Targeting mTORC1/2 by a mTOR Kinase Inhibitor (PP242) induces Apoptosis In AML Cells Under Conditions Mimicking Bone Marrow Microenvironment

Abstract Abstract 778 The prognosis of patients with acute myeloid leukemia (AML) remains poor. Our studies have demonstrated that chemoresistance of AML is not solely due to increased survival signaling in AML cells, but is also enhanced by microenvironment/leukemia interactions. Bone marrow-derived mesenchymal cells (MSC) comprise an essential component of the leukemia bone marrow microenvironment. MSC have the capacity to support normal and malignant hematopoiesis and protect leukemic cells from chemotherapy. We have previously reported that co-culture of AML cells with MSC results in activation of multiple pro-survival signaling pathways in leukemic cells, from which phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt)/mammalian target of rapamycin (mTOR) signaling is the key upstream regulator of survival and chemoresistance (Tabe et al., 2007 Cancer Res. 2007). In this study, we investigated the role of mTOR signaling in primary AML cells co-cultured with stroma and in the in vivo leukemia mouse model utilizing a novel TOR kinase inhibitor PP242 (Intellikine, La Jolla, CA). Unlike rapamycin and its analogs, which suppress TORC1 only partially and do not acutely inhibit TORC2, PP242 has been reported to achieve greater inhibition of both TOR complexes, resulting in broader suppression of the PI3K/AKT/TOR signaling in Ph+ B-ALL and T-cell lymphoma (Feldman, et al., PLoS Biol 2009; Janes, et al., Nat Med. 2010). We first employed reverse phase protein array (RPPA) technique profiling of 53 proteins to determine the changes in activation of signaling pathways in leukemic cells from 20 primary AML samples co-cultured with murine stromal line MS-5. Co-culture with stroma resulted in activation of multiple signaling pathways in primary AML cells, inducing upregulation of pAKT(Thr308) in 18, mTOR in 17, pERK(Thr202/204) in 14, and pSTAT3(Ser727) in 12 of the 20 pt samples. This resulted in significant decrease of spontaneous apoptosis in primary AML samples (average 33.7 ± 3.8% annexin V(+) cells in primary AML without co-culture vs. 19.6 ± 3.1% in primary AML co-cultured with MS5, p = 0.027, n = 20). In a next set of experiments, blockade of mTOR signaling with PP242, in a dose dependent fashion, effectively induced apoptosis in primary AML samples (n = 9) cultured with or without stroma: at 60nM, 6.4 ± 1.8% and 8.8 ± 2.4% specific apoptosis (annexin V+), respectively; at 190nM, 10.5% ± 52.8% and 14.9% ± 3.9%; at 560nM, 17.6.9 ± 5.7%; and 21.9 ± 4.9% at 1.67uM, 27.2 ± 6.1% and 27.3 ± 5.8%; at 5uM, 38.8 ± 6.5% and 37.1 ± 7.2%. Importantly, at low nanomolar concentrations, PP242 attenuates the activities of both TORC1 and TORC2, resulting in inhibition of phosphorylation of AKT at S473, S6K at S240/244 and 4EBP1 at T37/46 in both, primary AML cells and most importantly in MSC cultured alone or co-cultured with AML. In the in vivo leukemia mouse model utilizing GFP/luc-labeled Baf3-FLT3/ITD cells, PP242 (60mg/kg/QD gavage) exerted significantly greater anti-leukemia activity compared with TORC1 inhibitor rapamycin (0.1mg/kg/QD IP, p = 0.03). PP242 suppressed leukemia progression as determined by bioluminescence imaging (average luminescence intensity 5.65 ± 1.75 in control vs. average 2.75 ± 0.65 in PP242 group) and significantly extended survival (p = 0.005). In summary, our findings indicate a novel therapeutic strategy to target leukemia within the BM microenvironment through efficient blockade of mTOR/AKT signaling with novel selective TORC kinase inhibitor. This research is funded by Intellikine. Disclosures: Liu: Intellikine: Employment. Rommel:Intellikine: Employment. Fruman:Intellikine: Research Funding. Konopleva:Intellikine: Research Funding.

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Targeting of mTORC1/2 by the mTOR kinase inhibitor PP242 induces apoptosis in AML cells under conditions mimicking the bone marrow microenvironment

Blood ◽

10.1182/blood-2011-11-393934 ◽

2012 ◽

Vol 120 (13) ◽

pp. 2679-2689 ◽

Cited By ~ 75

Author(s):

Zhihong Zeng ◽

Yue Xi Shi ◽

Twee Tsao ◽

YiHua Qiu ◽

Steven M. Kornblau ◽

...

Keyword(s):

Bone Marrow ◽

Mouse Model ◽

Kinase Inhibitor ◽

Mammalian Target Of Rapamycin ◽

Leukemic Cells ◽

Phosphatidylinositol 3 Kinase ◽

Mtor Kinase ◽

Phosphorylated Akt ◽

Induced Apoptosis

Abstract The interactions between the bone marrow (BM) microenvironment and acute myeloid leukemia (AML) is known to promote survival of AML cells. In this study, we used reverse phase-protein array (RPPA) technology to measure changes in multiple proteins induced by stroma in leukemic cells. We then investigated the potential of an mTOR kinase inhibitor, PP242, to disrupt leukemia/stroma interactions, and examined the effects of PP242 in vivo using a mouse model. Using RPPA, we confirmed that multiple survival signaling pathways, including the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT)/mammalian target of rapamycin (mTOR), were up-regulated in primary AML cells cocultured with stroma. PP242 effectively induced apoptosis in primary samples cultured with or without stroma. Mechanistically, PP242 attenuated the activities of mTORC1 and mTORC2, sequentially inhibited phosphorylated AKT, S6K, and 4EBP1, and concurrently suppressed chemokine receptor CXCR4 expression in primary leukemic cells and in stromal cells cultured alone or cocultured with leukemic cells. In the in vivo leukemia mouse model, PP242 inhibited mTOR signaling in leukemic cells and demonstrated a greater antileukemia effect than rapamycin. Our findings indicate that disrupting mTOR/AKT signaling with a selective mTOR kinase inhibitor can effectively target leukemic cells within the BM microenvironment.

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Identification of Non-Cardiotoxic hERG1 Blockers to Overcome Chemoresistance in Acute Lymphoblastic Leukemias

Blood ◽

10.1182/blood.v120.21.1506.1506 ◽

2012 ◽

Vol 120 (21) ◽

pp. 1506-1506

Author(s):

Marika Masselli ◽

Serena Pillozzi ◽

Massimo D'Amico ◽

Luca Gasparoli ◽

Olivia Crociani ◽

...

Keyword(s):

Bone Marrow ◽

Mouse Model ◽

Map Kinases ◽

Conflicts Of Interest ◽

Lymphoblastic Leukemia ◽

Significant Proportion ◽

Leukemic Cells ◽

K Channel

Abstract Abstract 1506 Although cure rates for children with acute lymphoblastic leukemia (ALL), the most common pediatric malignancy, have markedly improved over the last two decades, chemotherapy resistance remains a major obstacle to successful treatment in a significant proportion of patients (Pui CH et al. N Engl J Med., 360:2730–2741, 2009). Increasing evidence indicates that bone marrow mesenchymal cells (MSCs) contribute to generate drug resistance in leukemic cells (Konopleva M et al., Leukemia, 16:1713–1724, 2002). We contributed to this topic, describing a novel mechanism through which MSCs protect leukemic cells from chemotherapy (Pillozzi S. et al., Blood, 117:902–914, 2011.). This protection depends on the formation of a macromolecular membrane complex, on the plasma membrane of leukemic cells, the major players being i) the human ether-a-gò-gò-related gene 1 (hERG1) K+ channel, ii) the β1integrin subunit and iii) the SDF-1α receptor CXCR4. In leukemic blasts, the formation of this protein complex activates both the ERK 1/2 MAP kinases and the PI3K/Akt signalling pathways triggering antiapoptotic effects. hERG1 exerts a pivotal role in the complex, as clearly indicated by the effect of hERG1 inhibitors to abrogate MSCs protection against chemotherapeutic drugs. Indeed, E4031, a class III antiarrhythmic that specifically blocks hERG1, enhances the cytotoxicity of drugs commonly used to treat leukemia, both in vitro and in vivo. The latter was tested in a human ALL mouse model, consisting of NOD/SCID mice injected with REH cells, which are relatively resistant to corticosteroids. Mice were treated for 2 weeks with dexamethasone, E4031, or both. Treatment with dexamethasone and E4031 in combination nearly abolished bone marrow engraftment while producing marked apoptosis, and strongly reducing the proportion of leukemic cells in peripheral blood and leukemia infiltration of extramedullary sites. These effects were significantly superior to those obtained by treatment with either dexamethasone alone or E4031 alone. This model corroborated the idea that hERG1 blockers significantly increase the rate of leukemic cell apoptosis in bone marrow and reduced leukemic infiltration of peripheral organs. From a therapeutic viewpoint, to develop a pharmacological strategy based on hERG1 targeting we must consider to circumvent the side effects exerted by hERG1 blockers. Indeed, hERG1 blockers are known to retard the cardiac repolarization, thus lengthening the electrocardiographic QT interval, an effect that in some cases leads to life threatening ventricular arrhythmias (torsades de points). On the whole, it is mandatory to design and test non-cardiotoxic hERG1 blockers as a new strategy to overcome chemoresistance in ALL. On these bases, we tested compounds with potent anti-hERG1 effects, besides E4031, but devoid of cardiotoxicity (e.g. non-torsadogenic hERG1 blockers). Such compounds comprise erythromycin, sertindole and CD160130 (a newly developed drug by BlackSwanPharma GmbH, Leipzig, Germany). We found that such compounds exert a strong anti-leukemic activity both in vitro and in vivo, in the ALL mouse model described above. This is the first study describing the chemotherapeutic effects of non-torsadogenic hERG1 blockers in mouse models of human ALL. This work was supported by grants from the Associazione Genitori contro le Leucemie e Tumori Infantili Noi per Voi, Associazione Italiana per la Ricerca sul Cancro (AIRC) and Istituto Toscano Tumori. Disclosures: No relevant conflicts of interest to declare.

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mTOR Kinase Inhibitors Enhance Efficacy of TKIs in Preclinical Models of Ph-like B-ALL

Blood ◽

10.1182/blood.v128.22.2763.2763 ◽

2016 ◽

Vol 128 (22) ◽

pp. 2763-2763 ◽

Cited By ~ 1

Author(s):

Moran Gotesman ◽

Thanh-Trang T Vo ◽

Sharmila Mallya ◽

Qi Zhang ◽

Ce Shi ◽

...

Keyword(s):

Bone Marrow ◽

Research Funding ◽

Kinase Inhibitors ◽

Kinase Inhibitor ◽

Inhibitor Therapy ◽

Cellular Viability ◽

Murine Bone Marrow ◽

Pdx Models

Abstract Background and Rationale: B-lymphoblastic leukemia (B-ALL) is the most common cancer of childhood. While event-free survival (EFS) exceeds 85% for most patients treated with contemporary therapy, outcomes are very poor for children who relapse, highlighting a need for new treatments. In particular, children with Philadelphia chromosome-like (Ph-like) B-ALL (who lack BCR-ABL1 rearrangement) have high rates of relapse and mortality with conventional chemotherapy. Transcriptional profiling and genomic sequencing of Ph-like ALL specimens have identified a variety of alterations that activate oncogenic kinase signaling, including rearrangements (R) of CRLF2, ABL1, and PDGFRB. Addition of the tyrosine kinase inhibitor (TKI) imatinib to chemotherapy has dramatically improved EFS for patients with BCR-ABL1-rearranged (Ph+) B-ALL, and it is hypothesized that TKI addition to therapy will similarly improve outcomes for patients with Ph-like ALL. Our prior preclinical studies in Ph+ B-ALL demonstrated enhanced efficacy of combining TKIs (imatinib or dasatinib) with mTOR kinase inhibitors (TOR-KIs) (Janes et al., Nature Medicine 2010; Janes et al, Leukemia2013). In the current studies, we hypothesized that dual kinase inhibitor therapy would have superior anti-leukemia cytotoxicity in Ph-like ALL and thus investigated combined TKI and TOR-KI treatment using patient-derived xenograft (PDX) models of childhood Ph-like ALL. Methods: For in vitro studies, viably cryopreserved leukemia cells from established ABL1-R Ph-like ALL PDX models (2 ETV6-ABL1) were incubated with the TKI dasatinib, TOR-KIs, or both TKI + TOR-KI for 72 hours prior to flow cytometric assessment of cellular viability via Annexin V and propidium iodide staining. Two chemically distinct TOR-KIs (MLN0128 or AZD2014) were used to confirm on-target effects. Additional primary ABL1-R or PDGFRB-R Ph-like ALL specimens were plated in methylcellulose without or with inhibitors in colony-forming assays. Phosphoflow cytometry (PFC) analysis of ALL cells incubated with inhibitors was also performed to measure the ability of TKIs and TOR-KIs to inhibit intracellular ABL1 and PI3K/mTOR signaling pathways. For in vivo studies, Ph-like ALL PDX models were treated with dasatinib, the TOR-KI AZD8055, or both drugs via daily oral gavage for 8 days. Human CD19+ ALL was quantified in murine spleens and bone marrow at end of treatment with quantification of cycling cells by EdU incorporation. PFC analysis of murine bone marrow was also performed 2 hours after drugs were dosed, to measure in vivo inhibition of signaling proteins. Results: Combined in vitro treatment with dasatinib and MLN0128 or AZD2014 decreased cellular viability more than inhibitor monotherapy. Similarly, in a set of CRLF2-rearranged samples, mTOR inhibitors augmented killing by the JAK2 inhibitor BBT-594. Incubation of primary ABL1-R or PDGFRB-R ALL cells with both dasatinib and AZD2014 more robustly inhibited colony formation than did inhibitor monotherapy. In in vitro PFC analyses of ABL1-R samples, we observed expected dasatinib-induced inhibition of phosphorylated (p) STAT5. Inhibition of the mTOR substrate pS6 was observed with dasatinib, MLN0128, and AZD2014 with more complete inhibition achieved when dasatinib combined with either MLN0128 or AZD2014. Similarly, in vivo treatment of PDX models with dasatinib and AZD8055 reduced leukemia burden and pS6 signaling more completely than either inhibitor alone. Importantly, dual inhibition decreased the percentage of cycling human ALL cells in murine bone marrow, but preserved cycling in normal mouse bone marrow cells in the same animals. Our data thus provide additional compelling preclinical rationale for combined inhibitor therapy with TKIs and TOR-KIs in Ph-like ALL. Disclosures Weinstock: Novartis: Consultancy, Research Funding. Mullighan:Incyte: Membership on an entity's Board of Directors or advisory committees; Amgen: Speakers Bureau; Loxo Oncology: Research Funding. Konopleva:Reata Pharmaceuticals: Equity Ownership; Abbvie: Consultancy, Research Funding; Genentech: Consultancy, Research Funding; Stemline: Consultancy, Research Funding; Eli Lilly: Research Funding; Cellectis: Research Funding; Calithera: Research Funding.

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Imetelstat Significantly Reduces Leukemia Stem Cells in Patient-Derived Xenograft Models of Pediatric AML

Blood ◽

10.1182/blood-2021-151607 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 3352-3352

Author(s):

Sonali P. Barwe ◽

Fei Huang ◽

E. Anders Kolb ◽

Anilkumar Gopalakrishnapillai

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Research Funding ◽

Annexin V ◽

Telomerase Activity ◽

Leukemia Stem Cells ◽

Patient Derived Xenograft ◽

Pediatric Aml ◽

Pdx Models

Abstract Introduction Acute myeloid leukemia (AML) is the deadliest malignancy in children. Despite the use of maximally intensive therapy, 20% of patients experience recurrent disease. These patients are also burdened with significant treatment-related toxicities. To improve survival in pediatric AML, novel targeted therapies that are more effective and less toxic are needed. Telomerase inhibition has been shown to be effective in reducing leukemic burden and eradicating leukemia stem cells (LSCs) in syngeneic mouse models of AML and in patient-derived xenograft (PDX) models of adult AML (Bruedigam et al., 2014). Recent transcriptome analyses demonstrate that the genomic landscape of pediatric AML is distinct from adult AML (Bolouri et al., 2018). In fact, mutations in the telomerase complex components are infrequent in pediatric AML unlike adult AML patients (Aalbers et al., 2013). However, similar to what is seen in adult patients, Aalbers et al. identified that telomere lengths in pediatric AML cells were shortened compared to normal leukocytes, and pediatric AML patients with the shortest telomere length tend to have shorter overall survival. Furthermore, the 5-year survival rate was 88% for pediatric AML patients who had lower telomerase activity, and 43% for those patients with higher telomerase activity, suggesting telomerase activity could be an important prognostic factor in pediatric AML patients (Verstovsek et al., 2003). Imetelstat is an oligonucleotide that specifically binds with high affinity to the RNA template of telomerase and is a potent, competitive inhibitor of telomerase enzymatic activity (Asai et al., 2003; Herbert et al., 2005). In this study, we evaluated if imetelstat has anti-leukemia activity in pediatric AML PDX models. Results The PDX lines tested in this study were derived using samples from pediatric AML patients who were 1-14 years old, representing different FAB subtypes. Mouse passaged pediatric AML PDX lines (n=6) were treated ex vivo with imetelstat or mismatch oligo control and the viability of LSC (CD34+CD38low population) was determined at 48 or 96 h by staining with BV785-human CD45, APC-human CD34, Pacific blue-human CD38, FITC conjugated annexin V and propidium iodide (PI). Imetelstat treatment significantly increased apoptosis/death (PI+/annexin V+) of the LSC population in a dose-dependent manner in all PDX lines evaluated (Fig. 1A, B), while it had limited activity on LSCs in normal pediatric bone marrow samples (n=4). The efficacy of imetelstat either alone or in combination with chemotherapy or azacitidine was evaluated in two distinct PDX models of pediatric AML in vivo. Mice engrafted with both NTPL-377 and DF-2 lived longer when treated with imetelstat than the untreated mice (Fig. 1C, D, n=5 each, P<0.05). Mice receiving standard chemotherapy consisting of cytarabine and daunorubicin or azacitidine showed prolonged survival compared to the untreated mice. Interestingly, sequential administration of imetelstat following chemotherapy treatment provided additional benefit over chemotherapy alone (P<0.01). Concurrent treatment of azacitidine and imetelstat further extended survival of these mice compared to azacitidine alone (P<0.05). At the end of the in vivo studies, the percentage of LSC population was evaluated in the bone marrow of mice post euthanasia. There was a significant reduction of LSC population in mice treated with imetelstat compared to those treated with the mismatch oligo (Fig. 1E, F, P<0.05). Neither chemotherapy nor azacitidine alone affected LSC population compared to untreated mice. However, imetelstat significantly reduced the LSC population when combined with chemotherapy or azacitidine compared to single agent (P<0.05). These results were confirmed by secondary transplantation in mice, which showed delayed engraftment of cells isolated from imetelstat treated mice (Fig. 1G, H). Conclusions Imetelstat treatment of pediatric AML PDX samples showed significant dose- and time-dependent effects on the viability of the LSCs to induce cell apoptosis/death. These results were corroborated in vivo in two distinct PDX models which showed reduced LSC population and increased median survival in mice with imetelstat treatment. Combining imetelstat with chemotherapy or azacitidine further enhanced activity against LSCs, suggesting imetelstat could represent an effective therapeutic strategy for pediatric AML. Figure 1 Figure 1. Disclosures Barwe: Prelude Therapeutics: Research Funding. Huang: Geron Corp: Current Employment, Current equity holder in publicly-traded company. Gopalakrishnapillai: Geron: Research Funding.

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Inhibiting the IGF-1 Receptor Tyrosine Kinase with Picropodophillin: An In Vitro and In Vivo Study in the 5T33MM Mouse Model.

Blood ◽

10.1182/blood.v104.11.640.640 ◽

2004 ◽

Vol 104 (11) ◽

pp. 640-640

Author(s):

Karin Vanderkerken ◽

Eline Menu ◽

Thomas Stromberg ◽

Hendrik De Raeve ◽

Kewal Asosingh ◽

...

Keyword(s):

Multiple Myeloma ◽

Bone Marrow ◽

Mouse Model ◽

Tyrosine Kinase ◽

Receptor Tyrosine Kinase ◽

Microvessel Density ◽

Kinase Inhibitor ◽

High Dose

Abstract Multiple myeloma (MM) represents a B-cell malignancy, characterized by monoclonal proliferation of plasma cells in the bone marrow (BM) and is associated with osteolysis and angiogenesis. Insulin-like growth factor-1 (IGF-1), produced by the BM stromal cells, has been described as an important factor in the survival, proliferation and migration of MM cells. The latter process is involved in the homing of the MM cells to the BM. IGF-1 also induces VEGF secretion by the MM cells, thus stimulating angiogenesis in the BM. As IGF-1 is a pleiotropic factor in MM, therapeutic strategies targeting the IGF-1R may be effective as anti-tumor treatments. In this work we investigated the effect of an IGF-1 receptor tyrosine kinase inhibitor (picropodophyllin or PPP1) in the murine, syngeneic 5T33MM model of multiple myeloma. This mouse model is representative for the human disease and can combine in vitro and in vivo studies. We first investigated the effects of PPP on the MM cells in vitro. We and others have previously demonstrated that IGF-1 induced ERK activation, involved in VEGF secretion and proliferation. When the 5T33MM cells were preincubated with 1microM PPP, Western blot analysis demonstrated the blocking of this activation. Furthermore, when the 5T33MM cells were preincubated with PPP for 30 min, IGF-1 induced VEGF secretion and proliferation of the 5T33MM cells were completely blocked. Next, we used the tyrosine kinase inhibitor PPP in vivo. 5T33MM cells were injected intravenously in C57BLKaLwRij mice and the development of the disease was monitored by measuring the serum paraprotein concentration. Mice were either treated with a low (17mM, IP, twice a day) or a high dose of PPP (50mM, IP, twice a day) or with the vehicle (DMSO/oil 9/1) from the day of injection with 5T33MM onward. At week 3, vehicle controls showed signs of morbidity and were sacrificed. The presence of tumor was measured by assessing serum paraprotein concentrations and determining the proportion of idiotype positive cells in the BM by flow cytometry. Angiogenesis was assessed by measuring the microvessel density on CD31 stained paraffin sections. The tumor burden in the bone marrow in the PPP treated mice was 77% lower than in vehicle treated animals (p< 0,0001) and the serum paraprotein concentration was 90% lower (p< 0,0001). The microvessel density in the BM of the PPP treated group was reduced by 60% (p< 0,02). In a separate survival experiment the mice were either treated with the vehicle or with the high dose (50mM) of PPP, from the time of tumor injection. Kaplan-Meier analysis demonstrated a significant increase in survival after treatment with PPP when compared with vehicle (28 vs. 18 days, p<0,001). These data demonstrate that the IGF-1RTK inhibitor PPP possesses strong anti-tumor activity, as demonstrated both in vitro and in vivo in a syngeneic model of multiple myeloma, and may therefore be an effective therapeutic candidate for MM treatment.

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The Role of microRNA-155 in Mouse Models of MLL -AML

Blood ◽

10.1182/blood.v126.23.2446.2446 ◽

2015 ◽

Vol 126 (23) ◽

pp. 2446-2446

Author(s):

Anna Staffas ◽

Edith Schneider ◽

Linda Fogelstrand ◽

Linda Röhner ◽

Michael Heuser ◽

...

Keyword(s):

Bone Marrow ◽

Mouse Model ◽

Therapeutic Target ◽

Conflicts Of Interest ◽

Micro Rna ◽

Blood Analysis ◽

Leukemic Cells ◽

Mouse Bone Marrow ◽

Fusion Genes

Abstract Background: Genetic rearrangements that fuse the mixed lineage leukemia (MLL) gene, now termed KMT2A, to one of a variety of partners are seen in 5% - 20% of acute myeloid leukemia (AML). MLL -fusions are especially common in childhood AML and many of them are associated with poor prognosis. The MLL -fusions perturb transcription through different mechanisms and they are often associated with high expression of the transcription factors HOXA9 and MEIS1. Based on a micro-RNA screen in an AML mouse model mimicking the step-wise development of aggressive AML we have found that concurrent Hoxa9 and Meis1 overexpression is associated with upregulation of micro-RNA-155 (miR-155). Expression of miR-155 was also found to be higher in bone marrow samples from patients with MLL- AML compared with bone marrow from healthy donors (p <0.001), as were the expression of HOXA9 and MEIS1 (p <0.05). In lymphomas, miR-155 plays a pivotal role as an oncogene. It is frequently upregulated in samples from lymphoma patients and a mouse model of lymphoma showed a certain degree of miR-155-addiction which could be targeted by miR-155 inhibitors. Despite the differences in the pathobiology of AML and lymphoma, the upregulation of miR-155 in AML with high HOXA9 and MEIS1 expression may indicate miR-155 as a relevant therapeutic target also in MLL -AML. Methods: To test the importance of miR-155 and its potency as a drug target in MLL -AML we used a miR-155 knock-out mouse model (miR-155-/-) (Thai et al, Science, 2007). MLL -fusion genes of varying leukemic potential; MLL-AF5 (KMT2A-AFF4), MLL-ENL (KMT2A-MLLT1), MLL-AF9 (KMT2A-MLLT3) were retrovirally expressed in miR-155-/- mouse bone marrow (mbm) cells and in wild-type mbm cells (miR-155+/+). Results: In concordance with the previous findings in human AML patient samples, miR-155+/+ cells expressing MLL-AF5, MLL-ENL, or MLL-AF9 showed upregulation of miR-155 (p < 0.05). Also, Hoxa9 and Meis1 transcripts were increased (p<0.05). Interestingly, the magnitude of upregulation of both miR-155 and Meis1 correlated with the degree of aggressiveness based on disease latency and survival observed in these leukemia models with highest upregulation in MLL-ENL and MLL-AF9 and lowest in MLL-AF5 (p<0.05). Expression of the MLL-fusion genes in miR-155-/- mbm cells resulted in similar induction of Hoxa9 and Meis1 expression as in miR-155+/+ mbm cells, indicating that miR-155 is downstream of the Hoxa9/Meis1 axis. To determine the leukemic potential in vivo, we transplanted recipient mice with miR-155+/+ mbm cells and miR-155-/- mbm cells expressing MLL-ENL or MLL-AF9. Engraftment of leukemic cells, based on peripheral blood analysis, did not differ between mice transplanted with miR-155+/+ mbm cells and miR-155-/- mbm cells expressing MLL-fusions. Also, disease development induced by MLL-AF9 and MLL-ENL (4-8 weeks and 10-32 weeks, respectively) was similar in mice transplanted with miR-155-/- mbm cells and mice transplanted with miR-155+/+ mbm cells. In accordance with the in vivo results, functional studies in vitro showed that the proliferative capacity and colony forming ability of MLL -fusion expressing cells were similar in miR-155+/+ mbm cells and miR-155-/- mbm cells, indicating that miR-155 is not essential for MLL-ENL- or MLL-AF9-induced leukemic transformation. Conclusions: In summary, miR-155 is upregulated in MLL-AML in both mice and man, seemingly through an MLL>HOXA9/MEIS1>miR-155 axis. Since absence of miR-155 does not alter the leukemic potential induced by MLL-AF9 or MLL-ENL, miR-155 may contribute to, but is not pivotal for MLL leukemogenesis. We therefore conclude that miR-155 is not a therapeutic target in MLL- AML. Disclosures No relevant conflicts of interest to declare.

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Signaling Pathways of Mutant IDH1 Independent of R-2-Hydroxyglutarate

Blood ◽

10.1182/blood-2018-99-110159 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 2617-2617

Author(s):

Charu Gupta ◽

Michelle Maria Araujo Cruz ◽

Nidhi Jyotsana ◽

Amit Sharma ◽

Ramya Goparaju ◽

...

Keyword(s):

Cell Cycle ◽

Bone Marrow ◽

Signaling Pathways ◽

Splice Variant ◽

Research Funding ◽

Bone Marrow Cells ◽

Stat3 Phosphorylation ◽

Mutant Idh1 ◽

Marrow Cells

Abstract #Michael Heuser and Anuhar Chaturvedi share senior authorship Background: Isocitrate dehydrogenase-1 (IDH1) is mutated in about 6% of AML patients. Mutant IDH produces R-2-hydroxyglutarate (R-2HG), which induces histone and DNA hypermethylation through inhibition of epigenetic regulators, thereby linking metabolism to tumorigenesis. We recently reported that at comparable intracellular R-2HG levels, mice receiving transplants of IDH1 mutant cells died significantly earlier than R-2HG treated mice in the context of HOXA9 overexpression. This suggests oncogenic functions of mutant IDH1 beyond R-2HG production. We employed a splice variant of mutated IDH1 that does not produce R-2HG (IDH1mutantΔ7) to decipher R-2HG independent signaling pathways that may contribute towards leukemogenesis. Methods: Bone marrow cells from mice were immortalized with HoxA9, and IDH1wildtype (IDH1wt), IDH1mutant (IDH1mut), IDH1wildtypeΔ7 (IDH1wtΔ7) and IDH1mutΔ7, were constitutively expressed and the leukemogenic potential was evaluated in vivo. Intracellular R-2HG was measured by enantiomer-specific quantification. Deletion of exon 7 from IDH1mut leads to a frameshift that creates a premature stop codon in the 9th exon, finally producing a 119 amino acids truncated protein, IDH1mutΔ7. This splice variant does not produce increased levels of R-2HG. The signaling pathways were explored by immunoblotting and immunofluorescence. Results: Mice receiving cells with IDH1mutΔ7 had the same short latency to leukemia as mice receiving cells with full-length mutant IDH1, while IDH1wt and IDH1wtΔ7 cells died with significantly longer latency. The WBC count increased over time in IDH1mutΔ7 mice similar to IDH1mut mice, whereas WBC counts in IDH1wtΔ7 mice remained normal. IDH1mutΔ7 mice died from monocytic leukemia that was phenotypically and morphologically indistinguishable from IDH1mut mice. HoxA9 IDH1mutΔ7 cells were readily transplantable into secondary recipients. During in vivo cell cycle analysis, we observed that the proportion of cells in S/G2/M phases was significantly higher in bone marrow cells transduced with IDH1mut or IDH1mutΔ7 when compared to cells transduced with IDH1wt or CTL. These data suggest that mutant IDH1 enhances myeloproliferation even in the absence of R-2HG. To identify R-2HG independent signaling pathways mediated by the mutant IDH1 protein, we first analyzed the gene expression of important regulators of cell cycle, differentiation, cell signaling and transcription by quantitative RT-PCR. Several genes (Ccnd1, Slc2a, Hdac3, Tgif2,and c-myc) were upregulated in IDH1mut and IDH1mutΔ7 cells compared to IDH1wt cells. Interestingly, we found a specific up-regulation of Ctnnb1 and Nfkb genes in IDH1mutΔ7 cells over both IDH1mut and IDH1wt cells. We next validated our mRNA expression results by immunoblotting and found that NFKB and ERK signaling were upregulated in both IDH1mut and IDH1mutΔ7 compared to IDH1wt and IDH1wtΔ7 cells. Interestingly, the protein level of β-catenin, STAT3 and STAT5 were many fold higher in IDH1mutΔ7 compared to IDH1mut and IDH1wt cells. β-catenin is known to be transactivated via c-Src, which is phosphorylated by EGFR to promote β-catenin nuclear localization and signaling. We traced this pathway for its relevance in our cells and found that IDH1mutΔ7 cells indeed showed higher levels of both EGFR and c-Src phosphorylation compared to IDH1mut cells. We performed immunofluorescence and cellular fractionation for β-catenin and found it to be partially localized in the nucleus in IDH1mutΔ7 but not in IDH1mut cells. We also observed an up-regulated STAT3 phosphorylation in IDH1mutΔ7 cells over IDH1mut. Conclusions: In summary, mutant IDH1 activates ERK and NFKB signaling, which is attributed to both R-2HG dependent and independent mechanisms of leukemogenesis. Interestingly, IDH1mutΔ7 employs R-2HG independent EGFR/β-catenin and JAK/STAT signaling for oncogenesis. This R-2HG-independent leukemogenesis reveals a novel signaling dynamic of IDH1mut which should be evaluated for its therapeutic potential. Disclosures Ganser: Novartis: Membership on an entity's Board of Directors or advisory committees. Heuser:Astellas: Research Funding; Karyopharm: Research Funding; Novartis: Consultancy, Honoraria, Research Funding; Pfizer: Consultancy, Honoraria, Research Funding; Janssen: Consultancy; StemLine Therapeutics: Consultancy; Bayer Pharma AG: Consultancy, Research Funding; Sunesis: Research Funding; BergenBio: Research Funding; Tetralogic: Research Funding; Daiichi Sankyo: Research Funding.

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Analysis of the Crosstalk Between TGF-β–VEGF-Angiogenesis in an In Vivo Model of Acute Promyelocytic Leukemia

Blood ◽

10.1182/blood.v116.21.1845.1845 ◽

2010 ◽

Vol 116 (21) ◽

pp. 1845-1845

Author(s):

Patricia Aparecida Assis ◽

Lorena Lobo Figueiredo Pontes ◽

Antonio Roberto Lucena Araújo ◽

Aglair Bergamo Garcia ◽

Roberto P. Falcao ◽

...

Keyword(s):

Bone Marrow ◽

Acute Promyelocytic Leukemia ◽

Research Funding ◽

Promyelocytic Leukemia ◽

Microvascular Density ◽

Leukemic Cells ◽

Lower Percentage ◽

Differential Count ◽

Leukemic Infiltration

Abstract Abstract 1845 Acute Promyelocytic Leukemia (APL) patients present increased bone marrow microvascular density (MVD) compared to normal bone marrow, which has been associated with the aberrant secretion of the proangiogenic factor VEGF by leukemic cells. The APL associated fusion protein PML-RARα is thought to deregulate the TGF-β pathway, through its dominant negative action on cytoplasmic PML, thus down regulating SMAD2/3 signaling, and VEGF transcription. However, PML-RARα expression was associated with increased TGF-β gene transcription and secretion. We used the low molecular weight quinazolinone alkaloid Halofuginone (HF), which has been shown to be a potent TGF-β inhibitor, to test the association between TGF-β/VEGF/angiogenesis. HF inhibited the VEGF secretion by NB4 (an APL cell lineage) and cell proliferation. To determine the effects of HF in vivo, irradiated NOD/SCID mice were transplanted with leukemic cell from hCG-PML-RARα transgenic mice. Twenty-four hours after transplantation, mice were treated with 150μg/kg of HF by intraperitoneal injections for 21 days. All recipients developed leukemia, however the leukemic infiltration of bone marrow and WBC were significantly lower in animals treated HF (4.2 ± 3.89 vs. 20.6 ± 21.9, p <0.0001) and hemoglobin and platelet counts higher in this treated group (Hb: 12.0 ± 1.40 vs. 9.6 ± 1.67, p <0.0001; and Platelets: 932.0 ± 122.5 vs. 552.0 ± 83.2, p <0.001). Accordingly, a lower leukemic infiltration of spleen was detected (ratio spleen/body of 0.006 vs. 0.012 in treated and untreated groups respectively, p=0.0415). Furthermore, the differential count and immunophenotyping of bone marrow showed a lower percentage of immature myeloid cells (27 ± 9.3 vs. 66.3 ± 17.9; p=0.0037 and 16.88 ± 6.27 vs. 44.06 ± 27.06, respectively). HF treatment did not induce molecular remission nor increased the survival of leukemic animals. However, the lack of difference in survival could be atributted to HF hepatoxicity, as a significant rise in AST and ALT serum levels was observed (661.2 ± 262.6/177.9 ± 107.4 vs. 89.88 ± 11.65/27.86 ± 4.47 U/L, prespectively). Phosphorilated SMAD2 levels were determined by ELISA in NB4 cells and a significant dose dependent decrease was observed in samples treated with HF at the doses of 50, 100 and 200 ng/mL. Gene expression analysis showed that the HF treatment inhibited the expression of TgfB, Smad3, Smad4, Myc, Vegf and Egf and the immunohistochemistry analysis of BM sections revealed a significant decrease of VEGF staining (30 vs. 80%, p =0.0227), but there was no decrease in the microvascular density. Taken together, these results showed that angiogenesis is an important therapeutic target in APL, and despite the toxicity, HF has antileukemic potential due to its antiproliferative and proangiogenic factors inhibitory capabilities. Disclosures: Assis: FAPESP: Research Funding; CNPq: Research Funding.

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A Novel Inhibitor of Mutant IDH1 Induces Differentiation in Vivo and Prolongs Survival in a Mouse Model of Leukemia

Blood ◽

10.1182/blood.v124.21.3598.3598 ◽

2014 ◽

Vol 124 (21) ◽

pp. 3598-3598 ◽

Cited By ~ 5

Author(s):

Anuhar Chaturvedi ◽

Michelle Maria Araujo Cruz ◽

Ramya Goparaju ◽

Nidhi Jyotsana ◽

Heike Baehre ◽

...

Keyword(s):

Bone Marrow ◽

Mouse Model ◽

Medical School ◽

Peripheral Blood ◽

Bone Marrow Cells ◽

Leukemic Cells ◽

Median Latency ◽

Mutant Idh1

Abstract Mutations in the metabolic enzymes isocitrate dehydrogenase 1 (IDH1) and 2 (IDH2) are frequently found in patients with glioma, acute myeloid leukemia (AML), melanoma, thyroid cancer, cholangiocellular carcinoma and chondrosarcoma. Mutant IDH produces R-2-hydroxyglutarate (R2HG), which induces histone- and DNA-hypermethylation through inhibition of epigenetic regulators, thus linking metabolism to tumorigenesis. We recently established an in vivo mouse model and investigated the function of mutant IDH1. By computational drug screening, we identified an inhibitor of mutant IDH1 (HMS-101), which inhibits mutant IDH1 cell proliferation, decreases R2HG levels in vitro, and efficiently blocks colony formation of AML cells from IDH1 mutated patients but not of normal CD34+ bone marrow cells. In the present study we investigated the effect of the inhibitor in our IDH1/HoxA9-induced mouse model of leukemia in vivo. To identify the maximally tolerated dose of HMS-101, we treated normal C57BL/6 mice with variable doses of HMS-101 for 9 days and measured the serum concentration. Mice receiving 0.5 mg and 1mg intraperitoneally once a day tolerated the drug well with mean plasma concentrations of 0.1 to 0.3 µM. To evaluate the effect of HMS-101 in the IDH1 mouse model, we transduced IDH1 R132C in HoxA9-immortalized murine bone marrow cells. Sorted transgene positive cells were then transplanted into lethally irradiated mice. After 5 days of transplantation, mice were treated with HMS-101 intraperitoneally for 5 days/week. The R/S-2HG ratio in serum was reduced 3-fold in HMS-101 treated mice after 8 weeks of treatment compared to control treated mice. HMS-101 or PBS treated mice had similar levels of transduced leukemic cells in peripheral blood at 2 and 6 weeks after transplantation. However, from week 6 to week 15 leukemic cells in peripheral blood decreased from 76% to 58, 63% to 29%, 67% to 7%, and 74% to 38% in 4/6 mice treated with HMS-101. In one mouse the percentage of leukemic cells was constant, and in one mouse it increased from week 6 to week 15 after transplantation. Leukemic cells increased constantly in peripheral blood until death in control treated mice. While the control cohort developed severe leukocytosis, anemia and thrombocytopenia around 8 to 10 weeks post transplantation, mice treated with HMS-101 still had normal WBC, RBC and platelet counts at 15 weeks after transplantation. Moreover, the HMS-101 treated mice had significantly more differentiated Gr1+CD11b+ cells in peripheral blood than control mice at 6 weeks and 15 weeks after transplantation and at death (P=.01). Morphologic evaluation of blood cells at 15 weeks or death from HMS-101 treated mice revealed a high proportion of mature neutrophils that were GFP positive and thus derived from IDH1 transduced cells, whereas control treated mice had monocytic morphology with a high proportion of immature cells. Importantly, HMS-101 treated mice survived significantly longer with a median latency of 87 days (range 80-118), whereas PBS-treated mice died with a median latency of 66 days (range 64-69) after transplantation (P<.001). Of note, HMS-101 was found to be specific for mutant IDH1, as mutant IDH2 cells were not preferentially inhibited over IDH2 wildtype cells in vitro. This data demonstrates that HMS-101 specifically inhibits R2HG-production of mutant IDH1 in vivo, inhibits proliferation, induces differentiation in leukemic cells, and thus prolongs survival of IDH1mutant leukemic mice. Therefore, HMS-101 - a novel inhibitor of mutant IDH1 - shows promising activity in vivo and warrants further development towards clinical use in IDH1 mutated patients. Disclosures Chaturvedi: Hannover Medical School: Patents & Royalties. Preller:Hannover Medical School: Patents & Royalties. Heuser:Hannover Medical School: Patents & Royalties.

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Potentiation of the Effects of Nilotinib by Combination with Plerixafor in a Mouse Model of BCR-ABL-Positive Residual Disease

Blood ◽

10.1182/blood.v118.21.2737.2737 ◽

2011 ◽

Vol 118 (21) ◽

pp. 2737-2737

Author(s):

Ellen L Weisberg ◽

Paul W. Manley ◽

Andrew L. Kung ◽

James D. Griffin

Keyword(s):

Bone Marrow ◽

Stem Cell ◽

Tyrosine Kinase ◽

Kinase Inhibitor ◽

Residual Disease ◽

Hematopoietic Cells ◽

Tumor Burden ◽

Leukemic Cells ◽

High Dose

Abstract Abstract 2737 Background: It has been shown that a small, residual pool of leukemic CD34+ progenitor cells can survive in the marrow microenvironment of chronic myeloid leukemia (CML) patients after years of kinase inhibitor treatment. Bone marrow stroma contributes to the expansion and proliferation of both transformed as well as normal hematopoietic cells, and has been implicated in the long-term survival of leukemic cells. We previously demonstrated the stromal protection of leukemic cells from the anti-proliferative effects of nilotinib, and identified stromal-derived viability factors, including IL-6 and GM-CSF, as possibly mediating stromal protection of tyrosine kinase inhibitor-treated leukemic cells (Weisberg et al. Mol Cancer Ther 2008;7:1121). Additionally, we have found high leukemia burden in the tissues of nilotinib-treated mice that have significant sources of hematopoiesis-promoting stroma, suggesting that significant reservoirs for tumor growth may be tissues that are able to support normal and malignant hematopoietic stem cell development. These studies revealed a leukemia distribution pattern consistent with that observed in imatinib or nilotinib-treated patients. One strategy for overriding stromal-mediated chemoresistance is the use of inhibitors of the stroma-derived factor (SDF-1a) receptor, CXCR4, which mediates the migration of hematopoietic cells to the bone marrow and plays a key role in leukemic cell-stromal cell interactions. Studies have shown small-molecule CXCR4 inhibitors, such as plerixafor, to be effective in mobilizing hematopoietic cells from bone marrow and enhancing chemotherapy- and tyrosine kinase inhibitor-induced apoptosis of bone marrow stroma-protected leukemic cells in vitro and in vivo. Thus, imatinib has been shown to up-regulate CXCR4, which induces CML migration to the bone marrow microenvironment and leads to stroma-mediated chemoresistance of quiescent CML progenitor cells. Methodology: Utilizing a functional in vivo assay system that allows monitoring of the growth of progressive disease, as well as baseline level (“residual” disease) resulting from treatment with a moderate-to-high dose of nilotinib, we investigated the ability of stem cell mobilization to enhance the efficacy of nilotinib by suppressing leukemia recurrence following nilotinib treatment. Specifically, 32D.p210-luc+ cells were injected into the tail-vein of mice, which were then imaged 10 days later to determine baseline bioluminescence and quantify tumor-burden. Mice were subsequently treated by oral gavage for 10 days with nilotinib (75 mg/kg qd) and reimaged (“Induction Phase” of treatment); mice were at this stage considered to have minimal residual disease, with reduced tumor burden > 2 logs. Mice were then divided into four treatment groups (“Consolidation Phase” of treatment addressing minimum residual disease) with similar mean bioluminescence (n=8 or 9 per group): Group 1: Vehicle (PEG300 po), Group 2: plerixafor (5 mg/kg sq qd), Group 3: nilotinib (75mg/kg po qd), and Group 4: Combination (plerixafor+nilotinib). Results: Nilotinib was highly efficacious in reducing disease burden in leukemia-engrafted mice. However, with continued treatment, animals developed resistance to nilotinib with increasing disease burden despite continued therapy. Although plerixafor had no single-agent activity, combination with nilotinib significantly delayed time to relapse, and significantly prolonged survival when compared to nilotinib monotherapy (p<0.0001). Since plerixafor had no monotherapeutic efficacy, there results demonstrate that plerixafor, at a well-tolerated dose, acts synergistically with nilotinib to suppress the growth of 32D.p210 leukemia. Conclusion: We demonstrated the ability of plerixafor to delay the onset of recurring BCR-ABL-positive disease in mice carrying an extremely low tumor burden following treatment with a moderate-to-high dose of nilotinib. These results support the idea of using stem cell mobilization in conjunction with targeted tyrosine kinase inhibition to override drug resistance and suppress or eradicate residual disease. Disclosures: Manley: Novartis Pharma AG: Employment. Kung:Novartis Pharmaceuticals: Consultancy, Research Funding. Griffin:Novartis Pharmaceuticals: Consultancy, Research Funding.

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