ALBERT

Description: Introduction: Wilms tumor protein 1 (WT1) is a transcription factor that is universally expressed in AML, and clinical studies have demonstrated a significant correlation between WT1 expression and disease progression. Heat shock proteins (HSP) are ubiquitous molecular chaperones that are involved in the folding, activation and assembly of many proteins. HSP90 may facilitate the maintenance of altered oncogenic client proteins such as flt-3 and bcr-abl in myeloid malignancies. It has been also shown that WT1 is a client protein of Hsp70, and this interaction may be important for the function of WT1. Since HSP70 and HSP90 have similar functions and often complement each other in engaging with client cellular proteins we wanted to determine if WT1 was chaperoned by HSP90 and if so could this HSP90-WT1 interaction be modulated. Materials and methods: We first looked for a potential interaction between HSP90 and WT1 by confocal microscopy colocalization and then by immunoprecipitation (IP) in the myeloid cell line K562. For confocal studies, cells were grown on coverslips, fixed and permeabilized and stained with the rabbit anti-WT1 and anti- HSP90 antibodies. Samples were examined by using a laser confocal/Zeiss axiovert microscope imaging system. This interaction was further confirmed by co-IP/Western and then confirmed using a GST-pulldown assay. A glutathione S-transferase (GST)-WT1 fusion protein was expressed in E. coli strain BL21(DE3) and immobilized onto glutathione sepharose beads. The full length Hsp90 and different deletion mutant proteins were in vitro translated and [35S] methionine-labeled; the bound proteins were eluted and then separated by SDS-PAGE. The gels were fixed, dried and subjected to fluorography. Results: Confocal microscopy revealed colocalization of HSP90 and WT1 mainly in the nucleus. Immunoprecipitation of K562 lysates with either anti-WT1 or anti-HSP90 antibody followed by Western analysis for WT1 and HSP90 demonstrated that WT1 and HSP90 could be co-immunoprecipitated. The GST pull down assay confirmed this interaction. Additional mutants of HSP90 from the C to N terminus were prepared to localize the WT1 interacting domain. These studies localized the WT1 interacting domain to the HSP90 region close to the binding pocket of the HSP inhibitor geldanamycin. Treatment of K562 with the geldanamycin analog 17-allylamino-17-demethoxygeldanamycin (17-AAG) resulted in downregulation of WT1 protein expression with an IC50 of 3μM. Conclusions: Understanding the mechanisms that regulate WT1 expression thus may lead to novel therapeutic strategies to inhibit this expression and result directly in myeloid blast cell killing. We have shown elsewhere that downmodulating WT1 gene expression with interferon or shRNA potentiates myeloid leukemic blast killing by chemotherapeutic agents. We demonstrate here that the WT1 protein interacts with the co-chaperone heat shock protein HSP90. Taken together, our findings support the hypothesis that the regulation of WT1 occurs at the protein level through an interaction with WT1-protein-HSP chaperone levels, and that pharmacologically targeting this interaction with the HSP inhibitor 17AAG results in a significant reduction in WT1 protein expression. Ongoing studies in primary AML cells will confirm these findings and define novel therapeutic strategies to manipulate WT1 protein expression levels and modulate leukemic blast survival.

Description: While a very high proportion of humans develop serious and fatal GVHD following bone marrow transplantation (BMT), mice typically do not develop lethal graft versus host disease (GVHD) when receiving bone marrow alone, even when the donor and recipient mice are MHC mismatched. Thus, the major pre-clinical model in which novel treatments for GVHD are being tested has a fundamental resistance to GVHD. Researchers therefore add donor-derived T cells from spleen or lymph node in order to generate a similar incidence and severity of GVHD as seen in humans, thus using a model system that has fundamental differences from the clinical situation. Recent work from our lab has revealed that the standard cool (sub-thermoneutral) temperature in which all laboratory mice must be housed (creating a chronic condition of metabolic cold stress) results in impaired CD8+ immune function, while bolstering the action of immunosuppressive cells. Current work suggests that the excessive stimulation of cold-stress induced sympathetic nerve activity, and subsequent production of norepinephrine is the cause of this immunosuppressive environment. Because GVHD depends upon the activation and function of donor CD8+ T cells in the host environment, we hypothesized that mouse models do not develop expected amounts of GVHD following a bone marrow only transplant because the host environment, which is metabolically cold-stressed, blocks the function of effector T cells. To test our hypothesis we first housed BALB/c mice at standard (22°C) and thermoneutral temperatures (30°C) for two weeks. To generate a MHC disparate model, we lethally irradiated the mice and transplanted C57BL/6 bone marrow without additional splenocytes. We used both weight loss and survival following BMT as a readout for GVHD severity. As expected, under standard conditions of housing at 22° mice were able to recover from the transplant and return to their initial weight within the same 17 day post-transplant timeframe (percent initial weight at 22°: 101% ± 1%; N=10). In support of our hypothesis we saw weight loss in the mice maintained at 30° as early as 17 days post-transplant (percent initial weight at 30°: 93% ± 2%; N=10). Interestingly, weight loss continued in the mice maintained at thermoneutrality (percent initial weight loss 48 days post-transplant at 30°: 0.84 ± 0.03, N=10). Beginning 49 days post-transplant some mice at thermoneutrality began to succumb to GVHD. Thus, housing mice at thermoneutrality decreased survival suggesting a greater incidence/more severe GVHD (Figure 1; p=0.01, N=10). As our previous data in solid tumor models suggests that numbers of immunosuppressive myeloid derived suppressor cells (MDSCs) are increased when mice are metabolically stressed at 22°, we next examined differences in this cell population in the spleens of transplanted mice. We found an obvious trend towards decreased splenic MDSCs in mice housed at 30° compared to 22°. To retest this hypothesis in a clinically relevant minor antigen mismatch model we housed male C57BL/6 mice at ST or TT for two weeks. We then lethally irradiated the mice and transplanted female 129/SvJ bone marrow. Again, we found a greater weight loss (p=0.02; N=10) in the mice receiving bone marrow only maintained at 30° compared to 22° as early as 28 days post-transplant (percent initial weight at 22°: 103% ± 2%, N=10; percent initial weight at 30°: 96% ± 2%, N=10). We also found increased numbers of MDSCs in the spleens of mice housed at 22° compared to 30° (p=0.09, N=10). The increasing number of patients receiving BMT for various malignancies clearly highlights the critical importance of improving mouse models for understanding GVHD as this problem remains a major barrier to achieving greater survival for patients. These data demonstrate the existence of a fundamental relationship between cool standard housing temperature used in all research institutes and a resistance to GVHD, which is likely linked to a reduced immune response and/or increased immunosuppression. Achieving a better understanding of this potential confounding environmental factor could help to improve the use of mouse models for identifying the best therapies to attenuate or eliminate GVHD in humans, while at the same time, promoting effective immune reconstitution and graft versus tumor effect. Disclosures: No relevant conflicts of interest to declare.

Description: β2 adrenergic receptor signaling is a key regulator of various immune cells, including T cells; however, its role in T cell function in the context of graft versus host disease (GvHD) is poorly understood. We previously showed that housing mice at thermoneutral temperature (TT; 30°C), which reduces systemic adrenergic stress, increased the incidence and severity of GvHD after allogeneic hematopoietic cell transplant (allo-HCT) compared to mice housed at standard temperature (ST; 22°C) which exerts a mild but chronic adrenergic stress (Leigh et al J Immunol 2015). The increased incidence and severity of GvHD in TT mice can be reversed by the administration of a β2-adrenergic receptor (β2-AR) agonist, suggesting an important role of epinephrine and norepinephrine in allo-HCT outcome (Leigh et al., J. Immunol 2015; Mohammadpour et al J Immunol 2018). We investigated the mechanisms and downstream events of β2-AR signaling in donor T cells after allo-HCT by using β2-AR knockout (β2-AR-/-) mice and commercially available β2-AR agonists. The main goal here was to explore whether signaling through β2-AR in donor T cells could control GvHD incidence and severity without minimizing the graft-versus leukemia (GvL) effect. We utilized both a major MHC-mismatch C57B6 (H-2kb) into BALB/c (H-2kd) model and a MHC-matched, multiple minor histocompatibility antigen (miHA) mismatched B6 (H-2kb) into C3H/SW (H-2kb) model. Recipient BALB/c and C3H/SW WT mice were lethally irradiated with 850 and 1100 cGy respectively and injected by tail vein with T cell depleted bone marrow (TCD-BM) alone (3 ×106) or TCD-BM and splenic T cells derived from allogeneic WT or β2-AR-/- B6 donors (0.7 × 106 T cells in B6 → BALB/c and 1.5 × 106 in B6 → C3H/SW). We found that donor T cells express β2-AR after allo-HCT and that β2-AR expression on WT T cells plays an important role in controlling GvHD, as evidenced by less severe weight loss, and increased survival compared to mice receiving β2-AR-/- donor T cells (Figure 1A). Histopathologic examination showed that β2-AR-/- T cells induced more damage in the small and large intestine. To explore further the mechanism(s) by which β2-AR signaling controls the severity of GvHD, we used NanoString analysis and discovered that β2-AR-/- T cells have the Th1 phenotype with an increase in Tbx21, Ifng, Irf8 and Emoes genes, while WT CD4+ T cells had higher levels of Th2 and Treg associated genes, including Foxp3, Ptgs5, Tgfb2, Il10, Il21 and Il22. We also observed a significant increase in the inflammatory cytokines IFN-γ and IL-17 in β2-AR-/- CD4+ T cells from the spleen and liver on days 7 and 14 after allo-HCT as compared to WT T cells (Figure 1B), while the expression of IL-10 was significantly higher in WT T cells compared to β2-AR-/- T cells (P〈 0.01). We next sought to determine whether GvL may be affected by use of long acting β2-AR agonist (Bambuterol) to control GvHD. Bambuterol was administered daily at a dose of 1mg/kg from day 0. We observed that Bambuterol controlled the severity and mortality of GvHD after allo-HCT in both major and minor mismatch mouse models, as evidenced by reduced weight loss and an improved clinical score and survival rate in mice receiving Bambuterol compared to vehicle (P