Search

Chronic lymphocytic leukemia (CLL) is effectively treated with targeted therapies including Bruton tyrosine kinase inhibitors and BCL2 antagonists. When these become ineffective, treatment options are limited. Positive transcription elongation factor complex (P-TEFb), a heterodimeric protein complex composed of cyclin dependent kinase 9 (CDK9) and cyclin T1, functions to regulate short half-life transcripts by phosphorylation of RNA Polymerase II (POLII). These transcripts are frequently dysregulated in hematologic malignancies; however, therapies targeting inhibition of P-TEFb have not yet achieved approval for cancer treatment. VIP152 kinome profiling revealed CDK9 as the main enzyme inhibited at 100 nM, with over a 10-fold increase in potency compared with other inhibitors currently in development for this target. VIP152 induced cell death in CLL cell lines and primary patient samples. Transcriptome analysis revealed inhibition of RNA degradation through the AU-Rich Element (ARE) dysregulation. Mechanistically, VIP152 inhibits the assembly of P-TEFb onto the transcription machinery and disturbs binding partners. Finally, immune competent mice engrafted with CLL-like cells of Eµ-MTCP1 over-expressing mice and treated with VIP152 demonstrated reduced disease burden and improvement in overall survival compared to vehicle-treated mice. These data suggest that VIP152 is a highly selective inhibitor of CDK9 that represents an attractive new therapy for CLL.

Purpose:To report on the first-in-human phase I study of VIP152 (NCT02635672), a potent and highly selective cyclin-dependent kinase 9 (CDK9) inhibitor.Patients and Methods:Adults with solid tumors or aggressive non–Hodgkin lymphoma who were refractory to or had exhausted all available therapies received VIP152 monotherapy as a 30-minute intravenous, once-weekly infusion, as escalating doses (5, 10, 15, 22.5, or 30 mg in 21-day cycles) until the MTD was determined.Results:Thirty-seven patients received ≥ 1 VIP152 dose, with 30 mg identified as the MTD based on dose-limiting toxicity of grade 3/4 neutropenia. The most common adverse events were nausea and vomiting (75.7% and 56.8%, respectively), all of grade 1/2 severity. Of the most common events, grade 3/4 events occurring in > 1 patient were neutropenia (22%), anemia (11%), abdominal pain (8%), increased alkaline phosphatase (8%), and hyponatremia (8%). Day 1 exposure for the MTD exceeded the predicted minimum therapeutic exposure and reproducibly achieved maximal pathway modulation; no accumulation occurred after multiple doses. Seven of 30 patients with solid tumors had stable disease (including 9.5 and 16.8 months in individual patients with pancreatic cancer and salivary gland cancer, respectively), and 2 of 7 patients with high-grade B-cell lymphoma with MYC and BCL2/BCL6 translocations (HGL) achieved durable complete metabolic remission (ongoing at study discontinuation, after 3.7 and 2.3 years of treatment).Conclusions:VIP152 monotherapy, administered intravenously once weekly, demonstrated a favorable safety profile and evidence of clinical benefit in patients with advanced HGL and solid tumors.

Supplementary Table from First-in-Human Dose-Escalation Study of Cyclin-Dependent Kinase 9 Inhibitor VIP152 in Patients with Advanced Malignancies Shows Early Signs of Clinical Efficacy

Supplementary Figure from First-in-Human Dose-Escalation Study of Cyclin-Dependent Kinase 9 Inhibitor VIP152 in Patients with Advanced Malignancies Shows Early Signs of Clinical Efficacy

Purpose: To report on the first-in-human phase I study of VIP152 (NCT02635672), a potent and highly selective cyclin-dependent kinase 9 (CDK9) inhibitor. Patients and Methods: Adults with solid tumors or aggressive non–Hodgkin lymphoma who were refractory to or had exhausted all available therapies received VIP152 monotherapy as a 30-minute intravenous, once-weekly infusion, as escalating doses (5, 10, 15, 22.5, or 30 mg in 21-day cycles) until the MTD was determined. Results: Thirty-seven patients received ≥ 1 VIP152 dose, with 30 mg identified as the MTD based on dose-limiting toxicity of grade 3/4 neutropenia. The most common adverse events were nausea and vomiting (75.7% and 56.8%, respectively), all of grade 1/2 severity. Of the most common events, grade 3/4 events occurring in > 1 patient were neutropenia (22%), anemia (11%), abdominal pain (8%), increased alkaline phosphatase (8%), and hyponatremia (8%). Day 1 exposure for the MTD exceeded the predicted minimum therapeutic exposure and reproducibly achieved maximal pathway modulation; no accumulation occurred after multiple doses. Seven of 30 patients with solid tumors had stable disease (including 9.5 and 16.8 months in individual patients with pancreatic cancer and salivary gland cancer, respectively), and 2 of 7 patients with high-grade B-cell lymphoma with MYC and BCL2/BCL6 translocations (HGL) achieved durable complete metabolic remission (ongoing at study discontinuation, after 3.7 and 2.3 years of treatment). Conclusions: VIP152 monotherapy, administered intravenously once weekly, demonstrated a favorable safety profile and evidence of clinical benefit in patients with advanced HGL and solid tumors.

Background: The Myc transcription factor has a short half-life of 20-30 minutes (Ramsay et al. 1984) and oncogenic activation and transcriptional addiction leads to sustained levels of Myc, (Gabay et al. 2014). Mechanisms of activation include MYC gene amplification, mutation or rearrangement and have been reported to be prevalent in gynecologic malignancies, such as serous ovarian (39.2%), uterine (28.1%) and endometrial (19.5%) (https://www.cancer.gov/tcga). Since CDK9 phosphorylation of RNA polymerase II is required for transcription of Myc mRNA, we evaluate VIP152, a highly selective CDK9 inhibitor (Lücking et al 2021), to deliver antitumor responses in preclinical models of gynecologic malignancies. Methods: Gynecologic cell lines were treated with 9 dose levels of VIP152 with DMSO and cisplatin as controls. IC50 were calculated using CellTiter-Glo luminescent cell viability assay after 72 hours treatment. A preliminary analysis of 19 cell lines was undertaken to determine whether response to VIP152 is associated with baseline mutation, gene expression, or copy number variation data. Monotherapy efficacy of VIP152 was evaluated in a cisplatin sensitive A2780 in vivo xenograft mouse model. Results: Screening of gynecologic cell lines demonstrates a 3-log range of sensitivity with VIP152 IC50s ranging from 38-593nM. Simultaneous cisplatin screening identified sensitive and resistant cell lines in the same panel. Low VIP152 IC50s were observed in cell lines sensitive or resistant to cisplatin. Cell lines were assigned as sensitive and resistant based on the VIP152 IC50 values. Preliminary analysis suggests a gene signature could predict response to VIP152 in gynecologic malignancies. VIP152 was evaluated in an ovarian cancer A2780 in vivo xenograft model. Monotherapy treatment with 4 doses of VIP152 across 5 dose levels from 5- to 15-mg/kg weekly regimens provided increasing tumor growth inhibition compared with vehicle control. Conclusions: VIP152 demonstrates sensitivity in gynecologic cell lines independent of cisplatin sensitivity. An interim gene signature that is associated with VIP152 sensitivity was defined and we plan to optimize this signature with a larger cell line panel. Dose-dependent tumor growth inhibition in an in vivo xenograft model is demonstrated. VIP152 is currently being evaluated in the clinic (ClinicalTrials.gov Identifiers: NCT02635672 and NCT04978779). References: Gabay et al. 2014. Cold Spring Harb Perspect Med. 4(6):a014241. Lücking et al. 2021. J Med Chem. 64(15):11651-11674. Ramsay et al. 1984. PNAS. 81(24):7742-7746. Citation Format: Melanie M. Frigault, Hermes Garban, Joy M. Greer, Stuart Hwang, Raquel Izumi, Amy J. Johnson, Beatrix Stelte-Ludwig, Ahmed Hamdy. VIP152, a selective CDK9 inhibitor, demonstrates sensitivity in gynecologic cell lines that are cisplatin sensitive or resistant and delivers in vivo antitumor efficacy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1859.

Background: Chronic Lymphocytic Leukemia (CLL) is a genetically heterogeneous disease characterized by clonal expansion of B-lymphocytes that induce secondary immune suppression. CLL is now treated with inhibitors of Bruton tyrosine kinase (BTK) and BCL2. Virtually all patients respond to therapy, however resistance to these therapies has been described justifying the need for novel CLL therapies. Broad inhibition of cyclin dependent kinases (CDK) and associated alternative target enzymes with agents such as flavopiridol or dinaciclib have demonstrated significant clinical activity in CLL but are hindered by a relatively narrow therapeutic window. VIP152 is a highly specific inhibitor of CDK9 - considered the most important CDK kinase member for CLL clinical activity. VIP152 has favorable pharmacokinetic properties and has demonstrated durable, preliminary single-agent clinical activity in double-hit diffuse large B-cell lymphoma. Herein, we report the efficacy of VIP152 preclinically in CLL. Methods: On-target activity of VIP152 was measured using a KinomeScan from DiscoveryRx at 100nM and 1000nM. Kinase profiling for VIP152 was performed on 6 kinases in a 10-dose assay at ReactionBio. Cell-based viability and proliferation assays (MTS, Annexin-V (AV), and propidium iodine (PI)) were performed in primary CLL cells and the CLL cell lines, HG3 and MEC1. Transcriptional activity after VIP152 exposure was measured via qPCR and limiting-cell RNA sequencing (lcRNAseq). Proteomic and immunoblot studies were performed to measure perturbations in CDK9 binding partners and on-target activity of VIP152. A genome-wide CRISPR/CAS9 knockout screen was performed to identify any synthetically lethal targets and pathways. Results: The KinomeScan identified CDK9 as the kinase with maximal inhibition upon VIP152 treatment and no other CDKs were identified at 100nM. Kinase profiling revealed the IC 50 of VIP152 was lowest for CDK9/Cyclin T1 and CDK9/Cyclin T2 with close similarity to dinaciclib and greater than 1 log superiority over KB-0742. Co-immunoprecipitation and proteomics experiments have identified a CDK9 specific mechanism of action relating to perturbations of CDK9 binding partners. Specifically, we showed that CDK9 nuclear immunoprecipitation resulted in decreased co-immunoprecipitation of 7SK RNA components (HEXIM1 & MEPCE) as well as decreased RNA Polymerase II (RNAP2). The decrease in RNAP2 CoIP was further seen via proteomics. A 2-hour exposure of VIP152 against HG3 and MEC1 demonstrated growth inhibition, with an IC 50 of 0.9814µM and 1.092µM respectively. Continuous exposure of the compound for 24 hours resulted in a statistically significant drop in relative viability of 30% across a 10-fold dose range (0.1µM to 1.0µM) as measured by AV/PI. Primary CLL cells (n=10) responded with similar dosing strategies with a 54% reduction in viability at 1µM; moreover, stromal cell co-culture experiments demonstrated VIP152's ability to induce cell death and overcome stromal protection with short exposure. Induction of apoptosis was observed with pro-caspase-3 and PARP cleavage on immunoblot. qPCR and immunoblot studies demonstrated a time dependence of phosphorylated serine 2 (pS2) RNAP2 decreases alongside diminishment of MYC and MCL1 mRNA and protein. pS2 was shown to decrease as early as 2 hours after VIP152 treatment with similar decreases in MCL1 and MYC at both the protein and mRNA levels. We identified several pathways which are disrupted following treatment via lcRNAseq, including TNFR1 and TNFR2 signaling as well as upregulation of autophagy signals. Finally, CRISPR screen identified several potentially synergistically lethal targets, including transcriptional co-activators, DNA binding proteins, and cell proliferation pathways. Validation of these is ongoing as is an in vivo study of VIP152 in a CLL mouse model. Conclusions: Our data demonstrate VIP152 to be a highly selective and potent CDK9 inhibitor that disrupts the CDK9 nuclear complex and mediates significant preclinical activity against CLL cell lines and primary CLL cells. VIP152 also demonstrates predictable and new pharmacodynamic markers to assess target engagement. Collectively, these data support the recently initiated CLL clinical trial (NCT04978779). Disclosures Johnson: Vincerx: Current Employment; Janssen: Divested equity in a private or publicly-traded company in the past 24 months, Ended employment in the past 24 months. Frigault: Vincerx Pharma Inc: Current Employment; AstraZeneca: Divested equity in a private or publicly-traded company in the past 24 months, Ended employment in the past 24 months, Patents & Royalties. Greer: Gilead: Current equity holder in publicly-traded company, Ended employment in the past 24 months; Vincerx Pharma Inc: Current Employment. Hamdy: Vincerx Pharma Inc: Current Employment, Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees; Acerta Pharma Inc: Current holder of individual stocks in a privately-held company, Current holder of stock options in a privately-held company, Ended employment in the past 24 months, Patents & Royalties. Izumi: Acerta Pharma Inc: Current holder of individual stocks in a privately-held company, Current holder of stock options in a privately-held company, Ended employment in the past 24 months, Patents & Royalties; Vincerx Pharma Inc: Current Employment, Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees. Hwang: Vincerx Pharma Inc: Current Employment, Current equity holder in publicly-traded company. Blachly: KITE: Consultancy, Honoraria; INNATE: Consultancy, Honoraria; AbbVie: Consultancy, Honoraria; AstraZeneca: Consultancy, Honoraria. Byrd: Vincerx Pharmaceuticals: Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees; Novartis, Trillium, Astellas, AstraZeneca, Pharmacyclics, Syndax: Consultancy, Honoraria; Newave: Membership on an entity's Board of Directors or advisory committees.

Background: High-grade B-cell lymphoma (HGBL), previously known as double-hit lymphoma (DHL), is an aggressive type of B-cell non-Hodgkin lymphoma characterized by rearrangements in MYC and BCL2 or BCL6, which drive oncogenic transcription and anti-apoptotic signaling, respectively, and is refractory to standard of care chemotherapy. VIP152 is a well-tolerated and clinically active CDK9 inhibitor leading to complete metabolic remissions in 2/7 HGBL patients treated with once weekly (QW) 30 mg intravenous administration (Lücking et al. J Med Chem 2021; Moreno et al. JCO 2021 (abstr 7538)). Herein, we demonstrate that once weekly inhibition of CDK9 provides an "oncogenic shock"-like (Jain et al. Science 2002) disruption of transcription of MYC and other short half-life mRNA gene transcripts, which provides durable control of oncogenic protein levels without continuous dosing, resulting in apoptosis and antitumor efficacy. Methods: To understand the mechanism of antitumor efficacy, an evaluation of the pharmacodynamic (PD) extent and duration of CDK9 pathway inhibition was performed in vitro using HGBL cell lines treated with 250 nM VIP152 4 hours (h) pulse and washout time course. PD biomarkers include pSer2 and total RNA Pol II, MYC, MCL1, PCNA and cleaved PARP measured by western blot and RNAseq for transcriptome analysis with confirmation of key short half-life gene transcripts MYC, MCL1 and PCNA by qPCR. The efficacy of VIP152 QW regimen and control following a 2-week regrowth period was evaluated in an SU-DHL-10 in vivo xenograft mouse model. Longitudinal levels of short-lived transcripts compared with the transcriptome were evaluated by RNAseq of whole blood collections predose and postdose on Cycle 1 Day1, Day 8 and Day 15 from 7 HGBL patients treated with 30mg VIP152 QW. Pharmacokinetic /pharmacodynamic (PK/PD) modeling was performed to characterize concentration-effect relationships in 7 HGBL patients for VIP152 on MYC, MCL1 and PCNA mRNA. Results: In vitro treatment with VIP152 shows ≤12h inhibition of p-Ser2 RNAPol II, the direct substrate of CDK9. Stalling of transcriptional activity by RNAPol II was confirmed with a 6-hour depletion of MYC, MCL1 and PCNA mRNA, as measured by qPCR. Transcriptome analysis demonstrates transient control of gene expression by VIP152 is selective for a subset of genes with a short mRNA half-life. Furthermore, ≤16h of depletion of MYC, MCL1 and PCNA protein confirms that a short pulse of VIP152 treatment is sufficient for durable clearance of oncogenic drivers when mRNA levels are transiently downregulated. VIP152-mediated oncogenic shock is characterized by protein depletion rendering cells susceptible to apoptosis with cPARP peaking at 4h after washout. To assess the relevance of MYC depletion, VIP152 was evaluated in a MYC fusion+ DLBCL in vivo model (SU-DHL-10). Dose dependent regressions are achieved following 3 doses of VIP152 with 10- and 15-mg/kg QW regimens providing 81% and 98% tumor growth inhibition, respectively, compared with vehicle control. After 2 weeks regrowth of the 15 mg/kg group, retreatment with 2 doses of VIP152 provides 82% tumor volume reduction demonstrating the QW schedule is sufficient to suppress outgrowth (Figure 1). In HGBL patients (n=7), biomarker modulation is transient with the average maximum postdose depletion of MYC, MCL1 and PCNA mRNA being 94% at 1.4h, 70% at 2.3h and 86% at 4.0h relative to baseline, respectively. Transcriptome analysis confirms that the mechanism of action elucidated from preclinical models translates to patients wherein short half-life transcripts of key oncogenic drivers such as MYC, MCL1 and PCNA are selectively controlled after each of the first 3 QW 30 mg doses of VIP152. Unbound VIP152 IC 50 for MYC, MCL1 and PCNA is 25.4, 30.7, and 20.5 nM, respectively. These are comparable to in vitro DLBCL cell line VIP152 unbound IC 50 of 47 nM. Conclusions: CDK9 inhibition with VIP152 selectively depletes short half-life transcripts including MYC, a known driver of HGBL. Clearance of MYC, MCL1 and PCNA protein demonstrates VIP152 control of multiple parallel oncogenic pathways in the clinic and in HGBL preclinical models. Dose-dependent regression and tumor-outgrowth control demonstrates that VIP152 QW treatment can drive antitumor efficacy. VIP152 is currently being evaluated in HGBL patients and other indications in the clinic (ClinicalTrials.gov Identifiers: NCT02635672 and NCT04978779). Figure 1 Figure 1. Disclosures Frigault: Vincerx Pharma Inc: Current Employment; AstraZeneca: Divested equity in a private or publicly-traded company in the past 24 months, Ended employment in the past 24 months, Patents & Royalties. Wong: Vincerx Pharma Inc: Consultancy; Genentech: Consultancy, Research Funding; Integrative Drug Discovery, ULC: Consultancy. Garban: ImmunityBio, Inc.: Ended employment in the past 24 months, Patents & Royalties; Vincerx Pharma Inc: Current Employment, Current equity holder in publicly-traded company. Greer: Vincerx Pharma Inc: Current Employment; Gilead: Current equity holder in publicly-traded company, Ended employment in the past 24 months. Hwang: Vincerx Pharma Inc: Current Employment, Current equity holder in publicly-traded company. Izumi: Acerta Pharma Inc: Current holder of individual stocks in a privately-held company, Current holder of stock options in a privately-held company, Ended employment in the past 24 months, Patents & Royalties; Vincerx Pharma Inc: Current Employment, Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees. Johnson: Janssen: Divested equity in a private or publicly-traded company in the past 24 months, Ended employment in the past 24 months; Vincerx: Current Employment. Stelte-Ludwig: Bayer: Ended employment in the past 24 months, Patents & Royalties; Vincerx Pharma Inc: Current Employment. Hamdy: Acerta Pharma Inc: Current holder of individual stocks in a privately-held company, Current holder of stock options in a privately-held company, Ended employment in the past 24 months, Patents & Royalties; Vincerx Pharma Inc: Current Employment, Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees.

The Janus kinase 1 (JAK1) pathway has been implicated in the pathogenesis of psoriatic arthritis. We aimed to investigate the efficacy and safety of filgotinib, a selective JAK1 inhibitor, for the treatment of psoriatic arthritis.The EQUATOR trial was a randomised, double-blind, placebo-controlled phase 2 trial that enrolled adults from 25 sites in seven countries (Belgium, Bulgaria, Czech Republic, Estonia, Poland, Spain, and Ukraine). Patients (aged ≥18 years) had active moderate-to-severe psoriatic arthritis (defined as at least five swollen joints and at least five tender joints) fulfilling Classification for psoriatic arthritis (CASPAR) criteria, active or a documented history of plaque psoriasis, and an insufficient response or intolerance to at least one conventional synthetic disease-modifying anti-rheumatic drug (csDMARD). Patients continued to take csDMARDs during the study if they had received this treatment for at least 12 weeks before screening and were on a stable dose for at least 4 weeks before baseline. Using an interactive web-based system, we randomly allocated patients (1:1) to filgotinib 200 mg or placebo orally once daily for 16 weeks (stratified by current use of csDMARDs and previous use of anti-tumour necrosis factor). Patients, study team, and sponsor were masked to treatment assignment. The primary endpoint was proportion of patients achieving 20% improvement in American College of Rheumatology response criteria (ACR20) at week 16 in the full analysis set (patients who received at least one dose of study drug), which was compared between groups with the Cochran-Mantel-Haenszel test and non-responder imputation method. This trial is registered with ClincalTrials.gov, number NCT03101670.Between March 9, and Sept 27, 2017, 191 patients were screened and 131 were randomly allocated to treatment (65 to filgotinib and 66 to placebo). 60 (92%) patients in the filgotinib group and 64 (97%) patients in the placebo group completed the study; five patients (8%) in the filgotinib group and two patients (3%) in the placebo group discontinued treatment. 52 (80%) of 65 patients in the filgotinib group and 22 (33%) of 66 in the placebo group achieved ACR20 at week 16 (treatment difference 47% [95% CI 30·2-59·6], p0·0001). 37 (57%) patients who received filgotinib and 39 (59%) patients who received placebo had at least one treatment-emergent adverse event. Six participants had an event that was grade 3 or worse. The most common events were nasopharyngitis and headache, occurring at similar proportions in each group. One serious treatment-emergent adverse event was reported in each group (pneumonia and hip fracture after a fall), one of which (pneumonia) was fatal in the filgotinib group.Filgotinib is efficacious for the treatment of active psoriatic arthritis, and no new safety signals were identified.Galapagos and Gilead Sciences.

Background: Filgotinib (FIL) is an orally administered, selective inhibitor of Janus Kinase 1 (JAK1) in Phase 3 development for the treatment of rheumatoid arthritis (RA). Objectives: Assess the long-term safety and efficacy of FIL in the DARWIN 3 open-label extension study. Methods: Two 24-week Phase 2b studies were completed in patients (pts) with moderately to severely active RA (DARWIN 1, DARWIN 2; Ref 1, 2). Following study completion, pts were offered FIL in the ongoing DARWIN 3 extension study: 100 mg QD (US males), 200 mg QD, or 100 mg BID. This report summarizes safety data from the first dose of FIL in the DARWIN program to 11 Oct 2017 and efficacy data from the DARWIN 3 baseline visit to Week 108, which all ongoing pts have completed. Results: Of 877 pts, 790 (90%) completed DARWIN 1/2, and 739 (84%) enrolled in DARWIN 3; 603 (82%) were female, mean age 53 years. At analysis, 491/739 (66%) were on study. Cumulative patient years of exposure (PYE) was 1931, median time on study drug was 1072 days. Key data are summarized in table 1. 87%, 68%, and 48% of pts achieved ACR20/50/70, respectively, and 72% achieved DAS28-CRP≤3.2 (by observed case analysis). Conclusions: Filgotinib long-term RA data demonstrates an acceptable safety and durable efficacy profile. References [1]Westhovens R, et al. Ann Rheum Dis2017;76:998–1008. [2]Kavanaugh A, et al. Ann Rheum Dis2017;76:1009–1019. Disclosure of Interest: R. Westhovens Grant/research support from: Galapagos and Celltrion, Roche and BMS, Consultant for: Galapagos and Celltrion, Roche and BMS, R. Alten Grant/research support from: Galapagos/Gilead, K. Winthrop Consultant for: Pfizer, Lilly, Galapagos, Gilead, AbbVie, M. Greenwald Grant/research support from: Gilead, L. Ponce: None declared, F. Enriquez-Sosa: None declared, M. Stanislavchuk: None declared, M. Mazur: None declared, A. Spindler: None declared, R. Cseuz: None declared, N. Nikulenkova: None declared, M. Glowacka-Kulesz: None declared, I. Szombati: None declared, A. Dudek: None declared, N. Mozaffarian Shareholder of: Gilead, Employee of: Gilead, J. Greer Shareholder of: Gilead, Employee of: Gilead, R. Kunder Shareholder of: Gilead, Employee of: Gilead, D. An Shareholder of: Gilead, Employee of: Gilead, P. Harrison Shareholder of: Galapagos, Employee of: Galapagos, A. Van der Aa Shareholder of: Galapagos, Employee of: Galapagos, A. Kavanaugh Consultant for: Galapagos, M. Genovese Consultant for: Gilead, Galapagos, Abbvie, Lilly, Pfizer

Background: At present, biological disease-modifying anti-rheumatic drugs (DMARDs) are the only treatment recommended for patients with ankylosing spondylitis who have not responded to first-line treatment with non-steroidal anti-inflammatory drugs (NSAIDs). The TORTUGA trial investigated the efficacy and safety of filgotinib, an oral selective Janus kinase 1 (JAK1) inhibitor, for the treatment of patients with active ankylosing spondylitis. Methods: In this completed, randomised, double-blind, placebo-controlled, phase 2 trial, we enrolled adult patients from 30 sites in seven countries (Belgium, Bulgaria, Czech Republic, Estonia, Poland, Spain, and Ukraine). Eligible patients had active ankylosing spondylitis and an inadequate response or intolerance to two or more NSAIDs. Patients were randomly assigned (1:1) with an interactive web-based response system to receive filgotinib 200 mg or placebo orally once daily for 12 weeks. Randomisation was stratified by current use of conventional synthetic DMARDs and previous receipt of anti-tumour necrosis factor therapy. The patients, study team, and study sponsor were masked to treatment assignment. The primary endpoint was the change from baseline in ankylosing spondylitis disease activity score (ASDAS) at week 12, which was assessed in the full analysis set (ie, all randomised patients who received at least one dose of study drug). Safety was assessed according to actual treatment received. This trial is registered with ClinicalTrials.gov, number NCT03117270. Findings: Between March 7, 2017, and July 2, 2018, 263 patients were screened and 116 randomly assigned to filgotinib (n=58) or placebo (n=58). 55 (95%) patients in the filgotinib group and 52 (90%) in the placebo group completed the study; three (5%) patients in the filgotinib group and six (10%) in the placebo group discontinued treatment. The mean ASDAS change from baseline to week 12 was −1·47 (SD 1·04) in the filgotinib group and −0·57 (0·82) in the placebo group, with a least squares mean difference between groups of −0·85 (95% CI −1·17 to −0·53; p

Background Filgotinib (FIL) is an oral, selective Janus kinase inhibitor, shown to be effective and well tolerated in patients (pt) with rheumatoid arthritis (RA) and other inflammatory diseases (IBD/AS/PsA). DARWIN 3, an ongoing, open-label, long-term extension of phase 2b studies evaluates the longer-term safety and efficacy of FIL in RA. Methods The study evaluated pt outcomes for methotrexate (MTX) inadequate responders completing the 24-week DARWIN 1 (FIL+MTX) and DARWIN 2 (FIL monotherapy) studies. We present an interim analysis at week 156 following FIL 200 or 100 mg/day treatment. Event rate: total events/total years of exposure of FIL; Exposure: until data cut-off in patients on the study at the time of analysis. Results Eight hundred and seventy-seven patients completed the parent studies. Seven hundred and thirty-nine enrolled in DARWIN 3 (497 DARWIN 1, 242 DARWIN 2); most DARWIN 1 and 2 patients were female (81.5%, 81.8%), white (75.3%, 74.8%); mean age was 53 and 52 years, respectively. Mean baseline MTX dose in the FIL+MTX group: 16.8mg/week. At week 156, 59.9% of patients remained on study. Most common reasons for discontinuation were adverse events (26.5%) and subject request (9.1%). Total exposure to FIL was 2203 pt-years; mean exposure ± standard deviation (SD): 3.04 ± 1.22 years for FIL+MTX and 2.86 ± 1.21 years for FIL monotherapy. Treatment-emergent adverse events (TEAEs): 419 (84.3%) patients on FIL+MTX and 203 (83.9%) patients on FIL monotherapy; serious TEAEs occurred in 45 (9.1%) and 33 (13.6%), respectively. Adverse event of special interest (AESI) rates remained low at week 156 (Table 1). Grade ≥3 toxicities in >1% of patients were decreased lymphocytes (4.2%/1%), decreased neutrophils (1.0/1.2%), increased fasting triglycerides (2.2/0%) and decreased fasting triglycerides (0/3.3%) for FIL+MTX/FIL monotherapy respectively. 5 deaths occurred (FIL+MTX: 2, FIL monotherapy: 3) none after week 132. Clinical efficacy was shown at week 156 in both FIL+MTX and FIL monotherapy groups, using observed cases as measured by ACR20 (87.2% and 89.7%)/ACR50 (72.4 and 63.0)/ACR70 (45.5 and 40.0) DAS28(CRP) ≤3.2 (69.0 and 64.7) and DAS28(CRP) Conclusion FIL was generally well tolerated; no new safety signals emerged. No safety differences observed in patients receiving FIL+MTX or FIL monotherapy. Efficacy was sustained up to week 156 in both treatment groups.

A procedure is described that directs the self-induced deletion of DNA sequences as they pass through the male germ line of mice. The testes-specific promoter from the angiotensin-converting enzyme gene was used to drive expression of the Cre-recombinase gene. Cre was linked to the selectable marker Neor, and the two genes flanked with loxP elements. This cassette was targeted to the Hoxa3 gene in mouse ES cells that were in turn used to generate chimeric mice. In these chimeras, somatic cells derived from the ES cells retained the cassette, but self-excision occurred in all ES-cell-derived sperm.

4130Background: The importance of the tumor microenvironment in the biology of mPC has been increasingly recognized. However, immune checkpoint blockade has previously shown limited clinical respon...

Dishevelled (Dvl) proteins are important signaling components of both the canonical β-catenin/Wnt pathway, which controls cell proliferation and patterning, and the planar cell polarity (PCP) pathway, which coordinates cell polarity within a sheet of cells and also directs convergent extension cell (CE) movements that produce narrowing and elongation of the tissue. Three mammalian Dvl genes have been identified and the developmental roles of Dvl1 and Dvl2 were previously determined. Here, we identify the functions of Dvl3 in development and provide evidence of functional redundancy among the three murine Dvls. Dvl3 −/− mice died perinatally with cardiac outflow tract abnormalities, including double outlet right ventricle and persistent truncus arteriosis. These mutants also displayed a misorientated stereocilia in the organ of Corti, a phenotype that was enhanced with the additional loss of a single allele of the PCP component Vangl2/Ltap (LtapLp/+). Although neurulation appeared normal in both Dvl3 −/− and LtapLp/+ mutants, Dvl3 +/−;LtapLp/+ combined mutants displayed incomplete neural tube closure. Importantly, we show that many of the roles of Dvl3 are also shared by Dvl1 and Dvl2. More severe phenotypes were observed in Dvl3 mutants with the deficiency of another Dvl, and increasing Dvl dosage genetically with Dvl transgenes demonstrated the ability of Dvls to compensate for each other to enable normal development. Interestingly, global canonical Wnt signaling appeared largely unaffected in the double Dvl mutants, suggesting that low Dvl levels are sufficient for functional canonical Wnt signals. In summary, we demonstrate that Dvl3 is required for cardiac outflow tract development and describe its importance in the PCP pathway during neurulation and cochlea development. Finally, we establish several developmental processes in which the three Dvls are functionally redundant., Author Summary Multi-gene families, comprising a set of very similar genes with shared nucleotide sequences, are common in mammals. Individual family members may be expressed in different places and perform separate functions. Alternatively, the genes may have redundant functions, but distinct dosage requirements. Mammals share three Dishevelled (Dvl) family members and while the roles of Dvl1 and Dvl2 have been described previously, the functions of Dvl3 have remained elusive. Here, we show that the lack of Dvl3 in mice affects the formation of the heart, neural tube, and inner ear. We further show that the defects in these tissues are much more severe when the mice are deficient in more than one Dvl family member, indicating redundant functions for these genes. Congenital heart disease affects approximately 75 in every 1,000 live human births, and approximately 30% of these diseases are due to disruptions in the outflow tract, the region affected in mice lacking Dvl genes. Neural tube defects, similar to those observed in the Dvl mutants, are also common in humans. The animal models described here provide useful tools to elucidate the genetic mechanisms that underlie these abnormalities and may provide novel ways of treating these disorders in the future.

Repertoires of grooming behaviors critical to survival are exhibited by most animal species, including humans. Genes that influence this complex behavior are unknown. We report that mice with disruptions of Hoxb8 show, with 100% penetrance, excessive grooming leading to hair removal and lesions. Additionally, these mice excessively groom normal cagemates. We have been unable to detect any skin or PNS abnormalities in Hoxb8 mutants. These observations suggest that the excessive, pathological grooming exhibited by these mice results from CNS abnormalities. Consistent with this interpretation, we demonstrate Hoxb8 expression in regions of the adult mouse CNS previously implicated in the control of grooming. The aberrant behavior observed in Hoxb8 mutants is not unlike that of humans suffering from the OC-spectrum disorder, trichotillomania. Interestingly, Hoxb8 is expressed in regions of the CNS known as the “OCD-circuit.”

Mice were generated with targeted disruptions in Hoxa7 and Hoxb7 , respectively. Mice carrying the Hoxa7 mutation are healthy. No abnormalities in the formation of the skeleton or other tissues were found in these mutants. Twelve percent of Hoxb7 −/− mutants show first and second rib defects similar to those observed in mice homozygous for a Hoxb9 mutation (Chen, F., Capecchi, M.R., 1997. Dev. Biol. 181, 186–196). Hoxb7 −/− mice are also fertile and were used to generate double mutants with Hoxa7 to reveal potential interactions between these two paralogous genes. Mice homozygous for both mutations have first and second rib defects with higher penetrance and increased expressivity, indicating a functional role for Hoxa7 in the patterning of the upper thoracic region. Although Hoxb6 , Hoxa7 , Hoxb7 , and Hoxb9 have distinctive anterior expression limits in axial mesoderm, the disruptions of these genes all yield first and second rib defects. A hypothesis is suggested to explain the observation that axial defects in these and other mouse Hox mutants appear to concentrate along the axial column at zones of transition between vertebral types.

The Leishmania GP63 major surface protein gene family encodes multiple isoforms which differ predominantly in the carboxyterminal region. We have isolated 4 full-length gp63 cDNA clones derived from stationary-phase promastigote RNA of a cloned isolate of Leishmania guyanensis , a member of the braziliensis complex. These genes, along with the previously published L. guyanensis gp63 gene sequence [15], appeared to be mosaics of different combination of 5′ and 3′ untranslated regions and sequences encoding the propeptide, internal, and C-terminal regions of GP63. The predicted L. guyanensis GP63 isoforms shared as little as 55% sequence identity, comparable to the inter-species diversity of Gp63. The genomic organization of gp63 genes in L. guyanensis is highly complex: there are at least 4 distinct polymorphic forms of tandemly linked gene clusters, with intra-gene cluster variation in gene sequence and in the number of gene repeats. Southern blot analysis suggested that the arrangement of gp63 genes in this L. guyanensis isolate did not differ from that in the parental lines.