Laurie A. Milner

Osteoblastic cells regulate the haematopoietic stem cell niche

Stem cell fate is influenced by specialized microenvironments that remain poorly defined in mammals. To explore the possibility that haematopoietic stem cells derive regulatory information from bone, accounting for the localization of haematopoiesis in bone marrow, we assessed mice that were genetically altered to produce osteoblast-specific, activated PTH/PTHrP receptors (PPRs). Here we show that PPR-stimulated osteoblastic cells that are increased in number produce high levels of the Notch ligand jagged 1 and support an increase in the number of haematopoietic stem cells with evidence of Notch1 activation in vivo. Furthermore, ligand-dependent activation of PPR with parathyroid hormone (PTH) increased the number of osteoblasts in stromal cultures, and augmented ex vivo primitive haematopoietic cell growth that was abrogated by γ-secretase inhibition of Notch activation. An increase in the number of stem cells was observed in wild-type animals after PTH injection, and survival after bone marrow transplantation was markedly improved. Therefore, osteoblastic cells are a regulatory component of the haematopoietic stem cell niche in vivo that influences stem cell function through Notch activation. Niche constituent cells or signalling pathways provide pharmacological targets with therapeutic potential for stem-cell-based therapies.

Embryonic lethality in mice homozygous for a processing-deficient allele of Notch1.

The Notch genes encode single-pass transmembrane receptors that transduce the extracellular signals responsible for cell fate determination during several steps of metazoan development. The mechanism by which extracellular signals affect gene transcription and ultimately cell fate decisions is beginning to emerge for the Notch signalling pathway. One paradigm is that ligand binding to Notch triggers a Presenilin1-dependent proteolytic release of the Notch intracellular domain from the membrane, resulting in low amounts of Notch intracellular domain which form a nuclear complex with CBF1/Su(H)/Lag1 to activate transcription of downstream targets. Not all observations clearly support this processing model, and the most rigorous test of it is to block processing in vivo and then determine the ability of unprocessed Notch to signal. Here we report that the phenotypes associated with a single point mutation at the intramembranous processing site of Notch1, Val1,744→Gly, resemble the null Notch1 phenotype. Our results show that efficient intramembranous processing of Notch1 is indispensable for embryonic viability and proper early embryonic development in vivo.

The Human Homolog of Rat Jagged1Expressed by Marrow Stroma Inhibits Differentiation of 32D Cells through Interaction with Notch1

A cDNA clone encoding the human homolog of rat Jagged1 was isolated from normal human marrow. Analyses of human stromal cell lines indicate that this gene, designated hJagged1, is expressed by marrow stromal cells typified by the cell line HS-27a, which supports the long-term maintenance of hematopoietic progenitor cells. G-CSF-induced differentiation of 32D cells expressing Notch1 was inhibited by coculturing with HS-27a. A peptide corresponding to the Delta/Serrate/LAG-2 domain of hJagged1 and supernatants from COS cells expressing a soluble form of the extracellular portion of hJagged1 were able to mimic this effect. These observations suggest that hJagged1 may function as a ligand for Notch1 and play a role in mediating cell fate decisions during hematopoiesis.

Notch1 and Notch2 Inhibit Myeloid Differentiation in Response to Different Cytokines

ABSTRACT We have compared the ability of two mammalian Notch homologs, mouse Notch1 and Notch2, to inhibit the granulocytic differentiation of 32D myeloid progenitor cells. 32D cells undergo granulocytic differentiation when stimulated with either granulocyte colony-stimulating factor (G-CSF) or granulocyte-macrophage colony-stimulating factor (GM-CSF). Expression of the activated intracellular domain of Notch1 inhibits the differentiation induced by G-CSF but not by GM-CSF; conversely, the corresponding domain of Notch2 inhibits differentiation in response to GM-CSF but not to G-CSF. The region immediately C-terminal to the cdc10 domain of Notch confers cytokine specificity on the cdc10 domain. The cytokine response patterns of Notch1 and Notch2 are transferred with this region, which we have termed the Notch cytokine response (NCR) region. The NCR region is also associated with differences in posttranslational modification and subcellular localization of the different Notch molecules. These findings suggest that the multiple forms of Notch found in mammals have structural differences that allow their function to be modulated by specific differentiation signals.

High-dose therapy and autologous stem cell transplant for transformed non-Hodgkin lymphoma in the rituximab era

Abstract The impact of rituximab on the outcome of high-dose therapy and autologous stem cell transplant (HD-ASCT) for transformed non-Hodgkin lymphoma (NHL) has not been previously described. We analyzed 18 consecutive patients with indolent NHL who transformed to diffuse large B-cell lymphoma (DLBCL), received rituximab-containing therapy either before or after transformation and underwent subsequent HD-ASCT. With a median follow-up of 40 months, the 2-year progression-free survival (PFS) was 59% and the 2-year overall survival (OS) was 82%. Six patients did not receive rituximab pre-transformation. This group had a significantly better PFS at 2 years post-HD-ASCT compared to 12 patients who were exposed to rituximab pre-transformation (p = 0.03). HD-ASCT remains an effective therapeutic option for transformed NHL in the rituximab era. However, patients exposed to rituximab pre-transformation appear to have inferior HD-ASCT outcomes, and thus may benefit from novel conditioning and maintenance regimens in the setting of HD-ASCT.

Use of eltrombopag, a thrombopoietin receptor agonist, in post‐transplantation thrombocytopenia

Persistent thrombocytopenia after stem cell transplantation can lead to increased morbidity and mortality [1,2]. The underlying causes are often multifactorial in this patient population [3,4]. In autologous transplantation, thrombocytopenia is usually a result of poor engraftment or a sign of impending disease relapse. In allogeneic stem cell transplantation, the etiology is often more complex with engraftment deficits, medication effects, graft versus host disease (GVHD), and other immunologic processes potentially contributing. Eltrombopag is an orally available nonpeptide thrombopoietin (TPO) receptor agonist which interacts with the transmembrane domain of the receptor on bone marrow megakaryocytes and upstream progenitor/stem cells. It has been studied in patients with chronic idiopathic thrombocytopenic purpura [5] and in patients with thrombocytopenia secondary to hepatitis C infection [6]. Unlike the case with recombinant human TPO, its use has not been associated with anti-platelet antibody production [7]. We report two cases of post-transplantation thrombocytopenia, one allogeneic and one autologous, where eltrombopag was given to treat prolonged thrombocytopenia. The use of eltrombopag in these two cases was effective in elevating platelet counts to levels that eliminated the need for platelet transfusions.

A Phase 1 Trial of Eltrombopag in Patients Undergoing Stem Cell Transplantation after Total Body Irradiation

Stem cell transplantation can be associated with significant periods of thrombocytopenia, necessitating platelet transfusions and contributing to the risk of bleeding. Thrombopoietin receptor agonists have been shown to enhance platelet counts in other clinical settings, and so a phase 1 clinical trial was conducted to assess the safety, pharmacokinetics, and maximum tolerated dose of once-daily eltrombopag in patients undergoing stem cell transplantation with conditioning regimens containing total body irradiation ≥400 cGy. Eltrombopag was examined at dosage levels of 75, 150, 225, and 300 mg given orally once daily for 27 days, starting at 24 to 48 hours post-transplantation. Pharmacokinetic sampling was performed over a 24-hour period after the first dose of eltrombopag, as well as during the second week of treatment (steady-state). Nineteen patients were enrolled, 15 of whom completed protocol treatments. Three patients completed each dose level up to 225 mg, and 6 completed treatment at the highest dose of 300 mg. Four patients were replaced because drug compliance was <75% of planned doses. No dose-limiting toxicities were observed in this heterogeneous post-transplantation patient population. Common adverse events were related to standard stem cell transplantation. One episode of pulmonary embolus occurred 9 days after discontinuation of eltrombopag, and the only other thromboembolic episode was a grade 2 catheter-related clot. We conclude that up to 27 days of once-daily dosing of eltrombopag after stem cell transplantation is well tolerated.

Improved outcomes in acute myeloid leukemia patients treated with washed transfusions.

how we treat. Am J Hematol 2016;91:76–89. 9. Steensma DP, Bejar R, Jaiswal S, et al. Clonal hematopoiesis of indeterminate potential and its distinction from myelodysplastic syndromes. Blood 2015;126:9–16. 10. Jerez A, Clemente MJ, Makishima H, et al. STAT3 mutations indicate the presence of subclinical T-cell clones in a subset of aplastic anemia and myelodysplastic syndrome patients. Blood 2013;122:2453–2459. 11. Couronne L, Bastard C, Bernard OA. TET2 and DNMT3A mutations in human T-cell lymphoma. N Engl J Med 2012;366:95–96. 12. Damm F, Mylonas E, Cosson A, et al. Acquired initiating mutations in early hematopoietic cells of CLL patients. Cancer Discov 2014;4:1088–1101. 13. Chung SS, Kim E, Park JH, et al. Hematopoietic stem cell origin of BRAFV600E mutations in hairy cell leukemia. Sci Transl Med 2014;6:238ra271. 14. Bejar R, Lord A, Stevenson K, et al. TET2 mutations predict response to hypomethylating agents in myelodysplastic syndrome patients. Blood 2014;124:2705–2712. 15. Metzeler KH, Walker A, Geyer S, et al. DNMT3A mutations and response to the hypomethylating agent decitabine in acute myeloid leukemia. Leukemia 2012;26:1106–1107.

The hematopoietic cell transplantation specific comorbidity index and survival after extracorporeal photopheresis, pentostatin, and reduced dose total body irradiation conditioning prior to allogeneic stem cell transplantation

Hematopoietic-cell-transplantation-specific-comorbidity-index (HCT-CI) has been reported as a predictor of survival in allogeneic-transplant recipients; however its validity has recently been challenged. We evaluated the association of HCT-CI with survival of transplant recipients who underwent reduced-intensity-conditioning (RIC) with photopheresis, pentostatin, and total-body-irradiation. Median age of 103 patients selected was 55 years. Most patients (58.3%) had high (≥ 3) HCT-CI. Median OS was 298 days. Age, disease-type, disease-status, HCT-CI correlated with survival on bivariate analysis. On multivariate analysis, only HCT-CI was significantly associated with OS (low HCT-CI HR=0.29, CI 0.091-0.886; intermediate HCT-CI HR=0.41, CI 0.226-0.752). Our findings suggest HCT-CI as an independent predictor of survival in the setting of RIC transplants.

A Phase I Trial: Dose Escalation of Melphalan in the “BEAM” Regimen Using Amifostine Cytoprotection

With the eventual goal of reducing relapse and thus improving overall survival in selected lymphoma patients, a Phase I study was performed using the cytoprotectant amifostine to permit safe dose-augmentation of melphalan in the carmustine (BCNU), etoposide, cytarabine (arabinosylcytosine), and melphalan (BEAM) regimen before autologous hematopoietic stem cell transplantation. Between 30 July 2003 and 25 November 2008, a total of 32 lymphoma patients were entered, of which 28 were evaluable. We found the melphalan dose in BEAM could be safely escalated to at least 260 mg/m², a substantial increase from the usual dose of 140 mg/m² in BEAM while the trial was terminated early due to poor accrual, no maximal tolerated dose or dose-limiting toxicity was found. A Phase II trial is planned.