Signaling Pathways In Leukemic Cells Research Articles

Abstract 5618: Evaluating anti-IL-2Ra in vivo efficacy on humanized knock-in mice

Abstract Interleukin-2 receptor alpha chain (also called as CD25) is a 55 kD glycoprotein, also known as IL-2Ra, Ly-43, p55, or Tac. It is expressed on activated T and B cells, thymocyte subset, pre-B cells, and T regulatory cells. IL-2Ra and IL-2Rβ (CD122), together with the common gamma chain (IL2Rg)(CD132), form the high affinity signaling IL-2 receptor. Its affinity for IL2 and cellular function are tightly regulated and vary in different cell types. The high frequency of IL-2Ra on the surface of many different haematological tumor cells is now well established and, apart from its prognostic significance, IL-2Ra may be present on leukemic stem cells and enable oncogenic signaling pathways in leukemic cells. Additionally, high IL-2Ra expression in activated circulating immune cells and Tregs is a factor that has already been exploited by IL2 immunotherapies for treatment of tumors and autoimmune disease. The relative clinical safety and efficacy of administering anti- IL-2Ra radioimmunoconjugates and immunotoxins in various haematological tumor indications has been established and clinical trials of a novel IL-2Ra-directed antibody drug conjugate are underway. Biocytogen has generated IL-2Ra humanized mouse for both in vitro functional validation of signaling pathway and in vivo efficacy evaluation of IL-2Ra antibodies. The targeting strategy is that the genomic DNA covering exons 2~6 of mouse IL-2Ra gene which encode the extracellular domain are replaced by the counterpart of human IL-2Ra genomic DNA covering exons 2~6. Human IL-2Ra is mainly detectable on the T cells in spleen of the IL-2Ra humanized mice. The humanized mice are highly similar to the wild-type mice in basal leukocyte subpopulations, including T/B cells, NK cells, DC, granulocytes and monocytes/macrophages. STAT-5 phosphorylation induced by mouse IL2 protein is blocked by anti-human IL-2Ra antibodies, suggesting that the functional pathways in IL-2Ra humanized mice work well. The colon cancer model established in IL-2Ra humanized mice is undergoing, anti-human IL-2Ra antibodies showed good efficacy in inhibiting tumor growth. Taken together, IL-2Ra humanized mice are a useful tool for in vivo efficacy evaluation of human IL-2Ra antibodies that can be advanced to human clinical trials. Citation Format: Yanan Guo, Xiaofei Zhou, Youhong Su, Gary Ng, Chaoshe Guo. Evaluating anti-IL-2Ra in vivo efficacy on humanized knock-in mice [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5618.

Experimental and integrative analyses identify an ETS1 network downstream of BCR-ABL in chronic myeloid leukemia (CML)

The BCR-ABL oncogene, the hallmark of chronic myeloid leukemia (CML), has been shown to activate several signaling pathways in leukemic cells. The natural history of this disease has been radically modified by tyrosine kinase inhibitors (TKIs). However, resistance to several lines of TKI therapies and progression to blast crisis (BC) remain significant concerns. To identify novel signaling pathways induced by BCR-ABL, we performed a transcriptome analysis in a BCR-ABL-expressing UT-7 cell line. More than 2000 genes differentially expressed between BCR-ABL-expressing and parental UT-7 cells were identified and ETS1 was found to be the most upregulated. ETS1 protein expression was also shown to be highly increased in UT-7 cells expressing BCR-ABL either constitutively or under the control of TET-inducible promoters. ETS1 expression is tyrosine-kinase dependent because it was reduced by TKIs. A significant increase of ETS1 messenger RNA (mRNA) expression was observed in blood cells from CML patients at diagnosis compared with healthy controls. Integration of publicly available chromatin immunoprecipitation sequencing and transcriptomic data with our results allowed us to identify potential ETS1 targets, some of which are involved in the progression of CML. The messenger RNA expression of two of these genes (DNM3 and LIMS1) was found to be associated with the absence of major cytogenetic response after 1 year of imatinib therapy. The present work demonstrates for the first time the involvement of the ETS1 transcriptional program in the experimental UT-7 model and a large cohort of CML patients.

IKK-dependent activation of NF-κB contributes to myeloid and lymphoid leukemogenesis by BCR-ABL1

AbstractThe product of the Ph chromosome, the BCR-ABL1 tyrosine kinase activates diverse signaling pathways in leukemic cells from patients with chronic myeloid leukemia (CML) and Ph+ B-cell acute lymphoblastic leukemia (B-ALL). Previous studies showed that nuclear factor κB (NF-κB) is activated in BCR-ABL1–expressing cells, but the mechanism of activation and importance of NF-κB to the pathogenesis of BCR-ABL1–positive myeloid and lymphoid leukemias are unknown. Coexpression of BCR-ABL1 and a superrepressor mutant of inhibitory NF-κB α (IκBαSR) blocked nuclear p65/RelA expression and inhibited the proliferation of Ba/F3 cells and primary BCR-ABL1–transformed B lymphoblasts without affecting cell survival. In retroviral mouse models of CML and B-ALL, coexpression of IκBαSR attenuated leukemogenesis, prolonged survival, and reduced myeloid leukemic stem cells. Coexpression of dominant-negative mutants of IκB kinase α (IKKα)/IKK1 or IKKβ/IKK2 also inhibited lymphoid and myeloid leukemogenesis by BCR-ABL1. Blockade of NF-κB decreased expression of the NF-κB targets c-MYC and BCL-X and increased the sensitivity of BCR-ABL1–transformed lymphoblasts to ABL1 kinase inhibitors. These results demonstrate that NF-κB is activated through the canonical IKK pathway and plays distinct roles in the pathogenesis of myeloid and lymphoid leukemias induced by BCR-ABL1, validating NF-κB and IKKs as targets for therapy of Ph+ leukemias.

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

AbstractAbstract 778The 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.

Distinct Roles for the NF-κB Pathway In Myeloid and Lymphoid Transformation and Leukemogenesis by BCR-ABL.

AbstractAbstract 1225The BCR-ABL tyrosine kinase, product of the t(9;22) Ph chromosome, activates multiple signaling pathways in leukemic cells from patients with chronic myeloid leukemia (CML) and Ph+ B-cell acute lymphoblastic leukemia (B-ALL). Previous studies have shown that NF-κB is activated in BCR-ABL-expressing cell lines and contributes to transformation of primary B-lymphoid cells by BCR-ABL (Reuther et al., Genes Dev. 1998;12:968), but the mechanism of activation has not been defined (Kirchner et al., Exp. Hematol. 2003;31:504), and importance of NF-kB to myeloid and lymphoid leukemogenesis by BCR-ABL is unknown.To interrogate the role of NF-κB in BCR-ABL-mediated transformation, we utilized a super-repressor mutant form of IκBα (IκBαSR), which has been used to block NF-κB nuclear localization and transactivation by constitutively sequestering NF-κB in the cytoplasm. Using retrovirus co-expressing BCR-ABL and IκBαSR, we found that IκBαSR blocked nuclear p65/RelA expression and inhibited the IL-3 independent growth of Ba/F3 cells and primary B-lymphoid cells transformed by BCR-ABL. The effect of NF-κB inhibition was primarily on proliferation rather than on cell survival, as there was no increase in apoptosis in cells expressing IκBαSR. When primary bone marrow cells were transduced and transplanted under conditions favoring induction of B-ALL or CML-like myeloproliferative neoplasm in recipient mice, co-expression of IκBαSR significantly attenuated disease development and prolonged survival of diseased mice. Molecular analysis of these leukemias demonstrated that NF-κB inhibition decreased the frequency of leukemia-initiating (“stem”) cells in the CML model, but not in the B-ALL model, and was associated with decreased expression of c-Myc, an NF-κB target. To clarify the mechanism of activation of NF-κB in BCR-ABL-expressing cells, we targeted two upstream kinases that negatively regulate IκBα, IKKα/IKK1 or IKKβ/IKK2. To accomplish this, we engineered retroviruses co-expressing BCR-ABL and kinase-inactive, dominant-negative mutants of IKK1 (IKK1KM) or IKK2 (IKK2KM). Co-expression of either IKK mutant inhibited both B-lymphoid transformation and leukemogenesis by BCR-ABL, as well as induction of CML-like MPN, with IKK1 inhibition more effective than IKK2.Together, these results demonstrate that NF-κB is activated in part through the canonical IKK pathway in BCR-ABL-expressing leukemia cells, and that NF-κB signaling plays distinct roles in the pathogenesis of myeloid and lymphoid leukemias induced by BCR-ABL. In CML, NF-κB may play a role for in generation and/or maintenance of leukemic stem cells. These results validate IKKs as targets for therapy in Ph+ leukemias, and motivate the evaluation of small molecule IKK inhibitors in these diseases. Disclosures:No relevant conflicts of interest to declare.

Targeting the leukemia microenvironment by CXCR4 inhibition overcomes resistance to kinase inhibitors and chemotherapy in AML

AbstractSDF-1α/CXCR4 signaling plays a key role in leukemia/bone marrow microenvironment interactions. We previously reported that bone marrow–derived stromal cells inhibit chemotherapy-induced apoptosis in acute myeloid leukemia (AML). Here we demonstrate that the CXCR4 inhibitor AMD3465 antagonized stromal-derived factor 1α (SDF-1α)–induced and stroma-induced chemotaxis and inhibited SDF-1α–induced activation of prosurvival signaling pathways in leukemic cells. Further, CXCR4 inhibition partially abrogated the protective effects of stromal cells on chemotherapy-induced apoptosis in AML cells. Fetal liver tyrosine kinase-3 (FLT3) gene mutations activate CXCR4 signaling, and coculture with stromal cells significantly diminished antileukemia effects of FLT3 inhibitors in cells with mutated FLT3. Notably, CXCR4 inhibition increased the sensitivity of FLT3-mutated leukemic cells to the apoptogenic effects of the FLT3 inhibitor sorafenib. In vivo studies demonstrated that AMD3465, alone or in combination with granulocyte colony-stimulating factor, induced mobilization of AML cells and progenitor cells into circulation and enhanced antileukemic effects of chemotherapy and sorafenib, resulting in markedly reduced leukemia burden and prolonged survival of the animals. These findings indicate that SDF-1α/CXCR4 interactions contribute to the resistance of leukemic cells to signal transduction inhibitor– and chemotherapy-induced apoptosis in systems mimicking the physiologic microenvironment. Disruption of these interactions with CXCR4 inhibitors represents a novel strategy of sensitizing leukemic cells by targeting their protective bone marrow microenvironment.