Hedin, Karen E.

Abstract Malignant B-cells characteristically home to the bone marrow and lymph nodes. However, the mechanisms by which cells are recruited into and mobilized from the bone marrow/lymph nodes into the peripheral blood are not well understood. Chemokines such as SDF-1 and it receptor CXCR4 play a central role for lymphocyte trafficking and homing. Downstream activation of the PI3K pathway by chemokines has been implicated in the migration of many cell types. We recently demonstrated that CXCR4 expression correlates significantly with disease progression in CLL. Patients with Rai stage 4 had a higher expression of CXCR4 when compared to patients with Rai stage 0. The expression of CXCR4 was not found on CLL B cells resident within the lymph nodes implying that there may have been downregulation of CXCR4. In this study, we explore the molecular mechanisms involved in homing and migration of B-CLL cells in response to CXCR4 and investigated the role of the PI3K pathway in migration of CLL cells in response to SDF-1. Boyden chamber in vitro migration assays were used with primary CLL peripheral blood cells and the MEK1 CLL cell line. In addition, we expressed fluorescent YFP-CXCR4 in the cells and used confocal microscopy to visualize changes in the subcellular location of the fluorescent CXCR4 before and after SDF-1 stimulation. Inhibitors of the PI3K pathway such as the PI3K inhibitor LY294002 (10uM, one hour pretreatment) and the mTOR inhibitor, rapamycin (200nM overnight) were used. SDF-1 induced a dose dependent migration of primary CLL cells and MEK1 cells, indicating functional CXCR4 receptors. MEK 1 cells transfected with YFP-CXCR4 demonstrated surface localization on the cells. 3-Dimensional and continuous live imaging after SDF-1 stimulation for 30 minutes demonstrated alterations in the CXCR4-YFP leading to its capping and internalization subcellularly. Pretreatment of the cells with PI3K pathway inhibitors led to inhibition of migration with LY294002 but not with rapamycin. Confocal microscopy demonstrated abrogation of subcellular localization in the presence of LY294002 but not with rapamycin with the addition of SDF-1. These results demonstrate that CLL cells express functional CXCR4 receptora and that the PI3K pathway is essential for SDF-1 dependent CLL migration. In addition, we demonstrate that the CXCR4 receptor is internalized in response to SDF-1 stimulation indicating that CLL cells downregulate the CXCR4 receptor once they home to the bone marrow or lymph nodes where there is abundant SDF-1 in the microenvironment. This leads to the confinement of the cells in the microenvironment preventing further migration of the cells outside of the bone marrow/lymph nodes. Supported in part by CA97274

Abstract Malignant plasma cells characteristically home to the bone marrow (BM). However, the mechanisms by which cells are recruited into and mobilized from the bone marrow into the peripheral blood (PB) are not well understood. In this study, we explore the molecular mechanisms involved in homing and migration of plasma cells in response to CXCR4 and investigated the role of the PI3K pathway in migration of MM cells in response to SDF-1. CXCR4 surface expression was determined by FACS analysis (PE CXCR4, Pharmingen) using samples from bone marrow and peripheral blood of patients diagnosed with multiple myeloma (MM), monoclonal gammopathy of undetermined significance (MGUS) and primary systemic amyloidosis (AL). All results were expressed as percent expression in gated CD38+ and CD45+ cells. Boyden chamber in vitro migration assays were performed using Kas6/1MM cells. In addition, live confocal microscopy was used to visualize changes in the subcellular location of YFP-fluorescent CXCR4 before and after SDF-1 stimulation. Cells were pretreated with 10mM LY294002 or 200nM rapamycin to assess the effect of inhibition of PI3K or mTOR on CXCR4 subcellular localization. Immunoblotting was performed to confirm inhibition of the PI3K pathway. Comparisons between groups was performed using Mann-Whitney testing. The median CXCR4 expression is described in table 1. There was a significant difference between expression of BM CXCR4 in MM and MGUS (p=0.02). SDF-1 induced dose dependent migration of Kas 6/1 cells indicating a functional CXCR4 receptor. MM cells transfected with YFP-CXCR4 demonstrated surface localization on the cells. 3-Dimensional and continuous live imaging after SDF-1 stimulation for 30 minutes demonstrated alterations in the CXCR4-YFP leading to its capping, internalization subcellularly, and production of pseudopodia in response to SDF-1. This process was abrogated with pretreatment with LY294002 and rapamycin. These data demonstrate for the first time that the surface expression of CXCR4 is markedly elevated in the peripheral blood as compared to the bone marrow. Once in the bone marrow, and with the presence of excess SDF-1, the receptor becomes internalized and downregulated. In contrast to MM, CXCR4 expression on plasma cells in the bone marrow of normal and MGUS patients was higher. In addition, we demonstrate that the process of CXCR4 subcellular localization is abrogated by the administration of PI3K inhibitors LY294002 and rapamycin. These data suggest that CXCR4 expression is required for MM cells to circulate, and that downregulation occurs in the BM leading to immobilization of the cells. Future clinical trials using CXCR4 inhibitors, or inhibitors of PI3K and mTOR to prevent the homing and migration of MM cells into the bone marrow may be explored. This was supported in part by CAP50 CA97274. CXCR4 expression N=45 Median CXCR4% expression of total plasma cells Range MM BM (n=23) 27 4.3–90.1 MM PB (n=5) 91 60–95.5 MGUS BM (n=6) 65 41–78.6 AL BM (n=7) 46 17.5–71.6 Normal BM (n=4) 59 6.3–69.5 Kas6/1 cell line 100 -

Abstract Chimeric antigen receptor T (CAR-T) cell therapy is a new pillar in cancer therapeutics; however, its application is limited by the associated toxicities. These include cytokine release syndrome (CRS) and neurotoxicity. Although the IL-6R antagonist tocilizumab is approved for treatment of CRS, there is no approved treatment of neurotoxicity associated with CD19-targeted CAR-T (CART19) cell therapy. Recent data suggest that monocytes and macrophages contribute to the development of CRS and neurotoxicity after CAR-T cell therapy. Therefore, we investigated neutralizing granulocyte-macrophage colony-stimulating factor (GM-CSF) as a potential strategy to manage CART19 cell–associated toxicities. In this study, we show that GM-CSF neutralization with lenzilumab does not inhibit CART19 cell function in vitro or in vivo. Moreover, CART19 cell proliferation was enhanced and durable control of leukemic disease was maintained better in patient-derived xenografts after GM-CSF neutralization with lenzilumab. In a patient acute lymphoblastic leukemia xenograft model of CRS and neuroinflammation (NI), GM-CSF neutralization resulted in a reduction of myeloid and T cell infiltration in the central nervous system and a significant reduction in NI and prevention of CRS. Finally, we generated GM-CSF–deficient CART19 cells through CRISPR/Cas9 disruption of GM-CSF during CAR-T cell manufacturing. These GM-CSFk/o CAR-T cells maintained normal functions and had enhanced antitumor activity in vivo, as well as improved overall survival, compared with CART19 cells. Together, these studies illuminate a novel approach to abrogate NI and CRS through GM-CSF neutralization, which may potentially enhance CAR-T cell function. Phase 2 studies with lenzilumab in combination with CART19 cell therapy are planned.

Abstract The endosteal niche is a component of the bone marrow microenvironment that can serve to protect hematological malignancies such as acute myeloid leukemia (AML) from standard chemotherapies such as cytarabine (Ara-C). Surviving AML cells harbored by this niche can eventually lead to relapse. The endosteal niche is rich in osteoblast lineage cells. U937 or KG1a AML cell lines were cultured with or without osteoblast lineage cells (MC3T3 or W-20-17 cell lines), challenged with doses of 0µM, 0.1µM, 0.5µM, 1µM, 5µM, or 10µM of Ara-C, and assayed for apoptosis via annexin-V staining and flow cytometry. Osteoblast lineage cells (MC3T3 or W-20-17 cell lines) were able to protect AML cells (U937 or KG1a cell lines) from Ara-C-induced apoptosis. Histone deacetylase inhibitors (HDACi) globally alter gene expression within cells. When we pre-treated osteoblast lineage cells (MC3T3) with the HDACi vorinostat (suberoylanilide hydroxamic acid, SAHA), it reduced the ability of the osteoblast lineage cells (MC3T3) to protect AML cells (U937) from Ara-C-induced apoptosis, which we have previously confirmed in the KG1a AML cell line as well. This indicates that osteoblast lineage cell-mediated protection of AML from Ara-C occurs via an HDACi sensitive mechanism. To begin to further explore the mechanism of action, we co-cultured AML cells (KG1a or U937) with and without osteoblast lineage cells (MC3T3) in the presence or absence of a transwell. We found that the presence of the transwell reduced the osteoblast lineage cell-mediated protection, indicating that osteoblast lineage cell-mediated protection of AML from Ara-C is cell contact dependent. Thus, osteoblast lineage cells can protect AML cells from Ara-C induced apoptosis, this protection can be reduced by pre-treatment of the osteoblast lineage cells with the HDACi vorinostat, and osteoblast lineage cell-mediated protection from Ara-C is cell contact dependent. These studies begin to characterize the mechanisms of osteoblast lineage cell-mediated protection of AML from Ara-C. Manipulating the protective properties of osteoblast lineage cells of the endosteal niche may help make AML cells more susceptible to chemotherapeutics. Therefore, developing combination therapies that target the protective mechanisms of osteoblast-lineage cells may help to further deplete the bone marrow microenvironment of AML cells and prevent relapse of disease. Citation Format: Rosalie M. Sterner, Kimberly N. Kremer, Meagan R. Rollins, Amel Dudakovic, Jennifer J. Westendorf, Andre J. van Wijnen, Karen E. Hedin. Osteoblast-lineage cells protect AML cells from cytarabine-induced apoptosis via a mechanism sensitive to HDACi and reduced cell-cell contact [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 2137.

Abstract The bone marrow microenvironment harbors and protects leukemic cells from apoptosis-inducing agents via mechanisms that are incompletely understood. We previously showed SDF-1 (CXCL-12), a chemokine readily abundant within the bone marrow microenvironment, induces apoptosis in acute myeloid leukemia (AML) cells that express high levels of the SDF-1 receptor CXCR4. However, differentiating osteoblasts found within this niche protect cocultured AML cells from apoptosis. Additionally, this protection was abrogated upon treatment of the differentiating osteoblasts with histone deacetylase inhibitors (HDACi). In this study, we begin to characterize and target the molecular mechanisms that mediate this osteoblast protection. Quantitative RT-PCR revealed that HDACi treatment of differentiating osteoblasts (mouse MC3T3 osteoblast cell line) reduced expression of multiple genes required for osteoblast differentiation, including genes important for producing mineralized bone matrix. Interestingly, pretreating differentiating osteoblasts with cyclosporine A, a drug known to inhibit osteoblast differentiation, similarly impaired osteoblast-mediated protection of cocultured AML cells (KG1a and U937 human AML cell lines). Both HDACi and cyclosporine A reduced osteoblast expression of the key mineralization enzyme tissue-nonspecific alkaline phosphatase (TNAP; encoded by Alpl). Moreover, specifically reducing TNAP expression or activity in differentiating osteoblasts significantly impaired the ability of the osteoblasts to protect cocultured AML cells. Together, our results indicate that inhibiting osteoblast matrix mineralization by specifically targeting TNAP is sufficient to significantly impair osteoblast-mediated protection of AML cells. Therefore, designing combination therapies that additionally target the osteoblast-produced mineralized bone matrix may improve treatment of AML by reducing the protection of leukemic cells within the bone marrow microenvironment.

Abstract The hematological malignancy acute myeloid leukemia (AML) interacts closely with osteoblasts within the protective bone marrow microenvironment. The bone marrow microenvironment protects tumor cells from chemotherapies, which can prevent sufficient eradication of tumor cells. To study the role of osteoblasts in the bone marrow microenvironment, our lab utilized a co-culture model of osteoblasts (MC3T3 osteoblast cell line) and acute myelogenous leukemia cells (KG1a AML cell line or AML patient samples from bone marrow aspirates). Osteoblasts were cultured with AML cells, or AML cells were cultured alone; AML cells were challenged with the standard chemotherapeutic agent cytarabine (Ara-C) at doses of 0µM, 0.1µM, 0.5µM, 1µM, 5µM, or 10µM in the presence or absence of osteoblasts; and AML cells were assayed by flow cytometry to assess cell death via annexin-V staining. Our lab has previously found that differentiating osteoblasts protect AML cells from an apoptosis inducing agent naturally present in the bone marrow. We now show that osteoblasts are also capable of protecting AML cells from the standard chemotherapeutic cytarabine. In addition, we have found that treatment of osteoblasts with the histone deacetylase inhibitor (HDACi) suberoylanilide hydroxamic acid (SAHA) prevents these treated osteoblasts from protecting AML cells from cytarabine treatment, which is consistent with our lab’s previous findings that HDACi treatment inhibits protection of AML cells from an apoptosis inducing agent naturally present in the bone marrow. We have preliminary data that indicates that TAZS89A over-expression, a constitutively active form of TAZ, which is a transcriptional modulator that regulates osteoblast differentiation, may be sufficient to inhibit osteoblast mediated protection of AML cells from cytarabine. This finding would be consistent with the HDACi manipulated Nherf1-protein phosphatase 1α-TAZ signaling pathway that we have previously found to be sufficient to inhibit protection of AML cells from an apoptosis inducing agent naturally present in the bone marrow. Overall, these studies have delivered insights into the role of osteoblasts in protecting AML cells from chemotherapy in the bone marrow microenvironment and begun the characterization of mechanisms and targets responsible for the protective effects of osteoblasts. Manipulating differentiating osteoblasts within the bone marrow microenvironment therapeutically could aid in more complete destruction of the tumor cell burden and improve patient survival. Citation Format: Rosalie M. Sterner, Kimberly N. Kremer, Amel Dudakovic, Jennifer J. Westendorf, Andre J. van Wijnen, Karen E. Hedin. Osteoblasts protect AML cells from cytarabine-induced death [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5930. doi:10.1158/1538-7445.AM2017-5930

Key Points T-cell activation induces TCR transactivation of CXCR4 to stabilize cytokine mRNA transcripts via a PREX1-Rac1–signaling pathway. Inhibition of the TCR-CXCR4–signaling pathway impairs TCR-dependent and TCR-independent cytokine secretion by CTCL cells.

Abstract While standard chemotherapeutics can initially induce remission in patients with acute myelogenous leukemia (AML), patients often relapse in part due to the protective bone marrow microenvironment, which promotes the survival of AML cells. Using an in vitro cell culture model of the bone marrow, our lab recently showed that differentiating osteoblasts potently protect AML cells from apoptosis. In addition, we found that differentiating osteoblasts treated with histone deacetylase inhibitors (HDACi) fail to protect AML cells in this model. Since HDACi decreased the expression of genes required for osteoblast differentiation, we hypothesize that blocking specific stage(s) of osteoblast differentiation may make AML cells within the bone marrow more susceptible to chemotherapy. We are currently disrupting specific osteoblast differentiation stages via molecular or therapeutic drug manipulation and exploring the impact on AML cell survival both in cell culture models of the bone marrow microenvironment and in a mouse model of AML. The results of our studies will characterize novel bone marrow molecular and cellular interactions that promote the survival of AML cells. Targeting these interactions in combination therapies may more completely eradicate the AML cell reservoir and better prevent relapse. Citation Format: Rosalie Sterner, Kimberly Kremer, Amel Dudakovic, Scott Kaufmann, Jennifer Westendorf, Andre van Wijnen, Karen Hedin. Manipulating osteoblast differentiation in order to inhibit protection of AML cells within the bone marrow. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3292.

Abstract The PD-1:PD-L1 immune signaling axis mediates suppression of T-cell–dependent tumor immunity. PD-1 expression was recently found to be upregulated on tumor-infiltrating murine (CD11c+CD11b+CD8−CD209a+) and human (CD1c+CD19−) myeloid dendritic cells (TIDC), an innate immune cell type also implicated in immune escape. However, there is little knowledge concerning how PD-1 regulates innate immune cells. In this study, we examined the role of PD-1 in TIDCs derived from mice bearing ovarian tumors. Similar to lymphocytes, TIDC expression of PD-1 was associated with expression of the adapter protein SHP-2, which signals to NF-κB; however, in contrast to its role in lymphocytes, we found that expression of PD-1 in TIDC tonically paralyzed NF-κB activation. Further mechanistic investigations showed that PD-1 blocked NF-κB–dependent cytokine release in a SHP-2–dependent manner. Conversely, inhibition of NF-κB–mediated antigen presentation by PD-1 occurred independently of SHP-2. Collectively, our findings revealed that PD-1 acts in a distinct manner in innate immune cells compared with adaptive immune cells, prompting further investigations of the signaling pathways controlled by this central mediator of immune escape in cancer. Cancer Res; 76(2); 239–50. ©2015 AACR.