ALBERT

Description: Background: Unlike mammals, zebrafish have the ability to regenerate damaged parts of their central nervous system (CNS) and regain functionality of the affected area. A better understanding of the molecular mechanisms involved in zebrafish regeneration may therefore provide insight into how CNS repair might be induced in mammals. Although many studies have described differences in gene expression in zebrafish during CNS regeneration, the regulatory mechanisms underpinning the differential expression of these genes have not been examined. Results: We used microarrays to analyse and integrate the mRNA and microRNA (miRNA) expression profiles of zebrafish retina after optic nerve crush to identify potential regulatory mechanisms that underpin central nerve regeneration. Bioinformatic analysis identified 3 miRNAs and 657 mRNAs that were differentially expressed after injury. We then combined inverse correlations between our miRNA expression and mRNA expression, and integrated these findings with target predictions from TargetScan Fish to identify putative miRNA-gene target pairs. We focused on two over-expressed miRNAs (miR-29b and miR-223), and functionally validated seven of their predicted gene targets using RT-qPCR and luciferase assays to confirm miRNA-mRNA binding. Gene ontology analysis placed the miRNA-regulated genes (eva1a, layna, nefmb, ina, si:ch211-51a6.2, smoc1, sb:cb252) in key biological processes that included cell survival/apoptosis, ECM-cytoskeleton signaling, and heparan sulfate proteoglycan binding, Conclusion: Our results suggest a key role for miR-29b and miR-223 in zebrafish regeneration. The identification of miRNA regulation in a zebrafish injury model provides a framework for future studies in which to investigate not only the cellular processes required for CNS regeneration, but also how these mechanisms might be regulated to promote successful repair and return of function in the injured mammalian brain.

Description: Background MF, PV, and ET are neoplasms characterized by activation of JAK2 signaling, and the majority of cases are associated with the gain of function JAK2 V617F mutation. Given that wild-type JAK2 plays a pivotal role in multiple stages of hematopoiesis it is desirable to treat JAK2V617F-positive cases by selectively inhibiting JAK2 V617F while minimizing inhibition of wild-type JAK2 in order not to impede normal marrow function. LY2784544 is a selective inhibitor of JAK2 which demonstrates dose dependent selectivity for JAK2 V617F/STAT5 signaling. LY2784544 entered clinical testing in a Phase I trial in MF, PV, and ET patients (I3X-MC-JHTA, NCT01134120). We have previously reported early interim analysis from this study and provide here an update following completion of study enrollment. Methods The study’s primary objectives were to determine the safety and tolerability of LY2784544 and define a recommended oral daily dose for further study in patients with JAK2 V617F-positive MF, ET, or PV. Secondary objectives included determination of pharmacokinetics and evaluation of response using the IWG-MRT response criteria (2006), European Leukemia Net response criteria for PV and ET (EULN 2009), and symptom burden using the Myeloproliferative Neoplasm Symptom Assessment Form (MPN-SAF) (Scherber et. al. Blood 2011). Study design included a standard 3+3 dose-escalation to define the maximum tolerated dose (MTD) and was amended to allow cohort expansions at multiple doses demonstrating potential clinical merit. Results Patients Prior to closing to enrollment, 47 patients were consented; 9 did not meet eligibility screening. The 38 treated patients included 31 MF (22 pMF, 6 post-PV MF and 3 post-ET MF), 6 PVs, and 1 ET patient(s), and 86% had received prior systemic therapy for their MPN. Dosing and tolerability of LY2784544: Systemic exposures were approximately dose proportional across the tested range from 30 to 300 mg. Daily doses of 200 mg or greater were associated with CTCAE Grade 3 increases in serum creatinine. In general, increases in serum creatinine occurred within the first 2 weeks of either starting treatment or after an increase in study drug dose. With the available data, the elevations in serum creatinine appear to be fully reversible after dose reduction or drug holiday in the majority of patients. The maximum tolerated daily dose of LY2784544 when given on a continuous daily basis was determined to be 120 mg per day. Serious AEs, across all grades and doses, included increased creatinine/acute renal insufficiency (4 grade 3, 5 grade 2, 1 grade 1), hyperuricemia (2 grade 4, 1 grade 1), hyperkalemia (1 grade 1), and anemia (1 grade 3). This study allowed intrapatient dose escalation, and 34 patients received 120 mg during their study participation. There were 4 patients dosed at 120 mg with SAEs but all were unrelated to study drug treatment (anemia, influenza, enterococcal bacteremia, hematuria and pneumonia). At 120 mg, the most frequently reported drug-related AEs across all grades were diarrhea (44%), nausea (29%), increased creatinine (21%), and anemia, vomiting, fatigue (9% each). Of these related AEs at 120 mg, there was 1 Grade 3 anemia, 3 Grade 3 increased creatinine, and 2 Grade 3 fatigue. There were no Grade 4 AEs. Efficacy Among a cohort of 10 MF patients who only received 120 mg, 3 achieved a clinical improvement. Analyzing across dose levels, a palpable spleen length reduction of ≥50% was seen in 15 of 27 evaluable patients (56%) at any time. The median duration of these responses was 18.3 weeks (range from 0.7 to 148.1 weeks). At 12 weeks, 56% of patients (15/27) reported a ≥50% improvement in Total Symptom Score on the MPN-SAF. Of the 6 PV patients, 3 achieved a clinicohematologic PR. Findings from a central pathology review of serial bone marrow samples will be presented at the meeting. Conclusions A recommended phase II dose for daily dosing of LY2784544 was identified as 120 mg. This dose has been well tolerated and associated with clinical improvements, supporting ongoing Phase II testing of LY2784544 in MPNs. Disclosures: Verstovsek: Eli Lilly and Co: Research Funding. Mesa:Eli Lilly and Co: Research Funding; Genetech: Research Funding; Gilead: Research Funding; Incyte: Research Funding; Sanofi: Research Funding; NS pharma: Research Funding; Celgene: Research Funding. Salama:Eli Lilly and Co: Research Funding. Giles:Eli Lilly and Co: Employment. Pitou:Eli Lilly and Co: Employment. Hayden Zimmermann:Eli Lilly and Co: Employment. Price:Eli Lilly and Co: Employment. Walgreen:Eli Lilly and Co: Employment. Prchal:Eli Lilly and Co: Research Funding.

Description: Abstract 2814 Background: The majority of primary and secondary MF, PV, and ET neoplasms are characterized by gain-of-function activation of JAK2 V617F/STAT5 signaling. Given that wild-type JAK2 plays a pivotal role in multiple stages of hematopoiesis it is desirable to treat JAK2 V617F-positive cases by selectively inhibiting JAK2 V617F while minimizing inhibition of wild-type JAK2 in order not to impede normal marrow function. In murine MPN models and in vitro cell based assays, a novel JAK2 inhibitor, LY2784544, demonstrates selective inhibition of JAK2 V617F/STAT5 signaling and mutant cell proliferation (Ma et al. Abstract 4087 ASH 2010). These results provided the basis for the LY2784544 Phase I trial in MF, PV, and ET patients (I3X-MC-JHTA, NCT01134120). Methods: The primary objectives are to determine the safety and tolerability of LY2784544 and define a recommended oral daily dose for further study in patients with JAK2 V617F-positive MF, ET, or PV. Secondary objectives include determination of pharmacokinetics and evaluation of response using the IWG-MRT response criteria, European Leukemia Net response criteria for PV and ET (EULN), and symptom burden using the Myeloproliferative Neoplasm Symptom Assessment Form (MPN-SAF) (Scherber et. al. Blood 2011). The study has a standard 3+3 dose-escalation design to define the maximum tolerated dose (MTD). The study was recently amended to allow cohort expansions at lower than MTD doses demonstrating potential clinical merit. Results: To date, 19 patients have been treated: 1 PV, 1 post-ET MF, and 17 MF patients. Four patients discontinued therapy, 1 for each of the following: Adverse Event, progressive disease, investigator decision, and subject's decision. Reported drug-related serious adverse events were seen in 4 patients, and include: creatinine increase (n=4 grade 2), hyperuricemia (2 grade 4), and hyperkalemia (1 grade 1). All resolved within 14 days with temporary treatment interruption. Analysis of collected laboratory studies using the definitions of Cairo and Bishop (BJH 2004, 2010) demonstrated that 1 instance of laboratory tumor lysis syndrome (LTLS) and 3 instances of Grade 2 clinical tumor lysis syndrome (CTLS) occurred. Hyperuricemia and creatinine increases in cases of TLS were reported as dose-limiting toxicities at 240 and 200 mg with 1 and 3 instances at each respective dose. The most frequently reported drug-related AEs across all grades were diarrhea (3 grade 2, 5 grade 1), nausea (3 grade 2, 4 grade 1), transient increased creatinine (4 grade 2), anemia (3 grade 2, 1 grade 1), and hyperuricemia (2 grade 4). A palpable spleen length reduction of at least 35% was seen in 13 of 17 evaluable patients (76% including 16 MF and 1PV). A partial response by EULN for PV involving 100% reduction in spleen size was observed for 〉10 months. In MF, 7 patients had a ≥50% reduction, including 4 with sufficient stability or duration of follow-up to qualify as a clinical improvement by IWG-MRT. After 5 cycles of therapy, a reduction in bone marrow fibrosis grade was observed in 3 of the 5 MF patients for whom follow-up biopsy results are available. Using the MPN-SAF questionnaire, patients reported improvements in symptom burden within the first 2 cycles of therapy. Fifty-nine percent (59%) reported a ≥50% improvement in the Key MPN Related Symptoms, and 47% reported a ≥50% improvement in the categories of Fatigue-Enjoyment of Life, Fatigue-Walking Ability, itching, and early satiety. A similar level of improvement was reported by 65% of patients for inactivity, while 35% had improvements in bone pain and night sweats. At tested doses, a reduction in mutant allele burden has not been observed. Conclusions: An MTD for daily dosing of LY2784544 was identified as 120 mg. Based on PK analysis, exposure from doses associated with TLS appear to overlap the lower boundary of the predicted biologically efficacious dose (BED) range established in murine JAK2 V617F models. To allow testing of doses within the BED, the dosing regimen has been amended to include a lower dose lead-in period, intended to reduce tumor burden, prior to further testing of dose escalation. This trial amendment will also further evaluate the bone marrow fibrosis response. Disclosures: Verstovsek: Eli Lilly: Research Funding. Off Label Use: Study of LY2784544 - Purpose is to present results of a study. Mesa:NS Pharma: Research Funding; Astra Zeneca: Research Funding; SBio: Research Funding; Lilly: Research Funding; Incyte: Research Funding; Celgene: Research Funding. Kloeker Rhoades:Eli Lilly: Employment, Equity Ownership. Giles:Eli Lilly: Employment, Equity Ownership. Pitou:Eli Lilly: Employment, Equity Ownership. Jones:Eli Lilly: Employment, Equity Ownership. Walgren:Eli Lilly: Employment, Equity Ownership.