IL1RAP Antibodies Block IL1-Induced Expansion of Primitive CML Cells and Display Therapeutic Effects in Xenograft Models

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 1118-1118
Author(s):  
Helena Ågerstam ◽  
Nils Hansen ◽  
Sofia Von Palffy ◽  
Carl Sandén ◽  
Kristian Reckzeh ◽  
...  
Keyword(s):  
Stem Cells ◽  
Research Funding ◽  
Effector Cell ◽  
Leukemic Cells ◽  
Nsg Mice ◽  
Xenograft Models ◽  
Equity Ownership ◽  
In Vivo Effects

Abstract Chronic myeloid leukemia (CML) is currently treated with tyrosine kinase inhibitors (TKIs) but these do not effectively eliminate the CML stem cells. As a consequence, CML stem cells persist and cause relapse in most patients upon drug discontinuation. Furthermore, no effective therapy exists for the advanced stages of the disease. Thus, there is still a need for novel treatment strategies in CML. We have previously shown that Interleukin 1 receptor accessory protein (IL1RAP), a co-receptor of IL1R1, is highly expressed on primitive CML cells and that a polyclonal IL1RAP antibody can direct natural killer (NK) cells to specifically target and destroy CD34+CD38- CML cells in an in vitro-based antibody dependent cell-mediated cytotoxicity (ADCC) assay (Järås et al, PNAS, 2010). The aim of the present study was to investigate the consequences of IL1RAP expression on primitive CML cells and the in vivo therapeutic efficacy of monoclonal IL1RAP antibodies against CML cells. Primary chronic phase (CP) CD34+ CML cells were cultured in medium supplemented with cytokines known to signal through receptor complexes involving IL1RAP. The addition of IL1 to the cultures resulted in a marked cellular expansion specifically for the primitive CD34+CD38- CML cells. Moreover, the CD34+CD38- cells showed phosphorylation of the downstream mediator of IL1-signaling NFKB. RNA-sequencing confirmed the activation of NFKB and of genes involved in cell cycling, indicating that IL1 stimulation of CD34+CD38- CML cells induced proliferation. Upon addition of an IL1RAP antibody capable of blocking IL1-signaling to the suspension cultures, the IL1-induced expansion and NFKB phosphorylation of CD34+CD38- CML cells was suppressed. Interestingly, both the IL1RAP expression and the response to IL1 as measured by NFKB phosphorylation was retained during TKI treatment of the cells. To assess the in vivo effects of IL1RAP antibodies in CML models, we first engrafted NOD/SCID mice with BCR/ABL1 expressing BV173 cells and treated the mice with the monoclonal IL1RAP antibody mAb81.2. Mice receiving treatment with mAb81.2 displayed a prolonged survival compared to controls, accompanied by reduced levels of leukemic cells in the BM. In vitro studies showed that mAb81.2 lacked a direct effect on cellular expansion or apoptosis. Instead, the IL1RAP antibody could direct NK cells to elicit killing of the leukemic cells, thereby suggesting effector cell mediated mechanisms to be an important in vivo mode-of-action. To validate the in vivo effects on primary CML cells, we next engrafted CP or blast phase (BP) CML cells into immunodeficient mice. Following engraftment of CP CD34+ CML cells into NSG mice and subsequent treatment with mAb81.2, a reduction of human myeloid cells was observed, suggesting that the treatment targeted the leukemic graft. Importantly, mAb81.2 treatment also reduced the levels of candidate CD34+CD38-IL1RAP+ CML stem cells. Finally, BP CML cells were engrafted into NOD/SCID mice that have a more intact effector cell function compared to NSG mice. Following treatment with mAb81.2 a significant reduction of leukemic cells in the BM as well as in the periphery was observed compared to control mice. Importantly, secondary transplantations revealed a therapeutic effect also on the BP CML stem cells. In vitro ADCC assays confirmed that CML BP cells, including a sample with the highly TKI-resistant T315I mutation, could be targeted and killed using mAb81.2. We conclude that IL1RAP antibodies can suppress IL1-induced expansion of primitive CML cells and that in vivo administration of IL1RAP antibodies in CML xenograft models has anti-leukemic effects that extend to the CML stem cells. These results show that an antibody-based therapy against IL1RAP can be used to efficiently target CML stem cells. Disclosures Richter: BMS: Honoraria, Research Funding; Pfizer: Honoraria, Research Funding; Ariad: Honoraria, Research Funding; Novartis: Honoraria, Research Funding. Järås:Cantargia AB: Equity Ownership. Fioretos:Cantargia AB: Equity Ownership.

Pronounced Anti-Leukemia Activity in Vivo of the Novel Peptidic CXCR4 Antagonist LY2510924 As a Single Agent and in Combination with Chemotherapy

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 3745-3745
Author(s):  
Byung-Sik Cho ◽  
Zhihong Zeng ◽  
Hong Mu ◽  
Zhiqiang Wang ◽  
Teresa McQueen ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Flow Cytometry ◽  
Cultured Cells ◽  
Single Agent ◽  
Leukemic Cells ◽  
Cxcr4 Antagonist ◽  
Nsg Mice ◽  
Xenograft Models

Abstract LY2510924 is a novel selective peptidic CXCR4 antagonist that blocks SDF-1α from binding to its receptor. We have demonstrated that LY2510924 at nanomolar concentrations durably disrupts the SDF-1α/CXCR4 axis in acute myeloid leukemia (AML) cells and exerts anti-leukemia effects as a single agent (AACR 2014: #4768). We further investigated the pronounced anti-leukemia activity of LY2510924 and the mechanisms underlying the anti-leukemia effect. To test the efficacy of LY2510924 in combination with chemotherapy, we injected OCI-AML3/luc/GFP cells into NSG mice. Mice were randomized into 4 groups (10 mice per group) on day 8: control, chemotherapy (cytarabine [50 mg/kg, daily for 5 days, intravenous or intraperitoneal]/doxorubicin [1.5 mg/kg, daily for 3 days, co-delivered intravenously]), LY2510924 (2.5 mg/kg, daily for 3 weeks, subcutaneously), or chemotherapy and LY2510924. Bioluminescence imaging demonstrated that LY2510924 exerted an anti-leukemia effect equal to that achieved with chemotherapy (P=0.249), and the combination therapy group had the lowest luciferase activity. LY2510924-treated mice had prolonged survival (Figure 1) compared to controls (52 days vs. 40 days, p=0.006), and combination therapy extended survival even further (62 days vs. 52 days, p=0.004). Next, we examined anti-leukemia efficacy of LY2510924 in primary human AML xenograft models. NSG mice were injected with primary AML cells and randomized into 2 groups on day 25, after engraftment was documented: control (n=13) and treatment with LY2510924 (n=15; 2.5 mg/kg subcutaneously, daily). First, we examined AML cell mobilization by measuring the proportion of circulating leukemic cells after daily LY2510924 administration. Mice treated with LY2519024 had a significant increase of circulating leukemic cells at 3 hours (2.1-fold, P=0.008), and further increases at 24 hours (2.7-fold, P=0.008) and 48 hours (3.0-fold, P=0.009) compared to controls. Flow cytometry showed a sustained inhibition of CXCR4 12G5 surface expression at 3 and 24 hours after the first LY2510924 injection. Thereafter, weekly examination of circulating leukemic cells in both groups revealed slower progression of leukemia in the LY2510924-treated group (54% vs. 86% circulating AML cells on day 45, P<0.001). Additionally, we sacrificed 3 mice per group on days 35 and 45 and demonstrated that LY2510924-treated mice had significantly lower leukemic cell burden in the spleen (22% vs. 51%, P=0.001) on day 35, and in both spleen (20% vs. 60%, P<0.001) and bone marrow (72% vs. 90%, P=0.012) on day 45 by flow cytometry. CXCR4 blockade with LY2510924 was associated with reduced AKT and/or ERK signaling in leukemic cells of spleen, bone marrow, and blood as measured by multi-parametric phospho-flow cytometry. This anti-leukemia effect translated into a significant prolongation of survival in LY2510924-treated mice (56 days vs. 44 days, p<0.001, Figure 2). Our previous study (AACR 2014:#4768) demonstrated that LY2510924 did not induce AML cell death in vitro on its own but inhibited AML cell growth in co-cultures with human marrow stromal cells (hMSC). To explore how CXCR4-mediated signaling in AML cells elicits anti-leukemia effects, we performed whole gene expression profiling of FACS-sorted OCI-AML3 cells co-cultured with hMSC for 48 hours and co-treated with LY2510924, in duplicates. Among genes modified by CXCR4 antagonist, we found that CTNNB1 (human beta-catenin), JARID1C (lysine-specific demethylase 5C), RARA (retinoic acid receptor alpha), RARRES2 (chemerin), and COQ4 (coenzyme Q) were downregulated in co-cultured OCI-AML3 cells treated with LY2510925, when compared to either mono-cultured cells or co-cultured cells without LY2510924. These findings are currently being validated by using functional in vitro assays. In conclusion, our findings demonstrate that CXCR4 antagonist LY2510924 inhibits AML progression in leukemia xenograft models in vivo and has a synergistic anti-leukemia effect in combination with chemotherapy. LY2510924 efficiently inhibits CXCR4 signaling in primary AML cells in vivo and induces mobilization of leukemic cells into circulation. This results in pronounced anti-leukemia activity as a single agent. LY2510924's potency and durable occupancy of CXCR4 receptors will likely translate into greater anti-leukemia potency in future clinical applications. Disclosures Peng: Eli Lilly & Company: Employment. Thornton:Eli Lilly & Company: Employment, stocks Other.

Identification of Small Molecules Selectively Targeting Leukemic Stem Cells within Their Stromal Niche

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 413-413
Author(s):  
Alissa R. Kahn ◽  
Kimberly A. Hartwell ◽  
Peter G. Miller ◽  
Benjamin L. Ebert ◽  
Todd R. Golub ◽  
...  
Keyword(s):  
Stem Cells ◽  
Flow Cytometry ◽  
Cord Blood ◽  
Leukemic Cells ◽  
Leukemic Stem Cells ◽  
Minimal Toxicity ◽  
Nsg Mice ◽  
Cd34 Cells

Abstract Abstract 413 Current therapies for acute myeloid leukemia (AML) are highly toxic, yet the relapse rate remains high. New therapies are needed to improve cure rates while decreasing toxicity. Because therapies may be affected by the tumor niche, we aimed to test new compounds on leukemic stem cells (LSCs) within their stromal microenvironment. A niche-based high throughput screen identified candidate small molecules potentially toxic to MLL-AF9 murine leukemic stem cells (LSCs) while sparing normal hematopoietic stem cells (HSCs) and bone marrow stroma (Hartwell et al, Blood 118, Abs 760, 2011.) Three such compounds, including a selective serotonin receptor antagonist highly specific for the 5-HT1B receptor, SB-216641, and two antihelminthics, parbendazole and methiazole, were found to be effective and selected for studies on human leukemias. We first examined SB-216641, studying the effects of this compound on 7 human primary AML samples. We began by assessing the compound's effect on LSCs using the week 5 cobblestone area forming cell (CAFC) assay, a standard in vitro stem cell assay. CD34+ cells were isolated with immunomagnetic beads. The leukemic cells were pulse treated for 18 hours and washed prior to placement on MS-5 murine stroma. We performed serial drug dilutions using the CAFC assay with the human primary samples as well as with HSCs derived from cord blood. All human leukemic samples formed cobblestone areas in the control setting (46-200 CAFCs/106 cells plated). IC50 for the human primary leukemia CAFCs was 630 nm, and at 10 μM all LSCs were killed while normal human HSCs had 100% survival. A combination of the AML cell line HL60 transduced with GFP-luciferase and normal cord blood CD34+ cells (1:200) were then pre-incubated overnight with SB-216641 at 5 and 10 μM and injected into Nod Scid IL2R-gamma null (NSG) mice. The control mice had leukemic engraftment by luciferase imaging and flow cytometry and the mice that received treated cells had no leukemic engraftment but normal multilineage engraftment of cord blood. Primary patient AML samples were also pre-incubated overnight with SB-216641 at 10 μM and injected into NSG mice. As shown by flow cytometry, control mice engrafted with leukemia and mice that received pre-treated cells had no engraftment following exposure to SB-216641. Finally, an in vivo study was completed on NSG mice injected intraperitoneally with 20 mg/kg/day beginning on day 1 or day 8 after inoculation with HL60 (500 cells). The mice were imaged at 2 and 3 week time points and both treatment groups had significantly less leukemia on imaging than the control group with minimal toxicity noted. Another specific 5-HT1B receptor antagonist, SB-224289, was found to have similar activity to SB-216641 against leukemic cells and to spare HSCs in preliminary studies. Similar CAFC studies with serial dilutions on primary AML samples were performed on the two anti-helminthic agents. IC50 for parbendazole was 1.25 μM and for methiazole 5 μM. As shown by luciferase imaging and flow cytometry, when injected with combined HL60 and cord blood pre-incubated overnight at 5 and 10 μM with each compound as described above, the control mice engrafted with leukemia and the mice that received treated cells had no leukemic engraftment but normal multilineage engraftment of cord blood. NSG mice were then injected with primary AML pretreated overnight with parbendazole at 10 μM. As shown by flow cytometry, control mice engrafted with leukemia and mice that received pre-treated cells had significantly lower engraftment following exposure to parbendazole (p = 0.01). Two new avenues of leukemia therapy were discovered warranting further investigation. SB-216641, an agent with a completely novel receptor target in leukemia therapy, has shown both in vitro success in human leukemia as well as preliminary success in vivo with minimal toxicity. We aim to move forward with this agent while also testing parbendazole in vivo, as this compound is already known to have good pharmacokinetics and minimal toxicity in animals. The high toxicity to LSCs and sparing of normal HSCs give both these agents an attractive profile for future clinical trials. Disclosures: Ebert: Genoptix: Consultancy; Celgene: Consultancy.

scholarly journals Phenotype Does Not Always Equal Function: HDAC Inhibitors and UM171, but Not SR1, Lead to Rapid Upregulation of CD90 on Non-Engrafting CD34+CD90-Negative Human Cells

Blood ◽  
2017 ◽  
Vol 130 (Suppl_1) ◽  
pp. 659-659
Author(s):  
Kevin A. Goncalves ◽  
Megan D. Hoban ◽  
Jennifer L. Proctor ◽  
Hillary L. Adams ◽  
Sharon L. Hyzy ◽  
...  
Keyword(s):  
In Vitro Culture ◽  
Small Molecule ◽  
Hdac Inhibitors ◽  
Employment Equity ◽  
Nsg Mice ◽  
Cd34 Cells ◽  
Equity Ownership ◽  
Human Hscs

Abstract Background. The ability to expand human hematopoietic stem cells (HSCs) has the potential to improve outcomes in HSC transplantation and increase the dose of gene-modified HSCs. While many approaches have been reported to expand HSCs, a direct comparison of the various methods to expand transplantable HSCs has not been published and clinical outcome data for the various methods is incomplete. In the present study, we compared several small molecule approaches reported to expand human HSCs including HDAC inhibitors, the aryl hydrocarbon antagonist, SR1, and UM171, a small molecule with unknown mechanism, for the ability to expand phenotypic HSC during in vitro culture and to expand cells that engraft NSG mice. Although all strategies increased the number of phenotypic HSC (CD34+CD90+CD45RA-) in vitro, SR1 was the most effective method to increase the number of NOD-SCID engrafting cells. Importantly, we found that HDAC inhibitors and UM171 upregulated phenotypic stem cell markers on downstream progenitors, suggesting that these compounds do not expand true HSCs. Methods. Small-molecules, SR1, HDAC inhibitors (BG45, CAY10398, CAY10433, CAY10603, Entinostat, HC Toxin, LMK235, PCI-34051, Pyroxamide, Romidepsin, SAHA, Scriptaid, TMP269, Trichostatin A, or Valproic Acid) and UM171 were titrated and then evaluated at their optimal concentrations in the presence of cytokines (TPO, SCF, FLT3L, and IL6) for the ability to expand human mobilized peripheral blood (mPB)-derived CD34+ cells ex vivo . Immunophenotype and cell numbers were assessed by flow cytometry following a 7-day expansion assay in 10-point dose-response (10 µM to 0.5 nM). HSC function was evaluated by enumeration of colony forming units in methylcellulose and a subset of the compounds were evaluated by transplanting expanded cells into sub-lethally irradiated NSG mice to assess engraftment potential in vivo . All cells expanded with compounds were compared to uncultured or vehicle-cultured cells. Results. Following 7 days of expansion, SR1 (5-fold), UM171 (4-fold), or HDAC inhibitors (&gt;3-35-fold) resulted in an increase in CD34+CD90+CD45RA- number relative to cells cultured with cytokines alone; however, only SR1 (18-fold) and UM171 (8-fold) demonstrated enhanced engraftment in NSG mice. Interestingly, while HDAC inhibitors and UM171 gave the most robust increase in the number and frequency of CD34+CD90+CD45RA- cells during in vitro culture, these methods were inferior to SR1 at increasing NSG engrafting cells. The increase in CD34+CD90+CD45RA- cells observed during in vitro culture suggested that these compounds may be generating a false phenotype by upregulating CD90 and down-regulating CD45RA on progenitors that were originally CD34+CD90-CD45RA+. We tested this hypothesis by sorting CD34+CD90-CD45RA+ cells and culturing these with the various compounds. These experiments confirmed that both HDAC inhibitors (33-100 fold) and UM171 (28-fold) led to upregulation of CD90 on CD34+CD90-CD45RA+ cells after 4 days in culture. Since approximately 90% of the starting CD34+ cells were CD90-, these data suggest that most of the CD34+CD90+CD45RA- cells in cultures with HDAC inhibitors and UM171 arise from upregulation of CD90 rather than expansion of true CD34+CD90+CD45RA- cells and may explain the disconnect between in vitro HSC phenotype and NSG engraftment in vivo . This was further confirmed by evaluation of colony forming unit frequency of CD34+CD90-CD45RA+ cells after culture with compounds. Conclusions. We have showed that AHR antagonism is optimal for expanding functional human HSCs using the NSG engraftment model. We also demonstrated that UM171 and HDAC inhibitors upregulate phenotypic HSC markers on downstream progenitors. This could explain the discrepancy between impressive in vitro phenotypic expansion and insufficient functional activity in the NSG mouse model. Therefore, these data suggest caution when interpreting in vitro expansion phenotypes without confirmatory functional transplantation data, especially as these approaches move into clinical trials in patients. Disclosures Goncalves: Magenta Therapeutics: Employment, Equity Ownership. Hoban: Magenta Therapeutics: Employment, Equity Ownership. Proctor: Magenta Therapeutics: Employment, Equity Ownership. Adams: Magenta Therapeutics: Employment, Equity Ownership. Hyzy: Magenta Therapeutics: Employment, Equity Ownership. Boitano: Magenta Therapeutics: Employment, Equity Ownership, Patents & Royalties. Cooke: Magenta Therapeutics: Employment, Equity Ownership, Patents & Royalties.

scholarly journals The in vitro and in vivo effects of mesenchymal stem cells on breast cancer cells

2017 ◽  
Vol 28 ◽  
pp. i15
Author(s):  
T. Herheliuk ◽  
O. Perepelytsina ◽  
O. Yakymchuk ◽  
L. Ostapchenko ◽  
M. Sydorenko
Keyword(s):  
Breast Cancer ◽  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Cancer Cells ◽  
Breast Cancer Cells ◽  
In Vivo Effects

Inhibition of Chronic Myeloid Leukemia Stem Cells by the Combination of the Hedgehog Pathway Inhibitor LDE225 with Nilotinib

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 514-514 ◽  
Author(s):  
Bin Zhang ◽  
David Irvine ◽  
Yin Wei Ho ◽  
Silvia Buonamici ◽  
Paul Manley ◽  
...  
Keyword(s):  
Stem Cells ◽  
Board Of Directors ◽  
Colony Formation ◽  
Research Funding ◽  
Leukemia Stem Cells ◽  
Advisory Committees ◽  
Nsg Mice ◽  
Cd34 Cells ◽  
Hh Signaling

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.

Preclinical Testing of a Novel Axl-Kinase Inhibitor in Chronic Lymphocytic Leukemia

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 1799-1799
Author(s):  
Maria Göbel ◽  
Michael Möllmann ◽  
Andre Görgens ◽  
Ulrich Dührsen ◽  
Andreas Hüttmann ◽  
...  
Keyword(s):  
Chronic Lymphocytic Leukemia ◽  
Tyrosine Kinase ◽  
Receptor Tyrosine Kinase ◽  
Cell Polarization ◽  
Lymphocytic Leukemia ◽  
Leukemic Cells ◽  
Blue Staining ◽  
Nsg Mice

Abstract Abstract 1799 The receptor tyrosine kinase Axl belongs to the TAM (Tyro-3, Axl and Mer) family and is involved in the progression of several human malignancies including chronic lymphocytic leukemia (CLL), where it is has been found to be overexpressed in comparison to normal B-cells. An increasing body of evidence suggests that Axl acts as an oncogene which increases the survival, proliferation, metastatic potential and chemotherapy resistance of tumor cells. Hence, it has been recently identified as a potential therapeutic target in a wide range of tumor entities with deregulated Axl expression including prostate cancer, glioma, lung cancer and CLL. Here, we investigated two different Axl inhibitors for their potential to inhibit the migratory capacity and survival of leukemic cells in preclinical CLL models. In vitro studies: Freshly isolated PBMC (>90% CD5+CD19+) from CLL patients were incubated in serum free medium for 48h containing concentrations series of 2 different Axl inhibitors: BMS777607, a previously published inhibitor of the MET kinase family, and LDC2636, a novel inhibitor of the TAM receptor tyrosine kinase (RTK) family with high affinity to Axl. Viability of CLL cells was assessed by trypan blue staining and flow cytometry employing annexin V staining. Since a polarized phenotype is required for migration, cell polarization was analyzed by time-lapse video-microscopy. We detected cytotoxic effects in a patient dependent manner that were more prevalent in LDC2636 as compared to BMS777607 treated cells (LD50= 1.4 μM vs. 5.2 μM, p<0.004, n=5). Cell polarization of the remaining viable cells was significantly reduced in a dose dependent fashion in comparison to vehicle only controls (LDC2636 IC50 = 7.2 μM, p<0.00001; BMS777607: IC50=6.2μM; p=0.0004). Of note, both Axl inhibitors exhibited significantly weaker effects on both, the viability and cell polarization of normal PBMC over the whole concentration range tested (p<0.05, n=5). In vivo studies: To verify our hypothesis that reduced cell polarization results in decreased homing of leukemic cells in vivo we employed a recently developed adoptive transfer model of CLL. In this model NOD/SCID/gcnull(NSG) mice were pre-treated with a single intraperitoneal bolus of LDC2636 or BMS777607 (20 mg/kg) and subsequently transplanted with primary CLL cells. Both Axl inhibitors significantly reduced the homing capacity of CLL cells to the bone marrow of NSG mice by 43% and 59%, respectively, compared to vehicle treated controls (LDC2636: p=0.046, BMS777607 p=0.0077; n=3). These data demonstrate that Axl inhibitors exert potent in vitro and in vivo activity against human CLL cells, which is caused at least in part by the suppression of CLL homing to their supportive stromal niches. Disclosures: No relevant conflicts of interest to declare.

Metastasis Suppressor 1 Is Downregulated in CML Stem Cells and Overexpression Impairs Early Leukemic Cell Propagation.

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 2776-2776
Author(s):  
Mirle Schemionek ◽  
Ulrich Steidl ◽  
Albrecht Schwab ◽  
Daniel G Tenen ◽  
Copland Mhairi ◽  
...  
Keyword(s):  
Stem Cells ◽  
Research Funding ◽  
Leukemic Cell ◽  
Metastasis Suppressor ◽  
Advisory Committees ◽  
Abl Kinase ◽  
Metastasis Suppressor 1 ◽  
Cell Propagation

Abstract Abstract 2776 The implementation of Bcr-Abl tyrosine kinase inhibitors (TKIs) has greatly improved the outcome of patients with chronic myeloid leukemia (CML). However, discontinuation of TKI therapy often results in relapse suggesting that leukemic stem cells (LSCs) survive despite treatment. More detailed investigations utilizing patient samples and murine CML models have confirmed that the leukemia-initiating cell population is usually not eradicated by inhibiting Bcr-Abl activity and that this is due to a lack of oncogene addiction of LSCs, showing that further research is required aiming to fully understand LSC biology. To identify new Bcr-Abl targets that are involved in LSC persistence, we performed a microarray analysis of the leukemia-initiating cell population in an inducible transgenic SCLtTAxBcr-Abl CML mouse model in which we had previously shown that these cells are not oncogene-addicted (Schemionek et al., BLOOD 2010; Hamilton et al., BLOOD 2012). One of the most downregulated genes in CML vs. normal stem cells was Metastasis Suppressor 1 (Mtss1/MIM). Although the multidomain protein Mtss1 may be involved in carcinogenesis of several solid tumors, its exact physiological role is still unknown. Current findings suggest that Mtss1 interacts with multiple partners and is involved in various signalling pathways that regulate actin dynamics and cell motility. Interestingly, Rac and Src are Mtss1 interacting partners, and both proteins are known to be involved in Bcr-Abl mediated leukemogenesis. We have previously shown that Mtss1 is downregulated in mouse LSCs and demonstrated that this is a Bcr-Abl kinase mediated effect in various human and murine CML cell lines. Moreover, we have shown that Mtss1 overexpression in 32D-Bcr-Abl cells induces a defect in Bcr-Abl mediated migration in vitro and reduces the potential to form solid tumors in vivo. Here we show that Bcr-Abl kinase-dependent regulation of Mtss1 expression was also evident in mononuclear cells and CD34+ progenitor cells from patients with CML upon IM or dasatinib treatment in vitro. Moreover, cells from IM-treated patients with chronic phase CML showed elevated Mtss1 expression levels within one to three weeks of treatment. Increasing Mtss1 expression upon 5-aza-2′-deoxycytidine-treatment of K562 and 32D-Bcr-Abl cells suggested that methylation might be involved in Mtss1 regulation. To determine a potential leukemia suppressing effect of Mtss1 overexpression, we performed colony assays using lineage negative SCLtTAxBcr-Abl (dtg) bone marrow (BM) cells that had been retrovirally infected to overexpress Mtss1 (dtg::Mtss1) or empty-vector (dtg::ev). Successfully transduced BM cells were FACS-sorted via GFP-expression, encoded by the retroviral vector. Mtss1 overexpression led to a 2.3-fold decrease in CFU numbers. In a second set of experiments we transplanted 1.3×105 GFP-FACS-sorted dtg::Mtss1 or dtg::ev cells into 9 Gy-irradiated recipients. While dtg::ev recipients contained 66% (+/−8%) of GFP-positive cells in the BM, these cells were decreased in dtg::Mtss1 transplanted mice to 23% (+/−21%), 12 days after transplantation. A similar effect was evident in the spleen [dtg::ev recipients: 90% (+/− 3%) versus dtg::Mtss1 recipients 59% (+/−20%)] suggesting that Mtss1 confers a disadvantage to Bcr-Abl positive BM cells in the early steps of leukemic cell propagation, compared to Bcr-Abl cells alone. Since the multidomain Mtss1 protein contains a putative Abl-SH2-binding site, we performed co-immunoprecipitations using 32D-Bcr-Abl-Flag-Mtss1 cells. These experiments showed that both proteins were direct binding partners and that Mtss1 was not phosphorylated by Bcr-Abl. Taken together, our data show that Mtss1 is downregulated via a Bcr-Abl kinase mediated mechanism and this might involve methylation. Moreover, additional inhibition of Mtss1 activity might be mediated through direct binding by Bcr-Abl. Forced expression of the potential tumor suppressor in CML stem and progenitor cells reduces leukemic cell propagation in vivo and may thus provide a rationale to contribute to LSC elimination in patients with CML. Disclosures: Mhairi: BMS: Honoraria, Research Funding; Novartis: Honoraria, Research Funding; Pfizer: Honoraria. Koschmieder:Novartis / Novartis Foundation: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Bristol-Myers Squibb: Honoraria, Research Funding; Pfizer: Honoraria, Membership on an entity's Board of Directors or advisory committees.

Stroma-Free Ex Vivo Expanded, CD34+ Cord Blood-Derived NK Cells Retain Lytic Activity After Long-Term Engraftment in NSGS Mice

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 580-580
Author(s):  
Mark Wunderlich ◽  
Mahesh Shrestha ◽  
Lin Kang ◽  
Eric Law ◽  
Vladimir Jankovic ◽  
...  
Keyword(s):  
Nk Cells ◽  
Research Funding ◽  
Nk Cell ◽  
Ex Vivo ◽  
Employment Equity ◽  
Cell Product ◽  
Equity Ownership ◽  
Cellular Therapeutics

Abstract Abstract 580 Generating a large number of pure, functional immune cells that can be used in human patients has been a major challenge for NK cell-based immunotherapy. We have successfully established a cultivation method to generate human NK cells from CD34+ cells isolated from donor-matched cord blood and human placental derived stem cells, which were obtained from full-term human placenta. This cultivation method is feeder-free, based on progenitor expansion followed by NK differentiation supported by cytokines including thrombopoietin, stem cell factor, Flt3 ligand, IL-7, IL-15 and IL-2. A graded progression from CD34+ hematopoietic progenitor cells (HSC) to committed NK progenitor cells ultimately results in ∼90% CD3-CD56+ phenotype and is associated with an average 10,000-fold expansion achieved over 35 days. The resulting cells are CD16- and express low level of KIRs, indicating an immature NK cell phenotype, but show active in vitro cytotoxicity against a broad range of tumor cell line targets. The in vivo persistence, maturation and functional activity of HSC-derived NK cells was assessed in NSG mice engineered to express the human cytokines SCF, GM-CSF and IL-3 (NSGS mice). Human IL-2 or IL-15 was injected intraperitoneally three times per week to test the effect of cytokine supplementation on the in vivo transferred NK cells. The presence and detailed immunophenotype of NK cells was assessed in peripheral blood (PB), bone marrow (BM), spleen and liver samples at 7-day intervals up to 28 days post-transfer. Without cytokine supplementation, very few NK cells were detectable at any time-point. Administration of IL-2 resulted in a detectable but modest enhancement of human NK cell persistence. The effect of IL-15 supplementation was significantly greater, leading to the robust persistence of transferred NK cells in circulation, and likely specific homing and expansion in the liver of recipient mice. The discrete response to IL-15 versus IL-2, as well as the preferential accumulation in the liver have not been previously described following adoptive transfer of mature NK cells, and may be unique for the HSC-derived immature NK cell product. Following the in vivo transfer, a significant fraction of human CD56+ cells expressed CD16 and KIRs indicating full physiologic NK differentiation, which appears to be a unique potential of HSC-derived cells. Consistent with this, human CD56+ cells isolated ex vivo efficiently killed K562 targets in in vitro cytotoxicity assays. In contrast to PB, spleen and liver, BM contained a substantial portion of human cells that were CD56/CD16 double negative (DN) but positive for CD244 and CD117, indicating a residual progenitor function in the CD56- fraction of the CD34+ derived cell product. The BM engrafting population was higher in NK cultures at earlier stages of expansion, but was preserved in the day 35- cultured product. The frequency of these cells in the BM increased over time, and showed continued cycling based on in vivo BrdU labeling 28 days post-transfer, suggesting a significant progenitor potential in vivo. Interestingly, DN cells isolated from BM could be efficiently differentiated ex vivo to mature CD56+CD16+ NK cells with in vitro cytotoxic activity against K562. We speculate that under the optimal in vivo conditions these BM engrafting cells may provide a progenitor population to produce a mature NK cell pool in humans, and therefore could contribute to the therapeutic potential of the HSC-derived NK cell product. The in vivo activity of HSC-derived NK cells was further explored using a genetically engineered human AML xenograft model of minimal residual disease (MRD) and initial data indicates significant suppression of AML relapse in animals receiving NK cells following chemotherapy. Collectively, our data demonstrate the utility of humanized mice and in vivo xenograft models in characterizing the biodistribution, persistence, differentiation and functional assessment of human HSC-derived cell therapy products, and characterize the potential of HSC-derived NK cells to be developed as an effective off-the-shelf product for use in adoptive cell therapy approaches in AML. Disclosures: Wunderlich: Celgene Cellular Therapeutics: Research Funding. Shrestha:C: Research Funding. Kang:Celgene Cellular Therapeutics: Employment, Equity Ownership, Patents & Royalties. Law:Celgene Cellular Therapeutics: Employment, Equity Ownership, Patents & Royalties. Jankovic:Celgene Cellular Therapeutics: Employment, Equity Ownership, Patents & Royalties. Zhang:Celgene Cellular Therapeutics: Employment, Equity Ownership, Patents & Royalties. Herzberg:Celgene Cellular Therapeutics: Employment, Equity Ownership, Patents & Royalties. Abbot:Celgene Cellular Therapeutics: Employment, Equity Ownership, Patents & Royalties. Hariri:Celgene Cellular Therapeutics: Employment, Equity Ownership, Patents & Royalties. Mulloy:Celgene Cellular Therapeutics: Research Funding.

Co-Stimulatory Blockade With CTLA4-Ig Permits Transplantation Of Human Hematopoietic Stem Cells and HLA Incompatible T Cells In NOD/SCID γ Null (NSG) Mouse Model

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 1999-1999
Author(s):  
Annie L. Oh ◽  
Dolores Mahmud ◽  
Benedetta Nicolini ◽  
Nadim Mahmud ◽  
Elisa Bonetti ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
T Cells ◽  
Stem Cell ◽  
T Cell ◽  
Nsg Mice ◽  
Human Cd34 ◽  
Cd34 Cells

Abstract Our previous studies have shown the ability of human CD34+ cells to stimulate T cell alloproliferative responses in-vitro. Here, we investigated anti-CD34 T cell alloreactivity in-vivo by co-transplanting human CD34+ cells and allogeneic T cells of an incompatible individual into NSG mice. Human CD34+ cells (2x105/animal) were transplanted with allogeneic T cells at different ratios ranging from 1:50 to 1:0.5, or without T cells as a control. No xenogeneic GVHD was detected at 1:1 CD34:T cell ratio. Engraftment of human CD45+ (huCD45+) cells in mice marrow and spleen was analyzed by flow cytometry. Marrow engraftment of huCD45+ cells at 4 or 8 weeks was significantly decreased in mice transplanted with T cells compared to control mice that did not receive T cells. More importantly, transplantation of T cells at CD34:T cell ratios from 1:50 to 1:0.5 resulted in stem cell rejection since >98% huCD45+ cells detected were CD3+. In mice with stem cell rejection, human T cells had a normal CD4:CD8 ratio and CD4+ cells were mostly CD45RA+. The kinetics of human cell engraftment in the bone marrow and spleen was then analyzed in mice transplanted with CD34+ and allogeneic T cells at 1:1 ratio and sacrificed at 1, 2, or 4 weeks. At 2 weeks post transplant, the bone marrow showed CD34-derived myeloid cells, whereas the spleen showed only allo-T cells. At 4 weeks, all myeloid cells had been rejected and only T cells were detected both in the bone marrow and spleen. Based on our previous in-vitro studies showing that T cell alloreactivity against CD34+ cells is mainly due to B7:CD28 costimulatory activation, we injected the mice with CTLA4-Ig (Abatacept, Bristol Myers Squibb, New York, NY) from d-1 to d+28 post transplantation of CD34+ and allogeneic T cells. Treatment of mice with CTLA4-Ig prevented rejection and allowed CD34+ cells to fully engraft the marrow of NSG mice at 4 weeks with an overall 13± 7% engraftment of huCD45+ marrow cells (n=5) which included: 53±9% CD33+ cells, 22±3% CD14+ monocytes, 7±2% CD1c myeloid dendritic cells, and 4±1% CD34+ cells, while CD19+ B cells were only 3±1% and CD3+ T cells were 0.5±1%. We hypothesize that CTLA4-Ig may induce the apoptotic deletion of alloreactive T cells early in the post transplant period although we could not detect T cells in the spleen as early as 7 or 10 days after transplant. Here we demonstrate that costimulatory blockade with CTLA4-Ig at the time of transplant of human CD34+ cells and incompatible allogeneic T cells can prevent T cell mediated rejection. We also show that the NSG model can be utilized to test immunotherapy strategies aimed at engrafting human stem cells across HLA barriers in-vivo. These results will prompt the design of future clinical trials of CD34+ cell transplantation for patients with severe non-malignant disorders, such as sickle cell anemia, thalassemia, immunodeficiencies or aplastic anemia. Disclosures: No relevant conflicts of interest to declare.

Combination of the glycoengineered Type II CD20 antibody obinutuzumab (GA101) and The novel Bcl-2 selective Inhibitor GDC-0199 Results in superior In Vitro and In Vivo Anti-tumor activity in models Of B-Cell Malignancies

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 4412-4412 ◽  
Author(s):  
Deepak Sampath ◽  
Sylvia Herter ◽  
Frank Herting ◽  
Ellen Ingalla ◽  
Michelle Nannini ◽  
...  
Keyword(s):  
Cell Death ◽  
Single Agent ◽  
Employment Equity ◽  
Direct Cell ◽  
Xenograft Models ◽  
Equity Ownership ◽  
Anti Cd20 ◽  
Agent Treatment

Introduction Obinutuzumab (GA101) is a novel glycoengineered type II, anti-CD20 monoclonal antibody induces a high level of direct cell death. As a result of glycoengineering, GA101 has increased affinity for FcgRIIIa on effector cells resulting in enhanced direct cell death and ADCC induction. GA101 is currently in pivotal clinical trials in CLL, indolent NHL and DLCBL. ABT-199 (GDC-0199) is a novel, orally bioavailable, selective Bcl-2 inhibitor that induces robust apoptosis in preclinical models of hematological malignancies and is currently in clinical trials for CLL, NHL and MM. Based on their complementary mechanisms of action involving increased apoptosis (GDC-0199) or direct cell death (GA101) the combination of anti-CD20 therapy with a Bcl-2 inhibitor has the potential for greater efficacy in treating B lymphoid malignancies. Experimental Methods The combination of GA101 or rituximab with GDC-0199 was studied in vitro utilizing assays that measure direct cell death induction/apoptosis (AxV/Pi positivity) on WSU-DLCL2, SU-DHL4 DLBCL and Z138 MCL cells by FACS and the impact of Bcl-2 inhibition on ADCC induction. In vivo efficacy of the combination of GA101 or rituximab and GDC-0199 was evaluated in SU-DHL4 and Z138 xenograft models. Results GA101 and rituximab enhanced cell death induction when combined with GDC-0199 in SU-DHL4, WSU-DLCL2 and Z138 cell lines. When combined at optimal doses an additive effect of the two drugs was observed. GDC-0199 did not negatively impact the capability of GA101 or rituximab to induce NK-cell mediated ADCC. Combination of GDC-0199 and GA101 induced a greater than additive anti-tumor effects in the SU-DHL4 and Z138 xenograft models resulting in tumor regressions and delay in tumor regrowth when compared to monotherapy. Moreover, continued single-agent treatment with GDC-0199 after combination with GA101 resulted in sustained in vivo efficacy in the SU-DHL4 model. Conclusions Our data demonstrate that the combination of GA101 with GDC-0199 results in enhanced cell death and robust anti-tumor efficacy in xenograft models representing NHL sub-types that is comparable to the combination of rituximab with GDC-0199. In addition, single-agent treatment with GDC-0199 following combination with GA101 sustains efficacy in vivo suggesting a potential benefit in continued maintenance therapy with GDC-0199. Collectively the preclinical data presented here supports clinical investigation of GA101 and GDC-0199 combination therapy, which is currently in a phase Ib clinical trial (clinical trial.gov identifier NCT01685892). Disclosures: Sampath: Genentech: Employment, Equity Ownership. Herter:Roche: Employment. Herting:Roche: Employment. Ingalla:Genentech: Employment. Nannini:Genentech: Employment. Bacac:Roche: Employment. Fairbrother:Genentech: Employment, Equity Ownership. Klein:Roche Glycart AG: Employment.

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