Mouse Model Of Chronic Myeloid Leukemia Research Articles

Structural and Biochemical Studies Confirming the Mechanism of Action of Asciminib, an Agent Specifically Targeting the ABL Myristoyl Pocket (STAMP)

Tyrosine kinase inhibitors (TKIs) that inhibit the transphosphorylation activity of the BCR-ABL1 oncoprotein by binding the ATP-binding site of the catalytic domain of protein kinases are well established as being effective drugs for the treatments of chronic myeloid leukemia (CML). However, the off-target kinase activities of these non-specific TKIs are associated with adverse events that can limit their suitability for the treatment of patients and can negatively impact quality of life. Therefore, a new drug combining high efficacy with minimal side-effects could provide substantial therapeutic advantages. Asciminib is a new investigational agent that at concentrations <10 micromolar has very little effect (residual activities ≥66%) on the transphosphorylation activity of a large panel comprising of 335wild-typeprotein kinases (including ABL1 and ABL2). In this study (33PanQinase® Activity Assay, ProQinase GmbH, Freiburg, Germany) the radiometric kinase assay employed recombinant kinase constructs of the catalytic domains including the ATP-binding site. However, in isothermal calorimetry studies asciminib showed strong binding affinity (KD 0.5 nM) to a much larger construct ofwild-typeABL1 that contains the catalytic, SH2 and SH3 domains (residues 46-534, ABL46-534), with thermodynamic parameters (ΔH = -72.8 kJ/mol, ΔS = -65.3 J/mol/K, resulting in ΔG = -53.2 kJ/mol) indicating strong enthalpy-driven, entropically unfavorable binding. This binding translates to inhibition of tyrosine phosphorylation catalysed by the ABL64-515 construct with a mean IC50 value of 2.6 ± 0.8 nM (radiometric filter binding assay) and 0.5 ± 0.1 nM (fluorescence resonance energy transfer assay). This data shows that asciminib inhibits the kinase activity of ABL1 by an allosteric mechanism which does not involve direct interaction with the ATP-binding pocket. X-ray crystallographic studies of a ternary complex between asciminib, nilotinib and the ABL146-534 protein possessing Thr315Ile and Asp382Asn substitutions show that asciminib binds to ABL1 in a deep-pocket on the C-lobe of the kinase domain, referred to as the myristate (or myristoyl) pocket. Nuclear magnetic resonance studies confirm that asciminib can also form asciminib-ABL1-TKI ternary complexes with thewild-typeABL83-534 protein in solution. Native ABL1 kinase is post-translationally myristoylated at the N-terminal glycine residue and this myristate group plays an important role in autoregulating the kinase by binding to a pocket (myristate pocket) on the catalytic domain of the protein to induce the formation of an inactive conformation of the enzyme. This regulatory mechanism is lost in BCR-ABL1 since the N-terminal region is replaced in the fusion protein with a fragment of the BCR protein, thus rendering the ABL1 kinase constitutively active. The binding of asciminib in this pocket therefore mimics that of myristate, thus stabilizing the inactive state of the ABL1 kinase. Consistent with this binding mode to the ABL1 kinase, asciminib potently inhibits BCR-ABL1 driven proliferation of leukemia-derived cell lines, while having little effect on cells that do not express BCR-ABL1 (Figure 1). Thein vitrodata translates into anti-tumor activity in mouse models of CML where asciminib dose-dependently inhibited the growth of subcutaneous KCL22 cell xenografts, with 30 mg/kg administered twice-daily by oral gavage affording 92% tumor regression (Figure 2). Asciminib, the first-in-class STAMP (Specifically Targeting the ABL Myristoyl Pocket) inhibitor, has several important features as a potential treatment of CML. At physiologically achievable concentrations asciminib can overcome mutations on the ATP-binding site of BCR-ABL1 that impede the binding of TKIs which lead to drug resistance in patients with CML. Secondly, by not inhibiting kinases such as EGFR, KIT, CSF1R, PDGFR or the sSRC-family kinases that are associated with off-target activities of TKIs such as bosutinib, dasatinib, imatinib, nilotinib and ponatinib, asciminib is not expected to be associated with cross-intolerance. Thirdly, asciminib can bind to the ABL1 kinase domain together with ATP-competitive TKIs to form ternary complexes, such that appropriate drug combinations should greatly impede the emergence of drug resistant kinase mutations. Disclosures Schuld: Novartis Pharma AG:Current Employment, Current equity holder in publicly-traded company.Grzesiek:Novartis Pharma:Research Funding.Jahnke:Novartis:Current Employment, Current equity holder in publicly-traded company, Patents & Royalties, Research Funding.Barys:Novartis Pharma AG:Current Employment.Cowan-Jacob:Novartis Pharma AG:Current Employment.Loo:Novartis Pharma AG:Current Employment.Wiget:Novartis Pharma AG:Current Employment.Manley:Novartis Pharma AG:Current Employment.

Discovery of Berberine that Targetedly Induces Autophagic Degradation of both BCR-ABL and BCR-ABL T315I through Recruiting LRSAM1 for Overcoming Imatinib Resistance.

Imatinib, the breakpoint cluster region protein (BCR)/Abelson murine leukemia viral oncogene homolog (ABL) inhibitor, is widely used to treat chronic myeloid leukemia (CML). However, imatinib resistance develops in many patients. Therefore, new drugs with improved therapeutic effects are urgently needed. Berberine (BBR) is a potent BCR-ABL inhibitor for imatinib-sensitive and -resistant CML. Protein structure analysis and virtual screening were used to identify BBR targets in CML. Molecular docking analysis, surface plasmon resonance imaging, nuclear magnetic resonance assays, and thermoshift assays were performed to confirm the BBR target. The change in BCR-ABL protein expression after BBR treatment was assessed by Western blotting. The effects of BBR were assessed in vitro in cell lines, in vivo in mice, and in human CML bone marrow cells as a potential strategy to overcome imatinib resistance. We discovered that BBR bound to the protein tyrosine kinase domain of BCR-ABL. BBR inhibited the activity of BCR-ABL and BCR-ABL with the T315I mutation, and it also degraded these proteins via the autophagic lysosome pathway by recruiting E3 ubiquitin-protein ligase LRSAM1. BBR inhibited the cell viability and colony formation of CML cells and prolonged survival in CML mouse models with imatinib sensitivity and resistance. The results show that BBR directly binds to and degrades BCR-ABL and BCR-ABL T315I via the autophagic lysosome pathway by recruiting LRSAM1. The use of BBR is a new strategy to improve the treatment of patients with CML with imatinib sensitivity or resistance.See related commentary by Elf, p. 3899.

Therapeutic inhibition of Fc\u03b3RIIb signaling targets leukemic stem cells in chronic myeloid leukemia

Despite the successes achieved with molecular targeted inhibition of the oncogenic driver Bcr-Abl in chronic myeloid leukemia (CML), the majority of patients still require lifelong tyrosine kinase inhibitor (TKI) therapy. This is primarily caused by resisting leukemic stem cells (LSCs), which prevent achievement of treatment-free remission in all patients. Here we describe the ITIM (immunoreceptor tyrosine-based inhibition motif)-containing Fc gamma receptor IIb (FcγRIIb, CD32b) for being critical in LSC resistance and show that targeting FcγRIIb downstream signaling, by using a Food and Drug Administration-approved BTK inhibitor, provides a successful therapeutic approach. First, we identified FcγRIIb upregulation in primary CML stem cells. FcγRIIb depletion caused reduced serial re-plaiting efficiency and cell proliferation in malignant cells. FcγRIIb targeting in both a transgenic and retroviral CML mouse model provided in vivo evidence for successful LSC reduction. Subsequently, we identified BTK as a main downstream mediator and targeting the Bcr-Abl-FcγRIIb-BTK axis in primary CML CD34+ cells using ibrutinib, in combination with standard TKI therapy, significantly increased apoptosis in quiescent CML stem cells thereby contributing to the eradication of LSCs.. As a potential curative therapeutic approach, we therefore suggest combining Bcr-Abl TKI therapy along with BTK inhibition.

Targeting of plasminogen activator inhibitor-1 activity promotes elimination of chronic myeloid leukemia stem cells.

Therapeutic strategies that target leukemic stem cells (LSC) provide potential advantages in the treatment of chronic myeloid leukemia (CML). Here we showed that selective blockade of plasminogen activator inhibitor-1 (PAI-1) enhances the susceptibility of CML-LSC to tyrosine kinase inhibitor (TKI), which facilitates the eradication of CML-LSC and leads to sustained remission of the disease. We demonstrated for the first time that the TGF-−PAI-1 axis was selectively augmented in CMLLSC in the bone marrow (BM), thereby protecting CML-LSC from TKI treatment. Furthermore, the combined administration of the TKI imatib plus a PAI-1 inhibitor, in a mouse model of CML, significantly enhanced the eradication of CML cells in the BM and prolonged the survival of CML mice. The combined therapy of imatinib and a PAI-1 inhibitor prevented the recurrence of CML-like disease in serially transplanted recipients, indicating the elimination of CML-LSC. Interestingly, PAI-1 inhibitor treatment augmented membrane-type matrix metalloprotease-1 (MT1-MMP)-dependent motility of CML-LSC, and the anti-CML effect of PAI-1 inhibitor was extinguished by the neutralizing antibody for MT1-MMP, underlining the mechanistic importance of MT1-MMP. Our findings provide evidence of, and a rationale for, a novel therapeutic tactic, based on the blockade of PAI- 1 activity, for CML patients.

Antitumor efficacy of arsenic/interferon in preclinical models of chronic myeloid leukemia resistant to tyrosine kinase inhibitors.

Tyrosine kinase inhibitors (TKIs) are the standard treatment for chronic myeloid leukemia (CML). Despite their clinical success, TKIs are faced with challenges such as treatment resistance, which may be driven by kinase domain mutations, and frequent disease relapse upon the cessation of treatment. The combination of arsenic trioxide (ATO) and interferon-α (IFN) was previously demonstrated to inhibit proliferation and induce apoptosis in CML cell lines, prolong the survival of primary wild-type CML mice, and dramatically decrease the activity of leukemia-initiating cells (LICs). The ATO/IFN combination was tested in vitro on imatinib (IMN)-resistant K562-R and Ar230-R cells. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide and terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling assays were used to evaluate proliferation and apoptosis, respectively. The acridine orange assay was used to assess autophagy, and quantitative reverse transcription-polymerase chain reaction was used to assess the involvement of the hedgehog (Hh) pathway. In vivo, a retroviral transduction/transplantation T315I BCR-ABL CML mouse model was used to assay the effect of the treatment on survival, tumor burden (histopathology and blood counts), and LIC activity (secondary transplantation). In vitro, ATO/IFN synergized to inhibit proliferation and induce apoptosis of IMN-resistant cells with variant modes of resistance. Furthermore, the preclinical effects of ATO/IFN were associated with induction of autophagy along with inhibition of the Hh pathway. Most remarkably, ATO/IFN significantly prolonged the survival of primary T315I-CML mice and displayed a dramatic impairment of disease engraftment in secondary mice, which reflected decreased LIC activity. Collectively, the ATO/IFN strategy has been demonstrated to have the potential to lead to durable remissions in TKI-resistant CML preclinical models and to overcome various TKI-specific mechanisms of resistance.

Abstract 5093: Bcr-Abl leads to mast cell differentiation and promotes degranulation of cells derived from a chronic myeloid leukemia mouse model in a Gab2 dependent manner

Abstract Chronic myeloid leukemia (CML) is driven by the fusion kinase Bcr-Abl, caused by a chromosomal translocation between chromosome 9 and 22. This tyrosine kinase organizes its own signaling network with various proteins, especially the docking protein Gab2. By being recruited to Bcr-Abl via Grb2, Gab2 plays a critical role in this network. We recently applied a transgenic approach to investigate the role of Gab2 in Bcr-Abl driven disease. Using Gab2 knock-out mice, we analyzed the in vivo role of Gab2 in a chronic-phase CML mouse model in which a tetracycline regulated Bcr-Abl transgene is expressed in hematopoietic stem cells in their native microenvironment. We demonstrated that Gab2 deficiency impairs disease development in a steady-state in vivo setting. In the course of this work, we also identified an abnormal number of mast cells infiltrating the kidneys of Bcr-Abl expressing Gab2 proficient mice, a phenotype associated with an inflammatory urothelium and hydronephrosis. Interestingly, this phenotype was completely absent in Gab2 deficient mice. Therefore, we aimed to analyze the role of mast cells in the CML mouse model in more detail. First, we performed bone marrow transplantations, using Bcr-Abl positive donor mice and C57/BL6N mice as recipients. Also in these mice, we observed high mast cell counts in the bone marrow and developed hydronephrosis in some cases, which demonstrates that these alterations were induced by cell-autonomous properties of the Bcr-Abl positive donor cells. Next, we were interested whether these cells are Bcr-Abl transformed mast cell precursors or reactive mast cells resulting from secondary effects of the leukemic disease. Therefore, we isolated bone marrow cells from Bcr-Abl transgenic mice and analyzed them for Bcr-Abl activity and mast cell properties. Strikingly, Bcr-Abl positive progenitors differentiated into mast cells under cytokine free conditions. Next, we assessed mast cell functionality of these cells by degranulation assays measuring β-hexosaminidase activity and cytokine release. Importantly, Bcr-Abl positive cells were more sensitive towards antigen stimulation and displayed a stronger degranulation und higher levels of secreted cytokines compared to Bcr-Abl negative controls. This suggests that Bcr-Abl positive mast cells could be responsible for the inflammatory urothelium and the hydronephrosis which we observed in our CML mouse model. In line with our previous results, Gab2 deficient cells from Bcr-Abl transgenic mice showed no elevated degranulation. In summary, we could show that Bcr-Abl can drive the expansion of mast cells. In addition, Bcr-Abl leads to stronger degranulation in a Gab2 dependent manner. This data and our previous work on Gab2 invite for the further evaluation of Gab2 as a biomarker and as a valuable target in the treatment of CML and, possibly, systemic mastocytosis. Citation Format: Julia Ellermann, Franziska Maria Uhl, Martin Köhler, Julia Huber, Robert Zeiser, Steffen Koschmieder, Konrad Aumann, Tilman Brummer, Sebastian Halbach. Bcr-Abl leads to mast cell differentiation and promotes degranulation of cells derived from a chronic myeloid leukemia mouse model in a Gab2 dependent manner [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5093.

Chronic Myeloid Leukemia: Immunobiology and Novel Immunotherapeutic Approaches.

Imatinib has revolutionized the treatment and prognosis of chronic myeloid leukemia (CML) with survival rates now approaching those of the age-matched healthy population. To be able to discontinue tyrosine kinase inhibitor (TKI) treatment, it is necessary to develop complementary therapies to target minimal residual disease. Recent findings by a number of investigators in both CML mouse models and CML patients offer evidence that many factors in the leukemic microenvironment can collectively contribute to immune escape, including expansion of myeloid-derived suppressor cells, programmed death-1/programmed death-1 ligand interactions resulting in T-cell impairment, expression ofsoluble suppressive factors such as soluble CD25, and down-regulation of MHC molecules by CML cells. Other investigators have studied the role of cytokines on the resistance to TKIs by leukemic stem cells (LSCs) and have highlighted the implication of the JAK/STAT pathway as well as the interleukin 1 (IL-1) signaling pathway. Distinct immunologic strategies have been considered to harness the immune system or trigger LSC death to allow more CML patients to discontinue TKI treatment (so-called functional cure). Successful immunotherapy and TKI combination and the optimal timing of immunotherapy determination represent major challenges for the future.

Telomerase Inhibition with Imetelstat Eradicates β-Catenin Activated Blast Crisis Chronic Myeloid Leukemia Stem Cells

IntroductionLeukemia stem cells (LSCs) in chronic myeloid leukemia (CML) are generated from progenitors that have aberrantly activated self-renewal pathways thereby resulting in tyrosine kinase inhibitor (TKI) resistance. The telomerase complex, consisting of a reverse transcriptase subunit (TERT), an RNA template subunit (TERC), and a protective shelterin scaffold, transcriptionally modulates the Wnt/b-catenin self-renewal pathway. Many malignancies, including BCR-ABL TKI resistant blast crisis CML (BC CML), exhibit robust telomerase activity thereby prompting the development of imetelstat, a competitive inhibitor of telomerase enzymatic activity. Imetelstat is a covalently lipidated 13-mer oligonucleotide that binds with high affinity to the TERC subunit. Recent clinical trials showed early signs of efficacy in myeloproliferative neoplasms. However, the role of imetelstat in selective self-renewing LSC inhibition in CML had not been elucidated. Thus, we performed progenitor RNA sequencing (RNA-seq), stromal co-cultures and humanized LSC primagraft studies to investigate the capacity of imetelstat to selectively inhibit LSC self-renewal and to determine the mechanism of action.Methods and ResultsCytoscape analysis of RNA-seq data derived from FACS-purified progenitors from human blast crisis (BC; n=9) compared with chronic phase (CP; n=8) CML and primary normal (n=6) samples revealed transcriptional upregulation of b-catenin, LEF1, TCF7L1, ABL1 and other key genes within the TERT interactome suggesting a role for TERT activation in human BC LSC generation. Human progenitor LSC-supportive SL/M2 stromal co-culture experiments revealed that combined treatment with a potent BCR-ABL TKI, dasatinib at 1 nM, and imetelstat at 1 or 5 mM significantly inhibited (p<0.001, ANOVA) in vitro self-renewal of BC CML (n=5) compared with age-matched normal bone marrow progenitors. Treatment of primagraft mouse models of human BC CML with 30 mg/kg of imetelstat three times a week for four weeks resulted in a significant reduction in bone marrow progenitor LSC burden compared with mismatch treated controls (p=0.04). Furthermore, qRT-PCR showed decreased β-catenin transcript levels in imetelstat compared with vehicle-treated samples. Finally, FACS analysis revealed a significant reduction in activated b-catenin protein levels in engrafted human myeloid progenitors following imetelstat treatment in the TKI resistant bone marrow niche.ConclusionsNiche responsive interactions between the telomerase complex and the Wnt/b-catenin self-renewal pathway sensitize b-catenin activated LSC to imetelstat in both in vitro and in vivo humanized pre-clinical BC CML models thereby providing a strong rationale for LSC eradication trials involving imetelstat. DisclosuresHuang:Janssen Research & Development, LLC: Employment, Other: I am an employee of Janssen and a stock owner . Jamieson:UC San Diego: Other: I received funding from Janssen Research & Development, LLC.

Dasatinib Exerts Differential Effects on Normal and BCR-ABL Positive Hematopoietic Cells in a Transgenic Mouse Model of Chronic Phase-CML

The second generation tyrosine kinase inhibitor dasatinib is a clinically approved drug for chronic myeloid leukemia (CML) as well as Ph+ acute lymphoblastic leukemia. Dasatinib is a dual-specific inhibitor of ABL and SRC family kinases but shows a broad inhibitory effect on various kinases, including Kit, EGFR, FAK and BTK. In addition to its antileukemic effects, dasatinib was shown to impact on the immune system, including alterations in B-cell development, T-cell differentiation, as well as a transient activation of quiescent hematopoietic stem cells (HSCs) in the bone marrow of wild-type mice. In CML patients treated with dasatinib, the development of pleural effusions and large granular lymphocyte (LGL) lymphocytosis during dasatinib treatment was associated with a favorable response. Non-hematologic adverse events included pulmonary artery hypertension and gastrointestinal symptoms.We investigated the influence of dasatinib (5 or 20 mg/kg p.o.) on the cellular composition of immune cells in our tetracycline-controlled transgenic BCR-ABL mice in comparison to control mice. We analyzed dasatinib-induced effects by flow cytometry in the bone marrow (BM) and spleen under steady-state conditions as well as after bone marrow transplantation. Furthermore, we conducted flow cytometry analysis of stem- and progenitor cells in the BM of BCR-ABL expressing mice treated with dasatinib or vehicle control and performed histophathology to evaluate effects on the BM, spleen, lung and intestine.Our results demonstrated that dasatinib dose-dependently increased Gr1/CD11b positive myeloid cells in the BM of normal mice, while B220 positive B cells and Ter119 positive erythrocytic cells were reduced. NK1.1 positive NK cells were decreased in BM but increased in the spleen. Interestingly, the lower dose (5 mg/kg) induced an increase of the percentage of CD41 positive megakaryocytic cells in BM and B cells in the spleen, while higher doses (20 mg/kg) decreased both percentages. The BCR-ABL-induced phenotype was strongly reversed by dasatinib in BCR-ABL expressing transgenic animals under steady-state conditions and in a transplantation setting: the oncogene-induced upregulation of granulocytes, megakaryocytes, and the reduction of erythroid cells as well as of B-lymphocytes was antagonized by dasatinib. Concurrently, a strong decrease of Linneg Sca1+ c-kit+ (LSK) HSCs and an increase of MEPs together with a reduction of GMPs were observed. Although the overall lymphocyte population was reduced in the BCR-ABL-expressing mice and replaced by granulocytes, we observed an increase of the proportion of CD3+, CD4+ and CD8+T cells among lymphocytes, and this increase was reverted by dasatinib treatment. Conversely, dasatinib did not significantly alter NK1.1 cells in these mice. Our observations in BCR-ABL-positive animals are in part contrary to the findings in wild-type animals, but this may be explained by the reversion of BCR-ABL effects in addition to the effects of dasatinib on normal hematopoietic cells. In addition to the above-mentioned changes, we observed a reduction of BCR-ABL mediated granulocyte infiltration of the small intestine indicating a beneficial effect in attenuating inflammation of the bowel. There was no significant alteration of the bronchial epithelium or bronchial arteries.Altogether, dasatinib robustly reduced the CML phenotype in vivo in our transgenic mouse model, and this effect included stem and progenitor populations and was stronger than the effects we have previously described for imatinib in this model. Thus, our transgenic CML mouse model is well suited to examine dasatinib effects on normal and malignant hematopoietic cells in vivo. Interestingly, there was a differential effect on myeloid cells in normal vs. CML mice, suggesting different targets in these cells. (CS and NC contributed equally to this study) DisclosuresBraunschweig:Bristol-Myers-Squibb: Honoraria; MSD Sharp & Dome: Honoraria. Brümmendorf:Pfizer: Consultancy, Honoraria; Ariad: Consultancy, Honoraria; Bristol-Myers Squibb: Consultancy, Honoraria; Novartis: Consultancy, Honoraria, Research Funding; Patent on the use of imatinib and hypusination inhibitors: Patents & Royalties. Mustjoki:Bristol-Myers Squibb: Honoraria, Research Funding; Ariad: Research Funding; Pfizer: Honoraria, Research Funding; Novartis: Honoraria, Research Funding. Koschmieder:Bristol-Myers Squibb: Honoraria, Membership on an entity’s Board of Directors or advisory committees, Other: Travel funding to conferences, Research Funding.

Potential role of Wnt/β-catenin signaling in blastic transformation of chronic myeloid leukemia: cross talk between β-catenin and BCR-ABL.

Chronic myeloid leukemia (CML) results from malignant transformation of hematopoietic stem cells induced by the BCR-ABL oncogene. Transformation from chronic to blastic phase is the lethal step in CML. Leukemic stem cells (LSCs) are the basic reason for blastic transformation. It has been shown that Wnt/β-catenin signaling contributes to the self-renewal capacity and proliferation of LSCs in CML. However, the role of Wnt/β-catenin signaling in blastic transformation of CML is still obscure. Here, we explored the relationship between BCR-ABL and β-catenin signaling in vitro and in vivo. We found that BCR-ABL stimulated β-catenin via activation of PI3K/AKT signaling in blastic phase CML cells. Inhibition of the kinase activity of BCR-ABL, PI3K, or AKT decreased the level of β-catenin in both K562 cells and a CML mouse model and suppressed the transcription of downstream target genes (c-myc and cyclin D1). In addition, inhibition of the BCR-ABL/PI3K/AKT pathway delayed the disease progression in the CML mouse model. To further explore the role of β-catenin in the self-renewal and survival of CML LSCs, we established a secondary transplantation CML mouse model. Our data revealed that inhibition of the BCR-ABL/PI3K/AKT pathway reduced the tumor-initiating ability of K562 cells, decreased leukemia cell infiltration into peripheral blood and bone marrow, and prolonged the survival of mice. In conclusion, our data indicate a close relationship between β-catenin and BCR-ABL/PI3K/AKT in blastic phase CML. β-Catenin inhibition may be of therapeutic value by targeting LSCs in combination with a tyrosine kinase inhibitor, which may delay blastic transformation of CML.

Distinct GAB2 signaling pathways are essential for myeloid and lymphoid transformation and leukemogenesis by BCR-ABL1

AbstractTyrosine kinase inhibitors (TKIs) directed against BCR-ABL1, the product of the Philadelphia (Ph) chromosome, have revolutionized treatment of patients with chronic myeloid leukemia (CML). However, acquired resistance to TKIs is a significant clinical problem in CML, and TKI therapy is much less effective against Ph+ B-cell acute lymphoblastic leukemia (B-ALL). BCR-ABL1, via phosphorylated Tyr177, recruits the adapter GRB2-associated binding protein 2 (GAB2) as part of a GRB2/GAB2 complex. We showed previously that GAB2 is essential for BCR-ABL1–evoked myeloid transformation in vitro. Using a genetic strategy and mouse models of CML and B-ALL, we show here that GAB2 is essential for myeloid and lymphoid leukemogenesis by BCR-ABL1. In the mouse model, recipients of BCR-ABL1–transduced Gab2−/− bone marrow failed to develop CML-like myeloproliferative neoplasia. Leukemogenesis was restored by expression of GAB2 but not by GAB2 mutants lacking binding sites for its effectors phosphatidylinositol 3-kinase (PI3K) or SRC homology 2–containing phosphotyrosine phosphatase 2 (SHP2). GAB2 deficiency also attenuated BCR-ABL1–induced B-ALL, but only the SHP2 binding site was required. The SHP2 and PI3K binding sites were differentially required for signaling downstream of GAB2. Hence, GAB2 transmits critical transforming signals from Tyr177 to PI3K and SHP2 for CML pathogenesis, whereas only the GAB2–SHP2 pathway is essential for lymphoid leukemogenesis. Given that GAB2 is dispensable for normal hematopoiesis, GAB2 and its effectors PI3K and SHP2 represent promising targets for therapy in Ph+ hematologic neoplasms.

CML Mouse Model Generated from Leukemia Stem Cells.

Chronic myeloid leukemia (CML) is a myeloproliferative disorder with a high number of well-differentiated neutrophils in peripheral blood and myeloid cells in bone marrow (BM). CML is derived from the hematopoietic stem cells (HSCs) with the Philadelphia chromosome (Ph(+), t(9;22)-(q34;q11)), resulting in generating a fusion oncogene, BCR/ABL1. HSCs with Ph(+) are defined as leukemia stem cells (LSCs), a subpopulation cell at the apex of hierarchies in leukemia cells and responsible for the disease continuous propagation. Several kinds of CML models have been developed to reveal the mechanism of CML pathogenesis and evaluate therapeutic drugs in the past three decades. Here, we describe the procedures to generate a CML mouse model by introducing BCR/ABL1 into Lin(-)Sca1(+) cKit(+) population cells purified from mouse bone marrow. In CML retroviral transduction/transplantation mouse models, this modified model can mimic CML pathogenesis on high fidelity.

New Mouse Models to Investigate the Efficacy of Drug Combinations in Human Chronic Myeloid Leukemia.

Chronic myeloid leukemia (CML) comprises a simple and effective paradigm for generating new insights into the cellular origin, pathogenesis, and treatment of many types of human cancer. In particular, mouse models of CML have greatly facilitated the understanding of the underlying molecular mechanisms and pathogenesis of this disease and have led to the identification of new drug targets that in some cases offer the possibility of functional cure. There are currently three established CML mouse models: the BCR-ABL transgenic model, the BCR-ABL retroviral transduction/transplantation model, and the xenotransplant immunodeficient model. Each has its own unique advantages and disadvantages. Depending on the question of interest, some models may be more appropriate than others. In this chapter, we describe a newly developed xenotransplant mouse model to determine the efficacy of novel therapeutic agents, either alone or in combination. The model facilitates the evaluation of the frequency of leukemic stem cells with long-term leukemia-initiating activity, a critical subcellular population that causes disease relapse and progression, through the utilization of primary CD34(+) CML stem/progenitor cells obtained from CML patients at diagnosis and prior to drug treatment. We have also investigated the effectiveness of new combination treatment strategies designed to prevent the development of leukemia in vivo using BCR-ABL (+) blast crisis cells as a model system. These types of in vivo studies are important for the prediction of individual patient responses to drug therapy, and have the potential to facilitate the design of personalized combination therapy strategies.

Chronic Myeloid Leukemia (CML) Mouse Model in Translational Research.

Chronic myeloid leukemia (CML) is a myeloproliferative disorder characterized by increased proliferation of granulocytic cells without the loss of their capability to differentiate. CML is a clonal disease, originated at the level of Hematopoietic Stem Cells with the Philadelphia chromosome resulting from a reciprocal translocation between the chromosomes 9 and 22t(9;22)-(q34;q11). This translocation produces a fusion gene known as BCR-ABL which acquires uncontrolled tyrosine kinase activity, constantly turning on its downstream signaling molecules/pathways, and promoting proliferation of leukemia cell through anti-apoptosis and acquisition of additional mutations. To evaluate the role of each critical downstream signaling molecule of BCR-ABL and test therapeutic drugs in vivo, it is important to use physiological mouse disease models. Here, we describe a mouse model of CML induced by BCR-ABL retrovirus (MSCV-BCR-ABL-GFP; MIG-BCR-ABL) and how to use this model in translational research.Moreover, to expand the application of this retrovirus induced CML model in a lot of conditional knockout mouse strain, we modified this vector to a triple gene coexpression vector in which we can co-express BCR-ABL, GFP, and a third gene which will be tested in different systems. To apply this triple gene system in conditional gene knockout strains, we can validate the CML development in the knockout mice and trace the leukemia cell following the GFP marker. In this protocol, we also describe how we utilize this triple gene system to prove the function of Pten as a tumor suppressor in leukemogenesis. Overall, this triple gene system expands our research spectrum in current conditional gene knockout strains and benefits our CML translational research.

A Novel TNF-Alpha Antibody Based Therapeutic Approach to Target Leukemic Stem Cells in Bcr-Abl Disease

Chronic myeloid leukemia (CML) reflects the biology of a stem cell driven neoplasm. The cytogenetic marker t(9;22) gives rise to Bcr-Abl protein that activates various signaling pathways resulting in increased proliferation and differentiation, protection from apoptosis and altered adhesion properties of CML cells. The implementation of Bcr-Abl tyrosine kinase inhibitors (TKI) has greatly improved therapy outcome of CML patients. However, more than one third of newly diagnosed CML patients develops primary or secondary resistance mostly due to Bcr-Abl mutations or intolerance to TKIs. Moreover, even in patients without mutations, mechanisms such as low persisting Bcr-Abl levels in patients on TKI and/or a lack of oncogene addiction have been described to induce CML stem cell persistence.We have previously demonstrated Bcr-Abl independent stem cell persistence using inducible SCLtTA/Bcr-Abl mice (BLOOD 2010, 115:3185; BLOOD 2012, 119:1501), and recent data suggest an important role for TNF-alpha in MPN stem cell persistence (BLOOD 2011, 118:6392), including CML (BLOOD 2013, 112:3335). Therefore, we have now interrogated our mouse model for a novel stem cell directed therapeutic approach, using pharmacologic inhibition of TNF-alpha.Analysis of TNF-alpha expression by qRT-PCR in Bcr-Abl infected 32Dcl3 and Ba/F3 cells revealed elevated cytokine expression upon Bcr-Abl transduction. Imatinib treatment of 32Dcl3:Bcr-Abl cells reverted elevated TNF-alpha levels suggesting that TNF-alpha upregulation is kinase mediated in this cell line. ChIP-Seq of TKI treated vs non-treated 32Dcl3:Bcr-Abl cells showed H3K9 acetylation of the TNF-alpha promoter in untreated cells, which decreased upon Bcr-Abl inhibition. Subsequently, we analyzed TNF-alpha expression in lin-;Sca-1+;c-kit+ (LSK) cells isolated from SCLtTA/Bcr-Abl mice that had been induced to express Bcr-Abl for 3 weeks. Leukemic LSK cells showed again significantly increased TNF-alpha expression. Interestingly, TNF-alpha RNA levels were not altered in total bone marrow (BM) cells isolated from transplanted leukemic Bcr-Abl mice compared to controls, suggesting a stem cell specific mechanism regulating TNF-alpha expression in vivo. Next, we tested the therapeutic effect of TNF-alpha inhibition combined with TKI therapy. We applied the chimeric antibody infliximab that blocks soluble as well as membrane-bound TNF-alpha. We transplanted 1.5x105 wildtype (wt) or Bcr-Abl BM cells expressing CD45.1 into 10 Gy irradiated CD45.2+ wt recipients (wt n=4; Bcr-Abl n=24) and allowed these cells to engraft and expand for 2 weeks. We then either treated Bcr-Abl transplanted mice with nilotinib alone (50mg/kg, daily) or combined the TKI therapy with infliximab (10mg/kg, weekly i.v.). As controls, we either treated Bcr-Abl transplanted mice with vehicle alone (daily) or together with IgG (10mg/kg, i.v., weekly). Wt recipients remained untreated. After 2.5 weeks of therapy, we sacrificed all mice for analyses. Spleen weight of vehicle and vehicle+IgG treated mice were increased compared to nilotinib and nilotinib+infliximab treated recipients (Table 1).Table 1Analyses of mice that were subjected to TNF-alpha and Bcr-Abl inhibitionwtBcr-Ablvehiclevehicle + IgGnilotinibnilotinib + infliximabspleen weight (mg)73 ± 5274 ± 63280 ± 55125 ± 5185 ± 31% of lin- cells4.8 ± 1.131.1 ± 11.025.0 ± 14.13.4 ± 1.03.1 ± 1.4% of LSK/CD45.1+ cells5.7 ± 0.741.2 ± 5.931.6 ± 11.720.3 ± 10.87.2 ± 3.4FACS analyses s that lin- cells were significantly elevated in vehicle and vehicle+IgG treated Bcr-Abl recipients compared to wt mice. Nilotinib and nilotinib+infliximab therapy decreased lin- cells to 3.4% and 3.1% respectively. As expected, leukemic CD45.1+ LSK cells were significantly increased in vehicle (7-fold) and vehicle+IgG (5.5-fold) treated mice compared to CD45.1+ wt LSK cells. Nilotinib treatment decreased the elevated levels of CD45.1+ LSK cells compared to wt controls to 3.6-fold but combining TKI+infliximab decreased the leukemic stem cell population further to 1.3-fold difference compared to wt mice.In conclusion, we show that TNF-alpha expression is elevated in Bcr-Abl positive leukemic stem cells and this cell population is significantly reduced by combining TKI therapy with an anti TNF-alpha approach. Our data reveal that this combinational therapy is a powerful tool to target the TKI resistant stem cell population in CML. DisclosuresKoschmieder:Janssen Cilag: Other: Travel reimbursement for scientific conferences; Pfizer: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel reimbursement for scientific conferences; Sanofi: Membership on an entity's Board of Directors or advisory committees; Baxalta/CTI: Membership on an entity's Board of Directors or advisory committees; Novartis Foundation: Research Funding; Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel reimbursement for scientific conferences, Research Funding. Brümmendorf:Ariad: Consultancy, Honoraria; Bristol-Meyers Squibb: Consultancy, Honoraria; Novartis: Consultancy, Honoraria, Research Funding; Pfizer: Consultancy, Honoraria. Schemionek:Novartis Foundation for Therapeutic Research: Research Funding. Off Label Use: Infliximab used in CML mouse model.

Inhibition of CML Stem Cell Renewal By the Porcupine Inhibitor WNT974

Treatment of chronic myeloid leukemia (CML) with BCR-ABL tyrosine kinase inhibitors (TKIs) fails to eradicate the leukemia stem cells (LSCs) from which the disease arises. Others and we have shown that extrinsic signals from the bone marrow (BM) microenvironment play an important role in the resistance of CML LSC to TKI treatment. Our studies indicate that microenvironmental Wnt signaling may play a role for in protecting CML LSC from TKI treatment (Blood. 2013; 121(10):1824-38). Wnt secretion and activity requires their palmitoylation by the Porcupine acyltransferase (PORCN). WNT974 is a potent PORCN inhibitor that inhibits Wnt signaling and demonstrates in vivo efficacy against Wnt-dependent tumors (PNAS. 2013; 110(50):20224-9). We have investigated whether WNT974 could sensitize CML stem/progenitor cells to TKI treatment. We observed that CML CD34+ cells show enhanced increased phospho-LRP6 (ser1490) expression, β-catenin protein levels, and colony-forming cell (CFC) growth following exposure to recombinant Wnt3a (200ng/ml) compared to normal CD34+ cells (isolated from cord blood of healthy donors) indicating an enhanced Wnt signaling response. We observed significant upregulation of FZD4 and FZD5 expression in CML compared to normal CD34+ cells on qPCR analysis, which could potentially contribute to their enhanced Wnt sensitivity. CML CD34+ cells enhanced Wnt signaling following co-culture with human bone marrow mesenchymal stromal cells (MSC) as evidenced by increased β-catenin expression and nuclear translocation, and increased expression of the Wnt target genes c-Myc, Cyclin-D1, PPARδ, and Axin2. WNT974 exposure reduced Wnt secretion from MSC and inhibited Wnt signaling in CML CD34+ cells both in the absence and presence of MSC. Treatment with WNT974 (1μM) in combination with nilotinib (Nil, 1μM) resulted in significant inhibition of CML CFC growth, both in the presence and absence of MSC, and to a significantly greater extent than the normal CFC growth. Although, no difference in engraftment of normal CD34+ cells treated with individual drugs or combination in immunodeficient mice after 16 weeks was observed, treatment of CML CD34+ cells with WNT974 + Nil resulted in significantly decreased engraftment of BCR-ABL+ CML LSC. We further investigated the effects of WNT974 and Nil on CML hematopoiesis in vivo using a transgenic BCR-ABL mouse model of CML. BM cells from CML mice were transplanted into congenic wild-type FVB/N mice to generate CML-like disease in recipient mice. Three weeks after transplantation, mice were treated with Vehicle, WNT974 (5mg/kg bid p.o), Nil (50mg/kg qd p.o), or the combination for 2 weeks. Treatment with WNT974 + Nil resulted in significantly greater reduction in WBC levels, %neutrophils and myeloid cells (Gr-1+ CD11b+) in the peripheral blood of CML mice compared with Nil alone. Long-term hematopoietic stem cells (LTHSC) and committed progenitors (MPP, CMP, GMP) were significantly reduced in the BM and spleen of mice treated with combination compared to individual drugs. Next, we evaluated subsequent survival of mice after completion of 3 weeks of drug treatment. Whereas all control mice died by 30 days, mice treated with the combination of WNT974 + Nil showed significantly prolonged survival after discontinuation of treatment when compared to individual drug-treated mice (p=0.0146). To assess the effects on LSC self-renewal potential, BM cells from primary drug-treated mice were transplanted into normal secondary recipient mice. At 12 weeks post-transplantation, there was a significant reduction in LTHSC, MPP, and GMP populations in the BM of mice transplanted with cells from primary mice treated with the combination compared with individual drugs, indicating reduced LSC self-renewal. Finally, we evaluated the effect of in vivo treatment on Wnt/β-catenin signaling in leukemic cells. Significant downregulation of c-Myc, Cyclin D1, and Axin2 was seen on qPCR performed on c-Kit+ BM progenitors from mice treated with WNT974 and Nil+WNT974 compared to vehicle treated mice. Therefore, we conclude that inhibition of Wnt secretion and signaling by treatment with the PORCN inhibitor WNT974 effectively and selectively inhibits the self-renewal capacity of CML LSC. These results support the further evaluation of combinations of PORCN inhibitors with TKI treatment for selective targeting of CML LSC. DisclosuresWang:Novartis Institute of Biomedical Research: Employment. Mclaughlin:Novartis Institute of Biomedical Research: Employment.

CD150(-) Side Population Defines Leukemia Stem Cells in a BALB/c Mouse Model of CML and Is Depleted by Genetic Loss of SIRT1.

Leukemia stem cells (LSCs) of chronic myeloid leukemia (CML) are refractory to tyrosine kinase inhibitor treatment, persist in the residual disease, and are important source for disease recurrence. Better understanding CML LSCs will help devise new strategies to eradicate these cells. The BALB/c mouse model of CML using retroviral bone marrow transduction and transplantation is a widely used mouse model system for CML, but LSCs in this model are poorly characterized. Here, we show that lineage negative CD150(-) side population (CD150(-)SP), but not CD150(+)SP, are CML LSCs in this model, although both CD150(-)SP and CD150(+)SP cells are enriched for long-term hematopoietic stem cells in normal BALB/c mice. We previously showed that BCR-ABL transformation activates protein lysine deacetylase SIRT1 and inhibition of SIRT1 sensitizes CML stem/progenitor cells to tyrosine kinase inhibitors by acetylating and activating p53. In this study, we demonstrate that SIRT1 homozygous knockout substantially reduces CD150(-)SP CML LSCs, and compromises the maintenance of CML LSCs in the BALB/c model. We identified several molecular alterations in CD150(-)SP LSCs that included the elevated expression of cyclin-dependent kinase Cdk6 facilitating LSC activation and significantly reduced p53 expression. SIRT1 knockout suppressed Cdk6 expression and likely increases p53 protein functions through deacetylation without increasing its expression. Our results shed novel insight into CML LSCs and support a crucial role of SIRT1 in CML LSCs. Our study also provides a novel means for assessing new agents to eradicate CML LSCs.

PKC Pathways Mediate BCR-ABL-Independent Imatinib Resistance in Chronic Myeloid Leukemia

Abstract Resistance to imatinib (IM) has posed a great obstacle in treating Chronic Myeloid Leukemia(CML). New generations of tyrosine kinase inhibitors (TKIs) have been developed for BCR-ABL dependent IM resistance, which is commonly due to BCR-ABL kinase-domain mutations. However, in 50% or more of IM-resistant CML patients there is no mutation in BCR-ABL, and intrinsic resistance of CML stem cells often causes disease relapse. Understanding the mechanism of BCR-ABL independent IM resistance is therefore essential for devising strategies to eradicate resistant and residual leukemia. Through a genome-wide shRNA screen in a IM-sensitive human CML cell line, K562 cells, we identified 16 imatinib-sensitizing genes (IMSGs), knockdown of which increased IM resistance of multiple human CML cell lines (e.g., K562, KYO-1 cells. &gt;2 folds increment), and primary CML mice bone marrow cells (&gt; 2 folds of CFUs). Knocking down 11 IMSGs increased IC50IM by more than 5 folds, and also conferred equivalent level of resistance to the second generation TKI, Dasatinib. Extensive signaling pathway analysis by immune-blotting for characteristic protein phosphorylation markers revealed co-activation of PKC and MEK/ERK pathway in majority of IMSG knockdown K562 cell lines. qRT-PCR analysis in IMSG knockdown cells identified PRKCH as the predominant deregulated PKC family member. Mild overexpression of PRKCH increased IC50IM by 10-20 folds, and in vitro phosphorylation confirmed that PRKCH could directly phosphorylate and activate CRAF, therefore MEK/ERK pathway. PRKCH is highly expressed in IM-resistant samples with wildtype BCR-ABL compared to those with mutant BCR-ABL (p&lt;0.05, Mann Whitney test) or IM-sensitive samples (p&lt;0.01, Mann Whitney test). Accordingly, multiple IMSGs were also significantly down-regulated (i.e.,CLEC5A, ELF5, WNT7B, p&lt;0.05), supporting a suppressive role of diverse IMSGs on PRKCH expression. Specifically, we found ELF5 acted as a transcription repressor of PRKCH. To investigate whether simultaneous inhibition of BCR-ABL and MEK/ERK signaling could more efficiently kill BCR-ABL-independent IM-resistant CML cells, we analyzed the effect of combining IM treatment with a FDA-approved MEK inhibitor, Trametinib, in multiple BCR-ABL independent IM-resistant CML mouse models, and primary resistant CML cells. Treatment with both drugs showed a substantially greater effect than either drug alone, and in many instances, the effect of combined drug treatment was synergistic. qRT-PCR analysis showed that PRKCH expression in IM-resistant CML stem cells is higher than that in IM-sensitive CML progenitors (4~25 folds, p&lt;0.01, Student’s t test), implying an important role of PRKCH in CML stem cell resistance. We established a PRKCH knockdown CML mouse model by co-expressing BCR-ABL and a PRKCH shRNA and performed stem cells analysis with or without IM treatment. Although both PRKCH knockdown and IM treatment abolishes MEK/ERK signaling in CML progenitors, only PRKCH controls MEK/ERK pathway in CML stem cells while IM barely has any effect. Combined IM treatment with PRKCH knockdown or MEK inhibition substantially increased the apoptosis rate of CML stem cells (p&lt;0.01, Student’s t test), and prolonged CML mice survival(n=5, p&lt;0.05, Log-rank test). In addition to MEK/ERK pathway, we found PRKCH could activate PKD2, a downstream effector of novel PKCs. PRKCH expression increases upon IM treatment, along with increased PKD2 activity (S916 phosphorylation) in multiple CML cell lines(e.g., K562, KYO-1, LAMA84). PKD2 expression was also induced by IM in primary human CML stem cells. PKD2 promotes cell survival by activating NF-kB pathway, and IM treatment increased p65/RelA phosphorylation at S536 and its nuclear translocation, together with over 4 folds increase in expression of anti-apoptotic p65 target genes (e.g., CIAP2, Bfl-1/A1). Pharmacological inhibition using pan-PKD inhibitor, CRT0066101, completely abolishes IM induced p65 phosphorylation and increased the killing of CML cells by IM. This study for the first time reveals the critical role of PKC pathway in BCR-ABL independent IM resistance in CML. The validation of MEK/ERK and PKD/NF-kB pathway as critical downstream effectors provided specific targets for small molecule therapy in TKI-resistant CML and other malignancies caused by deregulated PKC pathways. Disclosures Cerny: Cellerant Therapeutics: Honoraria, Research Funding.

Malignant Reprogramming of Progenitors into Leukemia Stem Cells Is Enhanced By Upregulation of CD44 Transcript Variant 3 in Malignant Microenvironments

IntroductionMalignant reprogramming, first described in chronic myeloid leukemia (CML), occurs upon activation of the Wnt/b-catenin pathway in granulocyte-macrophage progenitors (GMPs) that gain the capacity to self-renew and contribute to the emergence of BCR-ABL1 tyrosine kinase inhibitor (TKI) resistant blast crisis CML. Deregulation of the Wnt/b-catenin target gene, CD44, plays a vital role in leukemia stem cell (LSC) maintenance in the malignant microenvironment in mouse models of CML. However, extensive alternative mRNA splicing in humans results in expression of multiple CD44 isoforms, some of which have been implicated in cancer invasion and metastasis. In this study we investigated the role of CD44 splice variant expression on human blast crisis LSC maintenance in the malignant niche.Methods and ResultsCD44 Isoform Expression AnalysisTo investigate the splice isoform expression pattern of CD44, whole transcriptome RNA sequencing (RNA Seq; Illumina HiSeq 2000) was performed on FACS sorted chronic phase (CP; n=8) and blast crisis (BC; n=8) CML progenitors (CD34+CD38+Lin-) as well as their normal counterparts from cord blood (CB) (n=7) and adult peripheral blood (NPB; n=4). While whole gene expression analysis revealed upregulation of CD44 in blast crisis compared with chronic phase and normal progenitors, a plethora of CD44 transcript variants were also detected including variants 3, 4 (CD44s), 5, 6, 7, 8. Notably, RNA Seq isoform analysis detected a higher expression of CD44 transcript variant 3 in BC compared to CP and CB and NPB. Moreover, CD44 transcript variant 3 gene expression was highly expressed in undifferentied human embryonic stem cells (hESCs) while differentiated hESCs (embryoid bodies) had low expression, suggesting CD44 transcript variant 3 to be important for pluripotency.Lentiviral CD44 Variant 3 OverexpressionTo directly determine the impact of CD44 variant 3 expression on malignant reprogramming of CP progenitors into self-renewing LSC, we developed a lentiviral human CD44 variant 3 overexpression vector and transduced CP CML progenitors. Transduced CP progenitors harbored increased expression of migration specific markers, such as osteopontin and ICAM1, as well as an upregulation of the pro-survival long isoforms of BCL2 family members BCLX and MCL1, thereby enhancing survival and replating in hematopoietic progenitor assays. Moreover, hESCs transduced with CD44 transcript variant 3 showed upregulation of pro-survival BCL2 isoforms, enhanced proliferation and as well as maintenance of an undifferentiated state, suggesting that CD44 transcript variant 3 promotes pluripotency.Targeted Inhibition of CD44 variant 3 Expressing LSCHumanized RAG2-/-gc-/-mice engrafted with CD34+ BC CML patient samples showed a significant reduction of human progenitor cells post treatment with a clinical grade CD44 mAb, both alone and in combination with Dasatinib in all hematopoietic niches. Bone marrow and spleen samples from primary transplanted mice show a reduced gene expression level of CD44 and CD44 transcript variant 3 upon combination treatment of CD44 and Dasatinib. Most importantly, serial transplantation of progenitors treated with the CD44 mAb as well as in combination with Dasatinib revealed a significant reduction in LSC self-renewal capacity commensurate with a reduction in CD44 variant 3 expression.ConclusionsUpregulation of an embryonic splice variant of CD44, variant 3, expands pluripotent stem cell populations and promotes malignant reprogramming of CML progenitors into self-renewing LSC. Treatment with a humanized CD44 specific mAb sensitizes CML LSC residing in malignant niches to Dasatinib. From these results CD44 mAb appears to be an excellent antibody for future combination clinical studies aimed at eradicating therapy resistant blast crisis LSC in CML. In addition, these observations strongly suggest that CD44 transcript variant 3 upregulation serves as a biomarker of progression from CP to BC as well as the generation of TKI resistant LSCs, with the potential of being a more specific target for future combination therapies. DisclosuresNo relevant conflicts of interest to declare.

Genetic Depletion of Fc Gamma Receptor 2b Affects CML Stem Cell Biology

Chronic myeloid leukemia (CML) is caused by the acquisition of the Philadelphia chromosome t(9;22) at the hematopoietic stem cell (HSC) level. The resulting Bcr-Abl protein loses the Abl autoinhibitory function and gains the Bcr oligomerization domain resulting in the constitutive activation of the Abl tyrosine kinase. The implementation of Bcr-Abl tyrosine kinase inhibitors (TKI) has greatly improved therapy outcome of CML patients. However, various mechanisms such as the acquisition of tyrosine kinase domain mutations or kinase-independent alterations have been described to result in therapy resistance. Using an inducible transgenic SCLtTAxBcr-Abl CML mouse model, we previously demonstrated that Bcr-Abl inhibition leads to eradication of mature leukemic cells but spares disease-driving leukemic stem cells (LSC) due to a lack of oncogene-addiction (Schemionek et al., BLOOD 2010; Hamilton, Helgason, Schemionek et al., BLOOD 2012). Aiming to identify new Bcr-Abl targets that could overcome therapy resistance of stem cells, we previously performed a microarray analysis using murine LSC. Expression of FcγRIIb (Fc gamma receptor 2b; CD32) was significantly increased 2.79-fold in Bcr-Abl positive vs. control Lin-Sca1+c-kit+(LSK) cells. The FcγRIIb contains an immuno receptor tyrosine-based inhibitory motif (ITIM) and inhibits intracellular signaling upon activation. Phosphorylation of the receptor is mainly mediated via Src kinase Lyn and enables the receptor to recruit Src homology 2 domain-containing phosphatases.Using qRT-PCR, we confirmed 2.83-fold upregulation of FcγRIIb within the LSK population of CML mice. Retroviral infection of 32D cells and primary BM cells with Bcr-Abl significantly increased FcγRIIb expression level (90.3-fold and 13.2-fold, resp., p<0.005 each vs. emtpy vector). TKI treatment of Bcr-Abl positive 32D cells did not affect FcγRIIb mRNA levels or surface expression. Bcr-Abl infection of lineage-depleted BM cells decreased growth factor independent colony forming capability (5.5-fold, p<0.05) and the proliferation potential of FcγRIIb-/- lineage negative bone marrow (cells) compared to wild type (wt) in the absence of cytokines. Transplantation of Bcr-Abl infected FcγRIIb-/- BM cells significantly reduced white blood cell count (WBC) of the recipients as compared to wt Bcr-Abl positive cells by 2.4-fold (p<0.05). Interestingly, WBC of Bcr-Abl positive FcγRIIb-/- mice was similar to FcγRIIb-/- empty vector control animals (p=0.875) as well as wt control mice (p=0.241). CML development was analyzed upon autopsy by FACS of BM and spleen cells and showed a 2.0-fold reduction (p<0.05) of leukemic c-kit positive cells as well as a reduction in spleen weight in recipients of Bcr-Abl transduced FcγRIIb-/- cells (293mg ± 30) vs. Bcr-Abl transduced wt controls (381mg ± 13; p<0.05). Moreover, total numbers of leukemic LSK cells were significantly decreased by 3.5-fold in BM and 3-fold in spleen of Bcr-Abl transduced FcγRIIb-/- BM recipients compared to recipients of Bcr-Abl transduced wt cells. Transplantation of empty vector transduced FcγRIIb-/- vs. empty vector transduced wt control cells did not result in a decrease of knock-out cells showing that the observed effects were specific for impairment of Bcr-Abl transforming activity by loss of FcγRIIb but not due to FcγRIIb depletion alone.In conclusion, we show that FcγRIIb is upregulated in the stem cell compartment of CML mice and that this is due to Bcr-Abl expression. The absence of FcγRIIb impairs leukemic cell growth in vitro and in vivo. Moreover, genetic depletion of the receptor decreases LSC numbers and affects CML development in vivo suggesting that FcγRIIb plays an important role in Bcr-Abl leukemogenesis, making it an attractive novel stem cell specific target for CML therapy. (*Authors contributed equally) DisclosuresBrümmendorf:Novartis: Consultancy, Honoraria, Membership on an entity’s Board of Directors or advisory committees, Patents & Royalties, Research Funding. Koschmieder*:Novartis: Consultancy, Honoraria, Other, Research Funding.