Hedin, Karen E.

The bone marrow microenvironment acts as a protective niche for malignant cells. Within the bone marrow microenvironment is the endosteal niche, which is the area where the ossified surface of the bone and marrow cavity interact. The endosteal niche provides the opportunity for hematological malignancies such as acute myeloid leukemia (AML) of the bone marrow to interact with osteoblasts from the bone. To study the interaction between AML cells and osteoblasts, our lab developed a co‐culture model of osteoblasts (MC3T3 osteoblast cell line) and AML cells (KG1a cell line or patient samples from bone marrow aspirates). AML cells expressing high levels of CXCR4 were cultured alone or with osteoblasts and then challenged with SDF‐1, the ligand to CXCR4 and a chemokine readily abundant in the bone marrow microenvironment that our lab has previously shown actually induces apoptosis in AML cells expressing high levels of CXCR4 and that osteoblasts are able to protect AML cells from SDF‐1 induced apoptosis. Our lab now shows that treating osteoblasts with cyclosporine (CSA) prevents their ability to effectively protect AML cells from SDF‐1 induced cell death as assayed by annexin‐V staining of AML cells by flow cytometry. CSA treatment of osteoblasts decreased protein levels of tissue non‐specific alkaline phosphatase (ALPL), a phosphatase necessary for osteoblast mineralization, compared to vehicle control. As such, osteoblasts were treated with control siRNA or ALPL siRNA. CXCR4 expressing AML cells were then co‐cultured with the transfected osteoblasts and challenged with SDF‐1. siRNA knockdown of ALPL in osteoblasts was sufficient to prevent protection of AML cells from the apoptotic stimuli. These studies indicate that osteoblast differentiation plays a role in mediating the protection of AML cells. Therefore, targeting specific differentiation factors in osteoblasts may help to make to bone marrow microenvironment a more hostile environment for AML cells and aid in better malignancy eradication. Support or Funding Information This study was supported by NIH R21 CA194217 (to K.E.H.). R.M.S. is supported by the Mayo Clinic Medical Scientist Training Program Robert L. Howell Physician‐Scientist Scholarship.

Abstract The objective was to evaluate the impact of an interdisciplinary graduate level course on students’ knowledge and interests in immunotherapy and CAR-T cell therapy. It was a one week course conducted in November 2018. Students were surveyed before and after the course. The survey included questions regarding background, self-perceived knowledge/confidence in skills, and interests/predicted behaviors. Students were assigned to work in interdisciplinary teams to develop a research proposal. A total of 25 students taking the course for graduate level credit were surveyed. Of these, all 25 (100%) completed the surveys. Students came from variable backgrounds and were at different stages of graduate training. After completion of the course, there was a statistically significant increase in self-perceived knowledge of immunotherapy (mean±standard deviation score of 3.6±0.9 post-course vs 2.6±1.0 pre-course, on a 5 point Likert scale, P<0.001), knowledge of the benchtop research to clinic translational process (3.7±0.8 post-course vs 3.0±0.9 pre-course, P<0.001), confidence in critical reading skills (4.3±0.6 post-course vs 4.0±0.5 pre-course, P=0.008), confidence in immunotherapy focused grant writing skills (3.6±0.9 post-course vs 2.8±1.1 pre-course, P<0.001), interest in working in interdisciplinary teams (4.8±0.4 post-course vs 4.6±0.6 pre-course, P=0.02), and interest in attending immunotherapy educational events (3.5±1.3 post-course vs 3.2±1.3 pre-course, P≤0.05). In conclusion, this course helps trainees to build knowledge and interest in the fields of immunotherapy and CAR-T cell therapy and can potentially serve as a platform for future educational courses in interdisciplinary, translational research.

Abstract T cell cytokine production was recently shown to depend not only on the T cell antigen receptor (TCR), but also on the chemokine receptor CXCR4. Our lab recently published that stimulation of the TCR results in the TCR associating with and transactivating CXCR4, which, in turn, activates a PREX-1/Rac-1 pathway that stabilizes cytokine mRNA. This pathway therefore normally significantly increases IL-2, IL-4, and IL-10 production by activated T cells. Here, we further characterize the molecular mechanisms responsible for the first step of this pathway: TCR-mediated TCR-CXCR4 complex formation. First, we used FRET and proximity ligation assay (PLA) to define the required structural motifs of CXCR4, showing that this pathway requires the CXCR4 cytoplasmic tail domain and phosphorylation of CXCR4-S339. Second, we showed that TCR-induced TCR-CXCR4 complex formation requires TCR-induced activity of Src family and ZAP-70 tyrosine kinases, as well as one or more Ser/Thre kinases. Third, we found that the G protein-coupled receptor kinase-2 (GRK2) is required both for TCR-induced phosphorylation of CXCR4-S339 and for the formation of TCR-CXCR4 complexes. Finally, we found that GRK2 is required for robust IL-2, IL-4, and IL-10 cytokine secretion by normal human T cells. Together these results identify a novel role for TCR-induced GRK-2 in inducing the TCR-CXCR4 complex formation that subsequently signals to increase TCR-mediated cytokine production. Targeting GRK2 or other mechanisms required for TCR-induced TCR-CXCR4 formation may therefore be therapeutically useful for limiting production of T cell cytokines in humans, for example, during graft versus host disease or immunotherapy.

Abstract T cell cytokine production has been shown to be dependent not only on T cell antigen receptor (TCR), but also the chemokine receptor CXCR4. Evidence in the literature suggests that, in addition to stimulating migration, CXCR4 might also more directly regulate T cell immune activation. Recent studies from our lab have revealed that TCR stimulation in the absence of SDF-1 induces association of TCR and CXCR4, and that this is inhibited by pretreating cells with the specific CXCR4 antagonist AMD3100. Cytokine production, particularly cytokines IL-2, IL-4, and IL-10, by TCR-stimulated cells was inhibited by using either AMD3100 or CXCR4 siRNA to prevent TCR-CXCR4 formation. Interestingly, TCR-CXCR4 was not required for activation of many TCR-stimulated signaling pathways or for cytokine gene transcription. Instead, TCR-CXCR4 formation during TCR-mediated T cell immune activation was required for the post-transcriptional stabilization of cytokine mRNAs through a Rac-1 mediated pathway. Our results suggest that targeting the TCR-CXCR4 complex formation or downstream signaling pathways may provide therapeutic targets to regulate cytokine production.

Abstract The PD-1:PD-L1 axis is a major immune regulatory pathway that blunts T cell activation in the tumor microenvironment. We recently identified that PD-1 is also upregulated on ovarian mouse tumor-infiltrating myeloid dendritic cells (TIDCs) that exhibit an immune suppressive phenotype. Extending those observations in this study, we now report that PD-1 is also expressed on human myeloid TIDCs of ovarian cancer patients, on which it suppresses the expression of co-stimulatory molecules (CD40 and CD80) as well as production of inflammatory cytokines. Mechanistic studies revealed that PD-1 on TIDCs is associated with Src homology domain-containing phosphatase-2 (SHP-2) and that PD-1-mediated blockade of NFkB-dependent cytokine release is SHP-2 dependent. PD-1 also inhibits NFkB-mediated antigen presentation which appears, however, to be independent of SHP-2. Thus, suppression of NFkB is critical in mediating PD-1-induced paralysis of ovarian tumor-associated immune TIDCs and is mediated through multiple signaling circuits that are both SHP-2 dependent and independent.

Abstract IQGAP1 is a ubiquitous, cytoskeletal-interacting scaffold protein frequently overexpressed in cancer. CXCR4 is a chemokine receptor that signals upon binding stromal cell-derived factor 1 (SDF-1, CXCL12). CXCR4 regulates lymphocyte migration and development and cancer cell proliferation and metastasis. Here, we show that IQGAP1 potently enhances CXCR4’s expression, trafficking, and function in the Jurkat T cell line and epithelial cancer cells. The SDF-1-induced cell migration and activation of ERK MAPK were impaired in IQGAP1-deficient cells, even after forced CXCR4 overexpression. Exploring the mechanism, we found that SDF-1 treatment induced CXCR4 endocytosis and caused IQGAP1 to bind a-tubulin and localize to EEA-1 endosomes containing endocytosed CXCR4. In IQGAP1-deficient cells, CXCR4-containing EEA-1 endosomes accumulated at the distal ends of microtubules instead of moving towards the centrosome. This inhibited CXCR4’s signaling and cell-surface expression by preventing CXCR4 from trafficking into the Rab11 endosomes previously shown to be a critical site of CXCR4 signaling and recycling to the cell-surface. In contrast, IQGAP1 depletion had no effect on TCR trafficking, which does not require EEA-1 endosomes. Together, these results characterize a novel function of IQGAP1 as a regulator of EEA-1 endosomes, and in addition identify IQGAP1 as a critical enhancer of CXCR4 cell-surface expression and signaling in lymphocytes and epithelial cancer cells.

Abstract Osteoblasts are known to protect acute myeloid leukemia (AML) cells in the bone marrow microenvironment, however, the identity of these protective signals is not known. Because disrupting this protection could help prevent the relapse of AML patients following chemotherapy, we characterized the molecular mechanisms. We previously showed that SDF-1 (CXCL12), a chemokine abundant in the bone marrow, induces apoptosis of AML cells in cocultures unless the leukemic cells are protected via proximity to differentiating osteoblasts. Treating differentiating osteoblasts with either suberoylanilide hydroxamic acid (SAHA) or LBH-589 histone deacetylase (HDAC) inhibitor drugs inhibited their protection of AML cells. RNAseq revealed that SAHA globally inhibits osteoblast differentiation while inducing expression of the scaffold protein, Nherf-1. Remarkably, expressing Nherf-1 in differentiating osteoblasts was both required and sufficient to block osteoblast-mediated protection of AML cells. Together, these results indicate that disrupting the differentiation of osteoprogenitors in the bone marrow inhibits their ability to protect AML cells from apoptosis, and that this can be achieved via either inhibiting HDACs or overexpressing Nherf-1 in osteoblast-lineage cells. HDAC inhibitors or treatments specifically designed to induce Nherf-1 expression either during or immediately prior to chemotherapy may therefore be a useful strategy for reducing the risk of disease relapse in AML patients.

Stimulation of T lymphocytes with stromal cell‐derived factor‐1alpha (SDF‐1alpha/CXCL12) results in colocalization of the chemokine receptor, CXCR4, and the T cell receptor (TCR). Internalization of the colocalized complex can initiate prolonged activation of the extracellular signal‐regulated kinases (ERK) ERK1 and ERK2, gene transcription, cytokine expression and migration. The molecular mechanisms behind the internalization of the colocalized complex are incompletely characterized. In this study, we show that there are multiple sites in the CXCR4 C‐terminus that influence the internalization of the CXCR4/TCR complex in Jurkat and JRT3 T cells. A mutation of amino acids 328 and 329 in the CXCR4 C‐terminus, an important beta‐arrestin binding site, significantly reduced internalization in Jurkat cells. Deletion mutations in the CXCR4 C‐terminus also significantly reduced internalization in Jurkat cells. Basal internalization in Jurkat cells was decreased by an element(s) in amino acids 342–352. Basal internalization was induced by an element(s) in amino acids 334–342. Basal internalization in JRT3 cells was increased by an element in amino acids 322–334. SDF‐1alpha induced internalization was similarly regulated by the TCR. Research was supported by the Summer Undergraduate Research Fellowship at Mayo Clinic and Mayo Graduate School.

Abstract Angiogenesis plays an important role in tumor growth. Endothelial cells express the chemokine receptor CXCR4, and interact with its ligand SDF-1/CXCL12. Previous studies have demonstrated that the ERK MAP kinase and the PI3Kinase pathways are activated in response to SDF-1 stimulation. In this study, we investigate the effect of the role of inhibitors of CXCR4, ERK MAP kinase and PI3Kinase on angiogenesis. The AngioKit (TCS Cellworks, U.K) is a 24 well plate in which human endothelial cells are co-cultured with other human myoblasts and fibroblasts in a specially designed medium. Control wells in the kit include media alone, VEGF (+control) and suramin (-control). Test samples were added on the day the kits arrive, then changed on days 4, 7, and 9, and stained on day 11 with CD31 (PECAM). The wells are then photographed and subjected to image analysis. The software measures angiogenesis as total tubule length per well in microns. Test samples can then be compared to the control wells to determine the drugs affect on angiogenesis in vitro. The following drugs were tested in this angiogenesis model system, a human CXCR4 neutralizing antibody (MAB 171, R&D systems, MN), SDF-1, the MAP kinase inhibitor PD098059, and the PI3Kinase inhibitor LY294002. Treatment with the CXCR4 inhibitory antibody, PD098059, and LY294002 caused marked decrease in angiogenesis (below the level of the negative control suramin). Inhibition of angiogenesis below the level of suramin was first detected at 1mg/ml CXCR4 antibody, and10mg/ml CXCR4 antibody resulted in complete inhibition of angiogenesis. The effect of PD098059 on angiogenesis was dependent on its concentration; 20mM PD098059 inhibited angiogenesis while lower concentrations did not. These results are consistent with the drug’s known concentration-dependent inhibition of MEK-1 and indicate that the MEK-1 inhibitor leads to angiostasis secondary to its specific inhibitory effect on MEK-1. The lowest level tested of 1μM LY294002 led to inhibition of angiogenesis, and 50μM of LY294002 led to complete abrogation of angiogenesis. SDF-1 has been reported to be angiogenic. In this model system, the effect of SDF-1 alone on angiogenesis was subtle. However, the endothelial cells used in this model system may be secreting endogenous SDF-1 leading to the saturation of the CXCR4 receptor and minimal effects of exogenous SDF-1 stimulation. This was demonstrated by the significant effect of the CXCR4 inhibitor on angiogenesis without the addition of exogenous SDF-1. These results indicate that CXCR4 inhibition and its downstream pathways PI3K and ERK MAP kinase lead to significant inhibition of angiogenesis, and suggest that selective inhibitors of CXCR4 may be useful agents to inhibit angiogenesis.

Abstract Despite its efficacy, chimeric antigen receptor T-cell therapy (CART) is limited by the development of cytokine release syndrome (CRS) and neurotoxicity (NT). While CRS is related to extreme elevation of cytokines and massive T cell expansion, the exact mechanisms for NT have not yet been elucidated. Preliminary studies suggest that NT might be mediated by myeloid cells that cross the blood brain barrier. This is supported by correlative analysis from CART19 pivotal trials where CD14+ cell numbers were increased in the cerebrospinal fluid of patients that developed severe NT (Locke et al, ASH 2017). Therefore, we aimed to investigate the role of GM-CSF neutralization in preventing CRS and NT after CART cell therapy via monocyte control. First, we investigated the effect of GM-CSF blockade on CART cell effector functions. Here, we used the human GM-CSF neutralizing antibody (lenzilumab, Humanigen, Burlingame, California) that has been shown to be safe in phase II clinical trials. Lenzilumab (10 ug/kg) neutralizes GM-CSF when CART19 cells are stimulated with the CD19+ Luciferase+ acute lymphoblastic leukemia (ALL) cell line NALM6, but does not impair CART cell function in vitro. We have found that malignancy associated macrophages reduce CART proliferation. GM-CSF neutralization with lenzilumab results in enhanced CART cell antigen specific proliferation in the presence of monocytes. To confirm this in vivo, NOD-SCID-g-/- mice were engrafted with high disease burdens of NALM6 and treated with low doses of CART19 or control T cells (to induce tumor relapse), in combination with lenzilumab or isotype control antibody. The combination of CART19 and lenzilumab resulted in significant anti-tumor activity and overall survival benefit compared to control T cells (Fig 1A), similar to mice treated with CART19 combined with isotype control antibody, indicating that GM-CSF neutralization does not impair CART cell activity in vivo. This anti-tumor activity was validated in an ALL patient derived xenograft model. Next, we explored the impact of GM-CSF neutralization on CART cell related toxicities in a novel patient derived xenograft model. Here, NOD-SCID-g-/- mice were engrafted with leukemic blasts (1-3x106 cells) derived from patients with high risk ALL. Mice were then treated with high doses of CART19 cells (2-5x106 intravenously). Five days after CART19 treatment, mice began to develop progressive motor weakness, hunched bodies, and weight loss that correlated with massive elevation of circulating human cytokine levels. Magnetic Resonance Imaging (MRI) of the brain during this syndrome showed diffuse enhancement and edema, associated with central nervous system (CNS) infiltration of CART cells and murine activated myeloid cells. This is similar to what has been reported in CART19 clinical trials in patients with severe NT. The combination of CART19, lenzilumab (to neutralize human GM-CSF) and murine GM-CSF blocking antibody (to neutralize mouse GM-CSF) resulted in prevention of weight loss (Fig 1B), decrease in critical myeloid cytokines (Fig 1C-D), reduction of cerebral edema (Fig 1E), enhanced leukemic disease control in the brain (Fig 1F), and reduction in brain macrophages (Fig 1G). Finally, we hypothesized that disrupting GM-CSF through CRISPR/Cas9 gene editing during the process of CART cell manufacturing would result in functional CART cells with reduced secretion of GM-CSF. We designed guide RNA targeting exon 3 of the GM-CSF gene and generated GM-CSFk/o CART19 cells. Our preliminary data suggest that these CARTs produce significantly less GM-CSF upon activation but continue to exhibit similar production of other cytokines and exhibit normal effector functions in vitro (Fig 1H). Using the NALM6 high tumor burden relapse xenograft model as described above, GM-CSFk/o CART19 cells resulted in slightly enhanced disease control compared to CART19 cells (Fig 1I). Thus, modulating myeloid cell behavior through GM-CSF blockade can help control CART mediated toxicities and may reduce their immunosuppressive features to improve leukemic control. These studies illuminate a novel approach to abrogate NT and CRS through GM-CSF neutralization that also potentially enhances CART cell functions. Based on these results, we have designed a phase II clinical trial using lenzilumab as a modality to prevent CART related toxicities in patients with diffuse large B cell lymphoma. Disclosures Ahmed: Humanigen: Employment. Sahmoud:Humanigen: Employment. Durrant:Humanigen: Employment. Russell:Vyriad: Equity Ownership. Kay:Morpho-sys: Membership on an entity's Board of Directors or advisory committees; Pharmacyclics: Membership on an entity's Board of Directors or advisory committees, Research Funding; Tolero Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Research Funding; Gilead: Membership on an entity's Board of Directors or advisory committees; Agios Pharm: Membership on an entity's Board of Directors or advisory committees; Cytomx Therapeutics: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees; Infinity Pharm: Membership on an entity's Board of Directors or advisory committees; Acerta: Research Funding. Kenderian:Novartis: Patents & Royalties; Tolero Pharmaceuticals: Research Funding; Humanigen: Research Funding.