Research on platelets, on lenalidomide in myelodysplastic syndrome (MDS) and sorafenib in acute myeloid leukemia (AML), highlighted the plenary session held December 7, 2014, at the 56th Annual Meeting of the American Society of Hematology (ASH) in San Francisco. Presentations at the session were selected by the Program Committee from among the thousands of scientific abstracts that were accepted for the meeting.
Renata Grozovsky, PhD, a research fellow at Brigham and Women’s Hospital, Boston, delineated an in vitro and in vivo mechanism, regulated by the JAK2-STAT3 signaling pathway, which regulates platelet production. Thrombopoietin (TPO) was discovered in 1958 but only identified as a ligand of the c-MPL receptor in 1994. While TPO is a well-studied regulator of platelet production, supporting the survival, proliferation, and differentiation of platelet precursors, and bone marrow megakaryocytes, mechanisms regulating circulating TPO levels have been a topic of debate, Grozovsky said. Through a collaboration with scientists across Brigham and the Department of Biotechnology, University of Rijeka, Croatia, Grozovsky provided evidence that platelets lacking sialic acid (desialylated platelets) are removed by the hepatic Ashwell-Morell receptor (AMR), thereby regulating platelet survival and hepatic TPO levels.
“We hypothesized that clearance of desialylated platelets by AMR increases TPO in the liver,” said Grozovsky. Her research team found that preventing the expression of the asialoglycoprotein receptor (Asgr2) subunit of AMR in mice improved platelet survival compared to the control mice. In fact, Asgr2-/- mice had lower liver TPO mRNA levels, as well as expressed platelets that were desialylated. Mice lacking the sialyltransferaseST3GalIV, on the other hand, had an increase in liver TPO mRNA. Desialylated platelets isolated from St3gal4-/- or Asgr2-/- mice infused into WT mice increased hepatic TPO mRNA levels, plasma TPO levels, and bone marrow megakaryocytes. However, desialylated platelets infused into Asgr2-/- mice had no effect on TPO mRNA synthesis, TPO plasma levels, or bone marrow megakaryocyte numbers, researchers found.
To identify the underlying signaling mechanism, Grozovsky described conducting biochemical evaluation of the mouse liver cells and human HepG2 cells: within a half hour of ingesting desialylated platelets, increased phosphorylation of the tyrosine kinase JAK2 was observed in St3gal4-/- mice and a marked reduction in JAK2 phosphorylation in Asgr2-/- mice. “When we treated the cells with the JAK1/2-specific inhibitor, AZD1480, we observed reduced STAT3 phosphorylation and nuclear translocation, and reduced expression of both the TPO mRNA and protein in HepG2 cells incubated with desialylated platelets.” The results were translated in vivo in mice; treating the control wildtype mice with AZD1480 blocked TPO mRNA induction by the desialylated platelets. Grozovsky concluded that clearance of aging desialylated platelets by AMR has a substantial clinical significance, considering that the JAK1/2 inhibitor often causes thrombocytopenia.
All the presentations during the plenary session were delivered by senior research investigators, with the exception of the talk by Emma Fink, an MDPhD student in the laboratory of Benjamin L. Ebert, MD, PhD, Brigham and Women’s Hospital, Boston. Her talk was titled “Lenalidomide induced ubiquitination and degradation of CSNK1A1 in MDS with del(5q).”
Lenalidomide is a highly effective treatment for multiple myeloma and MDS with deletion of chromosome 5q (del(5q)). Recent work from their laboratory and others demonstrated that lenalidomide activates the CRBN-CRL4 E3 ubiquitin ligase to ubiquitinate IKZF1 and IKZF3. Degradation of these lymphoid transcription factors explains lenalidomide’s growth inhibition of multiple myeloma cells and increased IL-2 release from T cells. “We hypothesized that ubiquitination of a distinct CRBN substrate in myeloid cells could explain the efficacy of lenalidomide in del(5q) MDS,” Fink stated.
She went on to describe quantitative proteomic experiments in the myeloid KG-1 cells, which identified casein kinase 1A1 (CSNK1A1), a novel target that had increased ubiquitination and decreased protein abundance following lenalidomide treatment. Csnk1a1, being encoded in the del(5q) commonly deleted region, is a potential lenalidomide target in del(5q) MDS.
“We validated that lenalidomide treatment decreased CSNK1A1 protein levels in multiple human cell lines in a dose-dependent manner without altering Csnk1a1 mRNA levels,” said Fink.Moreover, lenalidomide treatment increased ubiquitination of CSNK1A1 in cell lines. Previous work from Ebert’s laboratory had shown that CSNK1A1 was a negative regulator of p53 and β-catenin. So their research group worked on the hypothesis that lenalidomide would induce degradation of CSNK1A1, resulting in the specific apoptosis of haploinsufficient del(5q) cells.
To tease out the mechanism, they conducted co-immunoprecipitation experiments, which pulled down CSNK1A1 with CRBN in the presence of lenalidomide, demonstrating a direct interaction of CSNK1A1 with the substrate adaptor for the ubiquitin ligase. Mixing CRBNCRL4 with CSNK1A1 demonstrated in vitro ubiquitination of CSK1A1. These results were further validated in a genetically defined CSNK1A1 conditional knockout mouse model. While murine cells were found resistant to the effects of IMiDs, murine Ba/F3 cells overexpressing human CRBN (hCRBN), but not murine CRBN, degraded CSNK1A1 in response to lenalidomide. “This could explain why thalidomide, which was found responsible for acute teratogenicity in humans, failed to show any toxicity in mouse experiments,” Fink explained. Finally, her group observed that CSK1A1 expression was sensitive to lenalidomide in del(5q) MDS patients. Fink concluded her talk by stating that lenalidomide provides the first example of an FDA-approved and clinically effective drug that derives its therapeutic window from specifically targeting a haploinsufficient gene.
The final presentation during the plenary session provided results from a multi-collaborative trial, the SORAML Trial (NCT00893373), conducted in Germany, presented by Christoph Röllig, MD, from Universitätsklinikum in Dresden, Germany. The trial was designed to evaluate the kinase inhibitor sorafenib as add-on to standard chemotherapy-as-backbone induction and consolidation treatment in patients with acute myeloid leukemia (AML). The primary eligibility criteria for the trial, which enrolled 276 patients from 25 centers over a period of 2.5 years, were newly diagnosed AML, aged 18 to 60 years, and suitability for intensive therapy. “Our exclusion criteria included heart disease, anaplastic leukemia, hypertension, surgery, or open wounds,” said Röllig. Röllig presented the following treatment plan:
Two cycles of induction with daunorubicin (DA) (DA 60 mg/m2: days 3 to 5, with cytarabine 100 mg/m2 as a continuous infusion: days 1 to 7), followed by 3 cycles of high-dose cytarabine consolidation (3 g/m2 once a day on days 1, 3, and 5). Nonresponders received a second induction with high-dose cytarabine and mitoxantrone (HAM) (cytarabine 3 g/m2 once a day on days 1 to 3, plus mitoxantrone 10 mg/m2: days 3 to 5). Allogeneic stem cell transplantation was scheduled for all intermediate-risk patients in first complete remission with a sibling donor and for all high-risk patients with a matched related or unrelated donor. At study inclusion, patients were randomized to receive either sorafenib (800 mg/day) or placebo as add-on to standard treatment in a double-blinded fashion. Study medication was administered on days 10 to 19 of DA I+II or HAM, from day 8 of each consolidation until 3 days before the start of the next consolidation, and as maintenance for 12 months after the end of consolidation.
The patients were nearly evenly split between the study arm and the placebo arm, and the median cumulative dose of study medication was similar between the arms. At the end of the trial, analysis of the results found no difference in the complete remission (CR) rates between the sorafenib arm and the placebo-controlled arm, showed Röllig: CR was 59% with placebo and 60% with sorafenib (P = .764). However, he presented a significant difference in event-free survival (EFS) rates between the 2 arms: 22% versus 40% EFS for placebo and sorafenib, respectively. He pointed out that the relapse-free survival (RFS) rate was 38% in patients treated with placebo following standard treatment, while patients treated with sorafenib had a RFS of 56%. Patients in the sorafenib arm were at an increased risk for fever, infections, and bleeding events.
Röllig concluded that in younger AML patients, the addition of sorafenib to standard chemotherapy in a sequential manner is feasible and associated with antileukemic efficacy. He emphasized, however, that confirmatory trials will be necessary to establish sorafenib as a new standard therapy for AML.