficacy of combined BET/MEK inhibition utilizing I-BET726 or I-BET762 and trametinib in high-risk neuroblastoma. PROCEDURE: Utilizing a panel of genomically annotated neuroblastoma cell line models, we investigated the in vitro effects of combined BET/MEK inhibition on cell proliferation and apoptosis. Furthermore, we evaluated the effects of combined inhibition in neuroblastoma xenograft models. RESULTS: Combined BET and MEK inhibition demonstrated synergistic effects on the growth and survival of a large panel of neuroblastoma cell lines through augmentation of apoptosis. A combination therapy slowed tumor growth in a non-MYCN-amplified, NRAS-mutated neuroblastoma xenograft model, but had no efficacy in an MYCN-amplified model harboring a loss-of-function mutation in NF1. CONCLUSIONS: Combinatorial BET and MEK inhibition was synergistic in the vast majority of neuroblastoma cell lines in the in vitro setting but showed limited antitumor activity in vivo. Collectively, these data do not support clinical development of this combination in high-risk neuroblastoma.

A Phase I/II Study of GSK525762 Combined with Fulvestrant in Patients with Hormone Receptor-positive/HER2-negative Advanced or Metastatic Breast Cancer.

PURPOSE: Endocrine-based therapy is the initial primary treatment option for hormone receptor-positive and human epidermal growth factor receptor 2-negative (HR+/HER2-) metastatic breast cancer (mBC). However, patients eventually experience disease progression due to resistance to endocrine therapy. Molibresib (GSK525762) is a small-molecule inhibitor of bromodomain and extraterminal (BET) family proteins (BRD2, BRD3, BRD4, and BRDT). Preclinical data suggested that the combination of molibresib with endocrine therapy might overcome endocrine resistance. This study aimed to investigate the safety, tolerability, pharmacokinetics, pharmacodynamics, and efficacy [objective response rate (ORR)] of molibresib combined with fulvestrant in women with HR+/HER2- mBC. PATIENTS AND METHODS: In this phase I/II dose-escalation and dose-expansion study, patients received oral molibresib 60 or 80 mg once daily in combination with intramuscular fulvestrant. Patients enrolled had relapsed/refractory, advanced/metastatic HR+/HER2- breast cancer with disease progression on prior treatment with an aromatase inhibitor, with or without a cyclin-dependent kinase 4/6 inhibitor. RESULTS: The study included 123 patients. The most common treatment-related adverse events (AE) were nausea (52%), dysgeusia (49%), and fatigue (45%). At a 60-mg dosage of molibresib, >90% of patients experienced treatment-related AE. Grade 3 or 4 treatment-related AE were observed in 47% and 48% of patients treated with molibresib 60 mg and molibresib 80 mg, respectively. The ORR was 13% [95% confidence interval (CI), 8-20], not meeting the 25% threshold for proceeding to phase II. Among 82 patients with detected circulating tumor DNA and clinical outcome at study enrollment, a strong association was observed between the detection of copy-number amplification and poor progression-free survival (HR, 2.89; 95% CI, 1.73-4.83; P < 0.0001). CONCLUSIONS: Molibresib in combination with fulvestrant did not demonstrate clinically meaningful activity in this study.

A Phase I/II Open-Label Study of Molibresib for the Treatment of Relapsed/Refractory Hematologic Malignancies.

PURPOSE: Molibresib is a selective, small molecule inhibitor of the bromodomain and extra-terminal (BET) protein family. This was an open-label, two-part, Phase I/II study investigating molibresib monotherapy for the treatment of hematological malignancies (NCT01943851). PATIENTS AND METHODS: Part 1 (dose escalation) determined the recommended Phase 2 dose (RP2D) of molibresib in patients with acute myeloid leukemia (AML), Non-Hodgkin lymphoma (NHL), or multiple myeloma. Part 2 (dose expansion) investigated the safety and efficacy of molibresib at the RP2D in patients with relapsed/refractory myelodysplastic syndrome (MDS; as well as AML evolved from antecedent MDS) or cutaneous T-cell lymphoma (CTCL). The primary endpoint in Part 1 was safety and the primary endpoint in Part 2 was objective response rate (ORR). RESULTS: There were 111 patients enrolled (87 in Part 1, 24 in Part 2). Molibresib RP2Ds of 75 mg daily (for MDS) and 60 mg daily (for CTCL) were selected. Most common Grade 3+ adverse events included thrombocytopenia (37%), anemia (15%), and febrile neutropenia (15%). Six patients achieved complete responses [3 in Part 1 (2 AML, 1 NHL), 3 in Part 2 (MDS)], and 7 patients achieved partial responses [6 in Part 1 (4 AML, 2 NHL), 1 in Part 2 (MDS)]. The ORRs for Part 1, Part 2, and the total study population were 10% [95% confidence interval (CI), 4.8-18.7], 25% (95% CI, 7.3-52.4), and 13% (95% CI, 6.9-20.6), respectively. CONCLUSIONS: While antitumor activity was observed with molibresib, use was limited by gastrointestinal and thrombocytopenia toxicities. Investigations of molibresib as part of combination regimens may be warranted.

Fragment-based Scaffold Hopping: Identification of Potent, Selective, and Highly Soluble Bromo and Extra Terminal Domain (BET) Second Bromodomain (BD2) Inhibitors.

The profound efficacy of pan-BET inhibitors is well documented, but these epigenetic agents have shown pharmacology-driven toxicity in oncology clinical trials. The opportunity to identify inhibitors with an improved safety profile by selective targeting of a subset of the eight bromodomains of the BET family has triggered extensive medicinal chemistry efforts. In this article, we disclose the identification of potent and selective drug-like pan-BD2 inhibitors such as pyrazole 23 (GSK809) and furan 24 (GSK743) that were derived from the pyrrole fragment 6. We transpose the key learnings from a previous pyridone series (GSK620 2 as a representative example) to this novel class of inhibitors, which are characterized by significantly improved solubility relative to our previous research.

Phase 1 Study of Molibresib (GSK525762), a Bromodomain and Extra-Terminal Domain Protein Inhibitor, in NUT Carcinoma and Other Solid Tumors.

BACKGROUND: Bromodomain and extra-terminal domain proteins are promising epigenetic anticancer drug targets. This first-in-human study evaluated the safety, recommended phase II dose, pharmacokinetics, pharmacodynamics, and preliminary antitumor activity of the bromodomain and extra-terminal domain inhibitor molibresib (GSK525762) in patients with nuclear protein in testis (NUT) carcinoma (NC) and other solid tumors. METHODS: This was a phase I and II, open-label, dose-escalation study. Molibresib was administered orally once daily. Single-patient dose escalation (from 2 mg/d) was conducted until the first instance of grade 2 or higher drug-related toxicity, followed by a 3 + 3 design. Pharmacokinetic parameters were obtained during weeks 1 and 3. Circulating monocyte chemoattractant protein-1 levels were measured as a pharmacodynamic biomarker. RESULTS: Sixty-five patients received molibresib. During dose escalation, 11% experienced dose-limiting toxicities, including six instances of grade 4 thrombocytopenia, all with molibresib 60-100 mg. The most frequent treatment-related adverse events of any grade were thrombocytopenia (51%) and gastrointestinal events, including nausea, vomiting, diarrhea, decreased appetite, and dysgeusia (22%-42%), anemia (22%), and fatigue (20%). Molibresib demonstrated an acceptable safety profile up to 100 mg; 80 mg once daily was selected as the recommended phase II dose. Following single and repeat dosing, molibresib showed rapid absorption and elimination (maximum plasma concentration: 2 hours; t1/2: 3-7 hours). Dose-dependent reductions in circulating monocyte chemoattractant protein-1 levels were observed. Among 19 patients with NC, four achieved either confirmed or unconfirmed partial response, eight had stable disease as best response, and four were progression-free for more than 6 months. CONCLUSIONS: Once-daily molibresib was tolerated at doses demonstrating target engagement. Preliminary data indicate proof-of-concept in NC.

H2B ubiquitin protease Ubp8 and Sgf11 constitute a discrete functional module within the Saccharomyces cerevisiae SAGA complex.

The SAGA complex is a multisubunit protein complex involved in transcriptional regulation in Saccharomyces cerevisiae. SAGA combines proteins involved in interactions with DNA-bound activators and TATA-binding protein (TBP), as well as enzymes for histone acetylation (Gcn5) and histone deubiquitylation (Ubp8). We recently showed that H2B ubiquitylation and Ubp8-mediated deubiquitylation are both required for transcriptional activation. For this study, we investigated the interaction of Ubp8 with SAGA. Using mutagenesis, we identified a putative zinc (Zn) binding domain within Ubp8 as being critical for the association with SAGA. The Zn binding domain is required for H2B deubiquitylation and for growth on media requiring Ubp8's function in gene activation. Furthermore, we identified an 11-kDa subunit of SAGA, Sgf11, and showed that it is required for the Ubp8 association with SAGA and for H2B deubiquitylation. Different approaches indicated that the functions of Ubp8 and Sgf11 are related and separable from those of other components of SAGA. In particular, the profiles of Ubp8 and Sgf11 deletions were remarkably similar in microarray analyses and synthetic genetic interactions and were distinct from those of the Spt3 and Spt8 subunits of SAGA, which are involved in TBP regulation. These data indicate that Ubp8 and Sgf11 likely represent a new functional module within SAGA that is involved in gene regulation through H2B deubiquitylation.

Structure and dimerization of the kinase domain from yeast Snf1, a member of the Snf1/AMPK protein family.

The Snf1/AMPK kinases are intracellular energy sensors, and the AMPK pathway has been implicated in a variety of metabolic human disorders. Here we report the crystal structure of the kinase domain from yeast Snf1, revealing a bilobe kinase fold with greatest homology to cyclin-dependant kinase-2. Unexpectedly, the crystal structure also reveals a novel homodimer that we show also forms in solution, as demonstrated by equilibrium sedimentation, and in yeast cells, as shown by coimmunoprecipitation of differentially tagged intact Snf1. A mapping of sequence conservation suggests that dimer formation is a conserved feature of the Snf1/AMPK kinases. The conformation of the conserved alphaC helix, and the burial of the activation segment and substrate binding site within the dimer, suggests that it represents an inactive form of the kinase. Taken together, these studies suggest another layer of kinase regulation within the Snf1/AMPK family, and an avenue for development of AMPK-specific activating compounds.