Abstract
Abstract 514
Background:
Tyrosine kinase inhibitors (TKI), although effective in inducing remissions and improving survival in CML patients, fail to eliminate leukemia stem cells (LSC), which remain a potential source of relapse on stopping treatment. Additional strategies to enhance elimination of LSC in TKI-treated CML patients are required. The Hedgehog (Hh) pathway, important for developmental hematopoiesis, has been shown to be activated in BCR-ABL-expressing LSC, in association with upregulation of Smoothened (SMO), and contributes to maintenance of BCR-ABL+ LSC. However the role of Hh signaling in chronic phase (CP) CML LSC is not clear. LDE225 (LDE, Novartis Pharma) is a small molecule SMO antagonist which is being clinically evaluated in patients with solid tumors. We have reported that LDE does not significantly affect proliferation and apoptosis of primary CP CML CD34+ cells, or reduce colony growth in CFC assays, but results in significant reduction in CML CFC replating efficiency and secondary colony formation. Treatment with LDE + Nilotinib resulted in significant reduction in colony formation from CD34+ CML cells in LTCIC assays compared to Nilotinib alone or untreated controls. These observations suggest that LDE may preferentially inhibit growth of primitive CML progenitors and progenitor self-renewal. We therefore further investigated the effect of LDE on growth of primitive CML LSC in vivo.
Methods and Results:
1) CP CML CD34+ cells were treated with LDE (10nM), Nilotinib (5μ M) or LDE + Nilotinib for 72 hours followed by transplantation into NOD-SCID γ-chain- (NSG) mice. Treatment with LDE + Nilotinib resulted in reduced engraftment of CML CD45+ cells (p=0.06) and CD34+ cells (p=0.02) compared with controls, and significantly reduced engraftment of CML cells with CFC capacity (p=0.005). In contrast LDE or Nilotinib alone did not reduce CML cell engraftment in the bone marrow (BM) compared with untreated controls. LDE, Nilotinib, or LDE + Nilotinib treatment did not significantly inhibit engraftment of normal human CD34+ cells in NSG mice compared to controls. 2) We also used the transgenic Scl-tTa-BCR-ABL mouse model of CP CML to investigate the effect of in vivo treatment with LDE on CML LSC. BM cells from GFP-SCL-tTA/BCR-ABL mice were transplanted into wild type congenic recipients to establish a cohort of mice with CML-like disease. Recipient mice developed CML-like disease 3–4 weeks after transplantation. Transplanted CML cells were identifiable through GFP expression. Mice were treated with LDE225 (80mg/kg/d by gavage), Nilotinib (50 mg/kg/d by gavage), LDE + Nilotinib, or vehicle alone (control) for 3 weeks. Treatment with Nilotinib, LDE, and LDE + Nilotinib resulted in normalization of WBC and neutrophil counts in peripheral blood. LDE + Nilotinib treatment significantly reduced the number of splenic long term hematopoietic stem cells (LT-HSC, Lin-Sca-1+Kit+Flt3-CD150+CD48-, p<0.01) and granulocyte-macrophage progenitors (GMP) compared to controls, but did not significantly alter LT-HSC numbers in the BM. LDE alone reduced splenic LT-HSC but not GMP, whereas Nilotinib alone did not reduce LT-HSC numbers in spleen or BM but significantly reduced splenic GMP numbers. The mechanisms underlying enhanced targeting of LSC in the spleen compared to the BM are not clear but could reflect greater dependence on Hh signaling in the context of the splenic microenvironment and/or relocalization of LDE treated LT-HSC to BM. Experiments in which BM and spleen cells from treated mice were transplanted into secondary recipients to determine functional stem cell capacity of remaining LT-HSC are ongoing. Importantly mice treated with LDE + Nilotinib demonstrated enhanced survival on follow up after discontinuation of treatment compared with control mice or mice treated with LDE or Nilotinib alone.
Conclusions:
We conclude that LDE225 can target LSC from CP CML patients and in a transgenic BCR-ABL model of CP CML, and that LDE + Nilotinib treatment may represent a promising strategy to enhance elimination of residual LSC in TKI-treated CML patients.
Disclosures:
Buonamici: Novartis: Employment. Manley:Novartis: Employment. Holyoake:Novartis: Consultancy, Research Funding. Copland:Novartis Pharma: Honoraria, Research Funding; Bristol-Myers Squibb: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Pfizer: Honoraria, Membership on an entity's Board of Directors or advisory committees. Bhatia:Novartis: Consultancy, Honoraria.
Synergistic interactions between interleukin-11 and interleukin-4 in support of proliferation of primitive hematopoietic progenitors of mice