Olga N. Uchakina

Targeting hyaluronic acid production for the treatment of leukemia: Treatment with 4-methylumbelliferone leads to induction of MAPK-mediated apoptosis in K562 leukemia

The current study examined the effect of modulation of hyaluronic acid (HA) synthesis on leukemia cell survival using the hyaluronic acid synthesis inhibitor 4-methylumbelliferone (4-MU). Treatment of CML cells with 4-MU led to caspase-dependent apoptosis characterized by decreased HA production, PARP cleavage, and increased phosphorylation of p38. Addition of exogenous HA, the pan caspase inhibitor Z-VAD-FMK or the p38 inhibitor SB203580 to 4-MU treated cells was able to protect cells from apoptosis. Treatment of tumor-bearing mice with 4-MU led to a significant reduction in tumor load which was mediated through the induction of apoptosis.

Fatigue in Medical Residents Leads to Reactivation of Herpes Virus Latency

The main objective of this study was to detect fatigue-induced clinical symptoms of immune suppression in medical residents. Samples were collected from the subjects at rest, following the first night (low-stress), and the last night (high-stress) of night float. Computerized reaction tests, Epworth Sleepiness Scale, and Wellness Profile questionnaires were used to quantify fatigue level. DNA of human herpes viruses HSV-1, VZV, EBV, as well as cortisol and melatonin concentrations, were measured in saliva. Residents at the high-stress interval reported being sleepier compared to the rest interval. EBV DNA level increased significantly at both stress intervals, while VZV DNA level increased only at low-stress. DNA levels of HSV-1 decreased at low-stress but increased at high-stress. Combined assessment of the viral DNA showed significant effect of stress on herpes virus reactivation at both stress intervals. Cortisol concentrations at both stress intervals were significantly higher than those at rest.

The role of hyaluronic acid in SEB-induced acute lung inflammation.

We investigated the role of the extracellular matrix component, hyaluronic acid (HA) in SEB-induced ALI/ARDS. Intranasal exposure of mice to SEB led to a significant increase in the level of soluble hyaluronic acid in the lungs. Similarly, in an endothelial cell/spleen cell co-culture, SEB exposure led to significant increases in soluble levels of hyaluronic acid, cellular proliferation, and cytokine production compared with SEB-exposed spleen cells or endothelial cells alone. Exposure of SEB-activated spleen cells to hyaluronic acid led to increased cellular proliferation and increased cytokine production. SEB-induced cytokine production and proliferation in vitro were significantly reduced by the hyaluronic acid blocking peptide, Pep-1. Finally, treatment of SEB-exposed mice with Pep-1 significantly reduced SEB-induced ALI/ARDS, through reduction of cytokine production and numbers of lung inflammatory cells, compared to mice treated with a control peptide. Together, these results suggest the possibility of targeting HA for the treatment of SEB-induced ALI/ARDS.

Treatment with the Hyaluronic Acid Synthesis Inhibitor 4-Methylumbelliferone Suppresses SEB-Induced Lung Inflammation

Exposure to bacterial superantigens, such as staphylococcal enterotoxin B (SEB), can lead to the induction of acute lung injury/acute respiratory distress syndrome (ALI/ARDS). To date, there are no known effective treatments for SEB-induced inflammation. In the current study we investigated the potential use of the hyaluronic acid synthase inhibitor 4-methylumbelliferone (4-MU) on staphylococcal enterotoxin B (SEB) induced acute lung inflammation. Culturing SEB-activated immune cells with 4-MU led to reduced proliferation, reduced cytokine production as well as an increase in apoptosis when compared to untreated cells. Treatment of mice with 4-MU led to protection from SEB-induced lung injury. Specifically, 4-MU treatment led to a reduction in SEB-induced HA levels, reduction in lung permeability, and reduced pro-inflammatory cytokine production. Taken together, these results suggest that use of 4-MU to target hyaluronic acid production may be an effective treatment for the inflammatory response following exposure to SEB.

Treatment with the Hyaluronic Acid Synthesis Inhibitor 4-Methylumbelliferone Suppresses LPS-Induced Lung Inflammation

Exposure to bacterial endotoxins, such as lipopolysaccharide (LPS), can lead to the induction of acute lung injury/acute respiratory distress syndrome (ALI/ARDS). To date, there are no known effective treatments for LPS-induced inflammation. In the current study, we investigated the potential use of the hyaluronic acid (HA) synthesis inhibitor 4-methylumbelliferone (4-MU) on LPS-induced acute lung inflammation. Culturing LPS-activated immune cells with 4-MU led to reduced proliferation, reduced cytokine production, and an increase in apoptosis when compared to untreated cells. Treatment of mice with 4-MU led to protection from LPS-induced lung injury. Specifically, 4-MU treatment led to a reduction in LPS-induced hyaluronic acid synthase (HAS) messenger RNA (mRNA) levels, reduction in lung permeability, and reduction in proinflammatory cytokine production. Taken together, these results suggest that use of 4-MU to target HA production may be an effective treatment for the inflammatory response following exposure to LPS.

Development of a novel treatment for leukemia directed at tumor-associated mRNA splicing

This report describes a novel approach to cancer therapy that targets genes that are preferentially alternatively spliced and expressed in leukemia. We developed CD44v6 and CD44v8 splicing constructs fused with GFP or a humanized fragment of Pseudomonas aeruginosa exotoxin A (hPE24). Transfection of K562 leukemia cells with the GFP-linked splicing constructs led to subsequent production of detectable levels of GFP. Transfection of K562 cells with the hPE24-linked splicing constructs led to significant reduction of cell viability and an increase in the induction of apoptosis. Normal human PBMCs were unaffected by following transfection with these constructs.

Inhibition of hyaluronic acid formation sensitizes chronic myelogenous leukemia to treatment with doxorubicin

In the current study we examined the ability of 4-methylumbelliferone (4-MU), which can inhibit hyaluronic acid synthesis, to sensitize K562 chronic myelogenous leukemia (CML) cells to doxorubicin therapy. Exposure of K562 cells to doxorubicin led to increased hyaluronic acid synthase (HAS) gene expression and increased levels of cell surface hyaluronic acid. Furthermore, exposure of K562 cells to exogenous HA caused resistance to doxorubicin-induced cell death. The combination of low dose 4-MU and doxorubicin led to increased apoptosis when compared to higher doses of any agent alone. Additionally, treatment with 4-MU led to a significant reduction in doxorubicin-induced increase in HA cell surface expression. Mechanistically, 4-MU treatment led to an increase in p38 activation and PARP cleavage. The role of p38 in 4-MU/doxorubicin-treated K562 cells was confirmed when p38 inhibitors led to protection from 4-MU/doxorubicin-induced apoptosis. Together, results from this study suggest that treatment with 4-MU increases the sensitivity of CML to chemotherapeutics by decreasing their HA-mediated resistance to apoptosis.

Abstract 3848: Targeting hyaluronidase reduces stromal cell protection of chronic myeloid leukemia to imatinib therapy

An estimated 20-30% of CML patients will become resistant to tyrosine kinase inhibitors (TKIs) including imatinib. Recent reports suggest that CML resistance to TKI9s is driven by interactions with protective microenvironment niches that influence their survival and self-renewal capacity. Hyaluronic acid (HA) has emerged as a key contributor in this phenomenon of CML resistance to TKI’s. HA influences cell viability through molecular weight dependent interactions with cell-surface receptors CD44 and RHAMM. Low molecular weight (LMW) HA, is typically found in association with many cancers including CML and promotes apoptotic resistance, proliferation, and migration of cancer cells. High molecular weight (HMW) HA is primarily produced by fibroblasts and its catabolism to LMW HA is mediated by hyaluronidase (HYAL) activity. HYAL activity may provide a potential therapeutic target in combination with current TKI treatments. Using a co-culture model, we investigated the role of HA produced by bone marrow stromal fibroblasts on CML viability when subject to imatinib (IM) treatment. Furthermore, we investigated the possibility of targeting hyaluronidase as an adjuvant in combination with IM for the treatment of CML. Co-culture with stromal fibroblasts protected CML cells from proliferation inhibition by IM. HA production and its catabolic products including LMW HA were elevated in the supernatant of co-cultures exposed to IM. The expression of genes encoding for HA metabolic proteins including hyaluronic acid synthases and HYALs, were significantly increased in co-cultures subject to IM stress. Additionally, HA treatment of CML mono-culture protected against chemotherapy-induced apoptosis. Treatment of CML co-culture with HYAL inhibitor, glycrrhizic acid (GA), inhibited HYAL activity, greatly reduced proliferation and increased apoptosis of CML cells alone and in combination with IM. This investigation provides additional evidence of the importance of stromal cell support in CML pathology and that resistance to TKI therapy may be mediated in part through upregulation of total HA production and its catabolism. Furthermore, we establish the potent therapeutic effects of GA on CML cells alone and in combination with current IM treatment strategies as a prospective method of circumventing TKI-resistant CML. For the first time we demonstrate GA9s effectiveness as a HYAL inhibitor in culture. HYALs are an attractive target not only in CML resistance but in many cancers due to the reported role of its product, LMW HA, in inflammation, proliferation, and apoptotic resistance. Citation Format: Bryan Hostetler, Olga Uchakina, Robert McKallip. Targeting hyaluronidase reduces stromal cell protection of chronic myeloid leukemia to imatinib therapy. [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 3848.