17-DMAG Treatment Research Articles

17-DMAG ameliorates neuroinflammation and BBB disruption via SOX5 mediated PI3K/Akt pathway after intracerebral hemorrhage in rats

Intracerebral hemorrhage (ICH) can result in secondary brain injury due to inflammation and breakdown of the blood–brain barrier (BBB), which are closely associated with patient prognosis. The potential of the heat shock protein 90 (Hsp90) inhibitor 17-DMAG in promoting neuroprotection has been observed in certain vascular diseases. However, the precise role of 17-DMAG treatment in ICH is not yet fully understood. In this study, we found that treatment with 17-DMAG (5 mg/kg) effectively reduced hematoma expansion and resulted in improved neurological outcomes. Meanwhile, the injection of 17-DMAG had a positive effect on reducing BBB disruption in rats with ICH. This effect was achieved by increasing the levels of BBB tight junction proteins (TJPs) such as zo-1, claudin-5, and occludin. As a result, the leakage of EB extravasation, brain edema and IgG in the peri-hematoma tissue were reduced. Furthermore, the injection of 17-DMAG decreased the infiltration of neutrophils into the brain tissues surrounding the hematoma in ICH rats and also reduced the production of proinflammatory cytokines IL-6 and TNF-α. Next, we used integrative mass spectrometry (MS) and molecular docking analysis to confirm that sex determining region Y-box protein 5 (SOX5) is a potential direct target of 17-DMAG in ICH. SOX5 encodes a positive regulator of the PI3K/Akt axis, and treatment with 17-DMAG resulted in a noticeable increase in SOX5 accumulation. To further investigate the role of SOX5, we employed virus-regulated SOX5 silencing and found that suppressing SOX5 blocked the ability of 17-DMAG to suppress neutrophil trafficking. Additionally, silencing SOX5 blocked the protective effects of 17-DMAG on the BBB by inhibiting PI3K, p-Akt, and BBB TJPs levels, which led to an increase in EB and IgG leakage in the peri-hematoma tissue after ICH. Similarly, when SOX5 was knocked down, the protective effects of 17-DMAG were lost. Overall, the results of our study indicate that the injection of 17-DMAG has the potential to mitigate neuroinflammation and prevent the disruption of the BBB caused by ICH, resulting in improved neurological outcomes in rats. These positive effects are attributed to the regulation of SOX5 and activation of the PI3K/Akt pathway. These findings highlight the possibility of targeting SOX5 and the PI3K/Akt pathway as a novel therapeutic approach for ICH.

Heat shock protein 90 inhibitors induce cell differentiation via the ubiquitin-dependent aurora kinase A degradation in a MPLW515L mouse model of primary myelofibrosis.

Primary myelofibrosis (PMF) is characterized by immature megakaryocytic hyperplasia, splenomegaly, extramedullary hematopoiesis and bone marrow fibrosis. Our preclinical study had demonstrated that aurora kinase A (AURKA) inhibitor MLN8237 reduced the mutation burden of PMF by inducing differentiation of immature megakaryocytes. However, it only slightly alleviated splenomegaly, reduced tissue fibrosis, and normalized megakaryocytes in PMF patients of the preliminary clinical study. So enhancing therapeutic efficacy of PMF is needed. In this study, we found that AURKA directly interacted with heat shock protein 90(HSP90) and HSP90 inhibitors promoted the ubiquitin-dependent AURKAdegradation. We demonstrated that HSP90 inhibitors 17-allylamino-17-demethoxygeldanamycin (17-AAG) and 17-dimethylaminoethylamino-17-demethoxygeldanamycin (17-DMAG), normalized peripheral blood counts, improved splenomegaly, attenuated extramedullary hematopoiesis, decreased tissue fibrosis and reduced mutant burden in a MPLW515L mouse model of PMF. Importantly, both 17-AAG and 17-DMAG treatment at effective doses in vivo did not influence on hematopoiesis in healthy mice. Collectively, the study demonstrates that HSP90 inhibitors induce cell differentiation via the ubiquitin-dependent AURKA and also are safe and effective for the treatment of a MPLW515L mouse model of PMF, which may provide a new strategy for PMF therapy. Further, we demonstrate that combined therapy shows superior activity in acute megakaryocytic leukemia mouse model than single therapy.

The HSP90 inhibitor 17-DMAG alleviates primary biliary cholangitis via cholangiocyte necroptosis prevention.

Cholangiocyte death accompanied by the progression of primary biliary cholangitis (PBC) has not yet been thoroughly investigated. Thus, we are aimed to explore the role of HSP90 and a potential treatment strategy in cholangiocyte necroptosis. First, we detected the expression of HSP90 and necroptotic markers in liver tissues from patients and mice with PBC by immunohistochemistry (IHC) and real-time polymerase chain reaction(PCR). Then, the HSP90 inhibitor, 17-dimethylaminoethylamino-17-demethoxygeldanamycin(17-DMAG), was administered by intraperitoneal injection to evaluate its therapeutic effect for PBC by IHC, real-timePCR, and western blotting. Human intrahepatic bile duct epithelial cells (HIBECs) were induced to necroptosis by toxic bile acid and lipopolysaccharide(LPS) treatment, and evaluated via Cell Counting Kit-8 and flow cytometry assays. Additionally, 17-DMAG, cycloheximide, and a proteasome inhibitor were used to evaluate the role of HSP90 in cholangiocyte necroptosis. We found that the expression of HSP90 was elevated in the cholangiocytes of patients and mice with PBC, along with higher expressions of receptor-interacting serine/threonine-protein kinase 1 (RIPK1), RIPK3, mixed lineage kinase domain-like protein (MLKL), andphosphorylated-MLKL(p-MLKL). Proinflammatory cytokines and antibody levels of the E2 subunit of pyruvate dehydrogenase complex decreased after treatment with 17-DMAG in PBC mice. Meanwhile, RIPK1, RIPK3, phosphorylated-RIPK3, MLKL, and p-MLKL protein expressions decreased with 17-DMAG treatment. In vitro, 17-DMAG and necrostatin-1 prevented glycochenodeoxycholic acid and LPS-induced necroptosis of HIBECs. Immunoprecipitation and high-performance liquid chromatography-mass spectrometry analysis showed that RIPK1 combined with HSP90. Additionally, the 17-DMAG treatment reduced the RIPK1 half-life. Overall, 17-DMAG might be a potential therapeutic agent for PBC via cholangiocyte necroptosis prevention by accelerating RIPK1 degradation.

Heat shock protein 90 is a new potential target of anti-rejection therapy in allotransplantation.

The critical roles of heat shock protein 90 (HSP90) in immune reactions associated with viral infection and autoimmune disease are well known. To date, however, its roles in the alloimmune response and the immunosuppressive effect of HSP90 inhibitors in allotransplantation have remained unknown. The purpose of this study was to examine the therapeutic efficacy of the HSP90 inhibitor 17-DMAG in allotransplantation models. C57BL/6 (H-2b) and BALB/c (H-2d) mice were used as donors for and recipients of skin and heart transplantation, respectively. Treatment with 17-DMAG (daily i.p.) or a vehicle was initiated 3days before transplantation. Immunological outcomes were assessed by histopathological examinations, flow cytometric analysis, quantitative RT-PCR, ELISA, ELISPOT assay, and MLR. 17-DMAG treatment significantly prolonged the survival of both skin and heart allografts. In 17-DMAG-treated mice, donor-reactive splenocytes producing IFN-γ were significantly reduced along with the intragraft mRNA expression level and serum concentration of IFN-γ. Intragraft mRNA expression of cytokines and chemokines associated with both innate and adaptive immunity was suppressed in 17-DMAG-treated group. MLR showed suppression of the donor-specific proliferation of CD4 + T and CD19 + B cells in the spleens of 17-DMAG-treated mice. 17-DMAG treatment also reduced the number of activated NK cells. Furthermore, the treatment lowered the titers of donor-specific antibodies in the serum and prolonged a second skin allograft in mice sensitized by previous skin transplantation. HSP90 inhibition by 17-DMAG can affect various immune responses, including innate immunity, adaptive immunity, and humoral immunity, suggesting its therapeutic potential against acute rejection in allotransplantation.

Hsp90α Mediates BMI1 Expression in Breast Cancer Stem/Progenitor Cells through Facilitating Nuclear Translocation of c-Myc and EZH2.

Heat shock protein 90 (Hsp90) is a molecular chaperone that facilitates the correct folding and functionality of its client protein. Numerous Hsp90-client proteins are involved in cancer development. Thus, Hsp90 inhibitors have potential applications as anti-cancer drugs. We previously discovered that Hsp90α expression increased in breast cancer stem cells (BCSCs), which can initiate tumorigenesis and metastasis and resist treatment. In the present study, we further demonstrated that 17-dimethylaminoethylamino-17-demethoxygeldanamycin (17-DMAG), an inhibitor of Hsp90, could suppress the self-renewal of BCSCs by downregulating B lymphoma Mo-MLV insertion region 1 homolog (BMI1), a polycomb family member with oncogenic activity in breast cancer. Through immunoprecipitation analysis, we found that BMI1 did not interact with Hsp90α and that the downregulation of BMI1 by 17-DMAG was mediated by the inhibition of c-Myc and enhancement of zeste homolog 2 (EZH2) expression. The transcriptional and BMI1 promoter-binding activities of c-Myc in BCSCs were inhibited by 17-DMAG treatment. The overexpression of EZH2 attenuated the inhibitory effect of 17-DMAG on BMI1 and c-Myc expression. Furthermore, Hsp90α could be co-immunoprecipitated with c-Myc and EZH2 and bind to the BMI1 promoter. Treatment with 17-DMAG decreased the nuclear expression of EZH2 and c-Myc but not that of Hsp90α. In conclusion, our data suggested that Hsp90α could positively regulate the self-renewal of BCSCs by facilitating the nuclear translocation of c-Myc and EZH2 to maintain BMI1 expression.

Preventative effects of a HIF inhibitor, 17-DMAG, on partial bladder outlet obstruction-induced bladder dysfunction.

Posterior urethral valves are the most common cause of partial bladder outlet obstruction (PBOO) in the pediatric population. Pathological changes in the bladder developed during PBOO are responsible for long-lasting voiding dysfunction in this population despite early surgical interventions. Increasing evidence showed PBOO induces an upregulation of hypoxia-inducible factors (HIFs) and their transcriptional target genes, and they play a role in pathophysiological changes in the obstructed bladders. We hypothesized that blocking HIF pathways can prevent PBOO-induced bladder dysfunction. PBOO was surgically created by ligation of the bladder neck in male C57BL/6J mice for 2 wk. PBOO mice received intraperitoneal injection of either saline or 17-DMAG (alvespimycin, 3 mg/kg) every 48 h starting from day 1 postsurgery. Sham-operated animals received injection of saline on the same schedule as PBOO mice and served as controls. The bladders were harvested after 2 wk, and basal activity and evoked contractility of the detrusor smooth muscle (DSM) were evaluated in vitro. Bladder function was assessed in vivo by void spot assay and cystometry in conscious, unrestrained mice. Results indicated the 17-DMAG treatment preserved DSM contractility and partially prevented the development of detrusor over activity in obstructed bladders. In addition, PBOO caused a significant increase in the frequency of micturition, which was significantly reduced by 17-DMAG treatment. The 17-DMAG treatment improved urodynamic parameters, including increases in the bladder pressure at micturition and nonvoid contractions observed in PBOO mice. These results demonstrate that treatment with 17-DMAG, a HIF inhibitor, significantly alleviated PBOO-induced bladder pathology in vivo.

The Hsp90 inhibitor 17-DMAG decreases infection of porcine circovirus type 2 in mice.

Although several factors affecting porcine circovirus type 2 (PCV2) infection have been reported, theirprecise roles are far from clear. The aim of this study was to determine whether 17-dimethylaminoethylamino-17-demethoxygeldanamycin (17-DMAG), an inhibitor of Hsp90, could significantly affect PCV2 infection and immune responses in BALB/c mice. Intraperitoneal injection of 17-DMAG significantly reduced viral loads in the blood and tissues of mice infected with PCV2, compared with control groups. The 17-DMAG treatment decreased serum interleukin (IL)-10 and tumor necrosis factor(TNF)-α levels, but it did not have a significant effect on the IL-1β level. These data demonstrate that 17-DMAG is highly effective in suppressing PCV2 replication in BALB/c mice, indicating that it has potential value as an antiviral drug against PCV2 infection.

Targeting HSP90-HDAC6 Regulating Network Implicates Precision Treatment of Breast Cancer.

Breast cancer is the leading cause of women death. Heat shock protein 90 (HSP90) and Histone deacetylase 6 (HDAC6) are promising anti-cancer drug targets. However, it's still unclear the applicability of anti-HSP90 and anti-HDAC6 strategies in precision treatment of breast cancer. In current study, we found that triple negative breast cancer (TNBC) cells, compared to T47D, an ERα+ breast cancer cell line, exhibited 7~40 times lower IC50 values, stronger cell cycle perturbation, increased cell apoptosis and stronger inhibition of cell migration upon 17-DMAG treatment, while T47D, compared to TNBC cells, expressed higher HDAC6 and showed stronger anti-cancer response upon treatment of Tubacin. Mechanically, 17-DMAG treatment inhibited a complex network consists at least ERK, AKT, and Hippo pathway in TNBC cells, and higher expression of HDAC6 inhibited HSP90 activity via deacetylating HSP90. Furthermore, we found higher HDAC6 expression level in tamoxifen-resistance T47D than that in T47D, and Tubacin treatment suppressed the growth of tamoxifen-resistant cells in vivo. Our data suggested that anti-HSP90 and anti-HDAC6 are promising strategies to treat TNBC and ERα+ breast cancers respectively, and anti-HDAC6 can be considered during treatment of tamoxifen-resistance breast cancers.

Inhibition of HIF Reduces Bladder Hypertrophy and Improves Bladder Function in Murine Model of Partial Bladder Outlet Obstruction

Posterior urethral valves are the most common cause of partial bladder outlet obstruction in the pediatric population. However, to our knowledge the etiology and the detailed mechanisms underlying pathological changes in the bladder following partial bladder outlet obstruction remain to be elucidated. Recent findings suggest that hypoxia and associated up-regulation of HIFs (hypoxia-inducible factors) have a key role in partial bladder outlet obstruction induced pathology in the bladder. We examined the effects of pharmacological inhibition of HIF pathways by 17-DMAG (17-(dimethylaminoethylamino)-17-demethoxygeldanamycin) in pathophysiological phenotypes after partial bladder outlet obstruction. Partial bladder outlet obstruction was surgically created in male C57BL/6J mice. The animals received oral administration of 17-DMAG or vehicle daily starting from the initiation of obstruction up to 5 days. Sham operated mice served as controls. Bladders were harvested from each group 2, 4 and 7 days postoperatively, and analyzed for histological and biochemical changes. Bladder function was assessed by invitro muscle contractility recordings. Partial bladder outlet obstruction caused a significant increase in the bladder mass accompanying enhanced collagen deposition in the bladder wall while 17-DMAG treatment suppressed those increases. Treatment with 17-DMAG attenuated the degree of up-regulation of HIFs and their target genes involving the development of tissue fibrosis in obstructed bladders. Treatment with 17-DMAG improved the decreased responses of obstructed bladder strips to electrical field stimulation and KCl. Invivo 17-DMAG treatment decreased partial bladder outlet obstruction induced pathophysiological changes in the bladder. HIF pathway inhibition has a potential clinical implication for the development of novel pharmacological therapies to treat bladder pathology associated with partial bladder outlet obstruction.

Hypoxia attenuates Hsp90 inhibitor 17-DMAG-induced cyclin B1 accumulation in hepatocellular carcinoma cells.

Hypoxia stress plays a pivotal role in tumor formation, proliferation, and invasion. Conventional chemotherapy is less effective in the hypoxia microenvironment of solid tumor. Heat shock protein 90 (Hsp90) is an important molecular chaperone in cancer cells and has been a pharmaceutical target for decades. However, Hsp90 inhibitors demonstrate limited effect on solid tumor and the mechanism underlying is not clear. To determine whether hypoxia impairs the therapeutic effect of Hsp90 N-terminal inhibitor, 17-demethoxygeldanamycin hydrochloride (17-DMAG), in live cancer cells, we measured cell proliferation and cell cycle distribution. Cell proliferation assay indicates that hypoxia obviously promotes the proliferation of HepG2 and Huh7 cells after 24, 48, and 72 h and impairs 17-DMAG-induced G2/M arrest in liver cancer cells. As a client protein of Hsp90, cyclin B1 is critical for the transition from G2 to M phase and is related to the prognosis of the patients. We further checked the cyclin B1 messenger RNA (mRNA) level, protein level, ubiquitination of cyclin B1, nuclear translocation, and degradation of cyclin B1 affected by hypoxia after 17-DMAG treatment. The results demonstrate that hypoxia decreases the transcription of cyclin B1 and accelerates the ubiquitination, nuclear translocation, and degradation of cyclin B1. Taken together, our results suggest that hypoxia attenuates cyclin B1 accumulation induced by 17-DMAG and, hence, alleviates 17-DMAG-induced G2/M arrest.

Heat shock protein 90 inhibition by 17-DMAG attenuates abdominal aortic aneurysm formation in mice

Abdominal aortic aneurysm (AAA) is a common degenerative vascular disease whose pathogenesis is associated with activation of multiple signaling pathways including Jun NH2-terminal kinases (JNK) and NF-κB. It is now well recognized that these pathways are chaperoned by the heat shock protein 90 (Hsp90), suggesting that inhibition of Hsp90 may be a novel strategy for inhibiting AAAs. The aim of this study is to investigate whether inhibition of Hsp90 by 17-DMAG (17-dimethyl-aminothylamino-17-demethoxy-geldanamycin) attenuates ANG II-induced AAA formation in mice, and, if so, to elucidate the mechanisms involved. Apolipoprotein E-null mice were infused with ANG II to induce AAA formation and simultaneously treated by intraperitoneal injection with either vehicle or 17-DMAG for 4 wk. ANG II infusion induced AAA formation in 80% of mice, which was accompanied by increased matrix metalloproteinase (MMP) activity, enhanced tissue inflammation, oxidative stress, and neovascularization. Importantly, these effects were inhibited by 17-DMAG treatment. Mechanistically, we showed that 17-DMAG prevented the formation and progression of AAA through its inhibitory effects on diverse biological pathways including 1) by blocking ANG II-induced phosphorylation of ERK1/2 and JNK that are critically involved in the regulation of MMPs in vascular smooth muscle cells, 2) by inhibiting IκB kinase expression and expression of MCP-1, and 3) by attenuating ANG II-stimulated angiogenic processes critical to AAA formation. Our results demonstrate that inhibition of Hsp90 by 17-DMAG effectively attenuates ANG II-induced AAA formation by simultaneously inhibiting vascular inflammation, extracellular matrix degradation, and angiogenesis, which are critical in the formation and progression of AAAs.

Glucose-regulated protein 78 mediates the therapeutic efficacy of 17-DMAG in colon cancer cells.

Glucose-regulated protein 78 (GRP78) is expressed as part of the molecular response to endoplasmic reticulum (ER) stress and mediates protein folding within the cell. GRP78 is also an important biomarker of cancer progression and the therapeutic response of patients with different cancer types. However, the role of GRP78 in the cytotoxic effect of 17-DMAG in colon cancer cells remains unclear. GRP78 expression was knocked down by small interfering RNA (siRNA). The anticancer effects of 17-DMAG were assessed by an 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, a flow cytometric cell-cycle analysis, and an Annexin V-propidium iodide (PI) apoptotic assay. We found that HT-29 cells expressed a lower level of GRP78 compared with DLD-1 cells. The MTT assay revealed that HT-29 cells were more sensitive to 17-DMAG treatment than DLD-1 cells. GRP78 knock down (GRP78KD) cells demonstrated an increased sensitivity to 17-DMAG treatment compared with the scrambled control cells. Based on the cell-cycle analysis and Annexin V-PI apoptotic assay, apoptosis dramatically increased in GRP78KD cells compared with scrambled control DLD-1 cells after these cells were treated with 17-DMAG. Finally, we observed a decrease in the level of Bcl-2 and an increase in the levels of Bad and Bax in GRP78KD cells treated with 17-DMAG. These results are consistent with an increased sensitivity to 17-DMAG after knock down of GRP78. The level of GRP78 expression may determine the therapeutic efficacy of 17-DMAG against colon cancer cells.

Immunomodulatory effects of heat shock protein 90 inhibition on humoral immune responses.

Heat shock protein 90 (Hsp90) inhibition blocks T-cell-linked inflammatory disease pathways and exhibits therapeutic activity in autoimmune disease mouse models, including the blistering disease epidermolysis bullosa acquisita. Although we previously showed that preformed autoreactive plasma cells do not seem to be directly affected by anti-Hsp90 treatment, immunomodulatory effects of Hsp90 inhibition on (auto-)antibody responses are not yet fully understood. In this study, the Hsp90 blocker 17-DMAG inhibited proliferation of activated total B cells and their IgG secretion in cultures of human peripheral B cells from healthy subjects, but IgG production was no longer affected when these activated B cells were allowed to differentiate prior to a deferred application of the inhibitor. 17-DMAG treatment was associated with induction of nuclear and cytoplasmic heat shock factor 1 and Hsp70 in stimulated human B cells, respectively. Type VII collagen (epidermolysis bullosa acquisita)-immunized mice early treated with 17-DMAG had reduced total B cells in spleens, a relative increase in splenic regulatory B cell fractions, higher serum IL-10 concentrations, and lower levels of circulating autoantibodies (paralleled by less pronounced disease induction) compared with vehicle-treated immunized mice. Autoantibody production was blunted in isolated and autoantigen-restimulated lymph node cells from immunized mice by either 17-DMAG or purified autologous splenic regulatory B cells. Thus, in addition to the previously described T cell inhibitory effects of Hsp90 blockade, this treatment potently modulates humoral immune responses at the B cell level, further supporting the introduction of Hsp90 inhibitors into the clinical setting for treatment of autoantibody-mediated disorders.

Hsp90 blockade modulates bullous pemphigoid IgG-induced IL-8 production by keratinocytes.

Bullous pemphigoid (BP) is the most common subepidermal autoimmune blistering skin disease characterized by autoantibodies against the hemidesmosomal proteins BP180 and BP230. The cell stress chaperone heat shock protein 90 (Hsp90) has been implicated in inflammatory responses, and recent evidence suggests that it represents a novel treatment target in autoimmune bullous diseases. The aim of the study was to investigate the contribution of Hsp90 to the proinflammatory cytokine production in keratinocytes induced by autoantibodies to BP180 from BP patient serum. HaCaT cells were treated with purified human BP or normal IgG in the absence or presence of the Hsp90 blocker 17-DMAG and effects on viability, interleukin 6 (IL-6) and IL-8 (cytokines critical for BP pathology), NFκB (their major transcription factor), and Hsp70 (marker of effective Hsp90 inhibition and potent negative regulator of inflammatory responses) were investigated. We found that BP IgG stimulated IL-6 and IL-8 release from HaCaT cells and that non-toxic doses of 17-DMAG inhibited this IL-8, but not IL-6 secretion in a dose- and time-dependent fashion. Inhibition of this IL-8 production was also observed at the transcriptional level. In addition, 17-DMAG treatment blunted BP IgG-mediated upregulation of NFκB activity and was associated with Hsp70 induction. This study provides important insights that Hsp90 is involved as crucial regulator in anti-BP180 IgG-induced production of keratinocyte-derived IL-8. By adding to the knowledge of the multimodal anti-inflammatory effects of Hsp90 blockade, our data further support the introduction of Hsp90 inhibitors into the clinical setting for treatment of autoimmune diseases, especially for BP.

The growth inhibitory effect of 17-DMAG on ALK and MYCN double-positive neuroblastoma cell line

Neuroblastoma (NB), an embryonal tumor derived from the neural crest, originates from sympathetic nerve system, manifests as thoracic, paraspinous or abdominal tumors, and metastases to bone in high-risk cases. Although NB stands as the most common solid tumor in early childhood and accounts for about 15% of total pediatric cancer death, there has been limited success in searching for novel therapeutic regimen for this lethal disease during the past two decades. Numerous epidemiological and clinical studies have pinpointed anaplastic lymphoma kinase (ALK) and MYCN as potent governors for NB malignant behavior. ALK and MYCN amplification and constitutive active mutations are common in high-risk NB patients. However, there is still lack of evidence showing that a small molecule compound could simultaneously inhibits ALK and MYCN and plays strong negative regulatory roles in NB. Here, we showed that 17-DMAG, a well-known HSP-90 inhibitor, significantly inhibits NB cell growth, arrests cell cycle, and strongly induces NB cell apoptosis. Interestingly, our data suggests that NB cells with both ALK and MYCN amplification/mutation are more sensitive to 17-DMAG treatment, while NB cells with only ALK or MYCN amplification are less sensitive and NB cells without ALK or MYCN amplification/mutation are least sensitive. Moreover, we also found that knocking down ALK and MYCN additively inhibits NB cell growth and that transduction of MYCN largely abolished the ALK-dependent NB cell growth, indicating that there is a cross-talk between MYCN and ALK signaling machinery. Our results provide proof-of-principle that 17-DMAG strongly inhibits NB cell growth by targeting both ALK and MYCN. Our findings might shed a light for further investigation of novel small molecule compound which is safe and exerts similar strong effects in vivo as novel approaches for management of high-risk NB.

Hsp90 Inhibitor 17-DMAG Decreases Expression of Conserved Herpesvirus Protein Kinases and Reduces Virus Production in Epstein-Barr Virus-Infected Cells

All eight human herpesviruses have a conserved herpesvirus protein kinase (CHPK) that is important for the lytic phase of the viral life cycle. In this study, we show that heat shock protein 90 (Hsp90) interacts directly with each of the eight CHPKs, and we demonstrate that an Hsp90 inhibitor drug, 17-dimethylaminoethylamino-17-demethoxygeldanamycin (17-DMAG), decreases expression of all eight CHPKs in transfected HeLa cells. 17-DMAG also decreases expression the of the endogenous Epstein-Barr virus protein kinase (EBV PK, encoded by the BGLF4 gene) in lytically infected EBV-positive cells and inhibits phosphorylation of several different known EBV PK target proteins. Furthermore, 17-DMAG treatment abrogates expression of the human cytomegalovirus (HCMV) kinase UL97 in HCMV-infected human fibroblasts. Importantly, 17-DMAG treatment decreased the EBV titer approximately 100-fold in lytically infected AGS-Akata cells without causing significant cellular toxicity during the same time frame. Increased EBV PK expression in 17-DMAG-treated AGS-Akata cells did not restore EBV titers, suggesting that 17-DMAG simultaneously targets multiple viral and/or cellular proteins required for efficient viral replication. These results suggest that Hsp90 inhibitors, including 17-DMAG, may be a promising group of drugs that could have profound antiviral effects on herpesviruses.

THU0057 Inhibition of Heat Shock Protein 90 (Hsp90) Prevents Fibrosis by Targeting Canonical TGF-B Signaling

BackgroundHeat shock protein 90 (Hsp90) has a crucial role in folding and conformational stabilization of TGF-β receptors and also of Src kinases, which are intracellular mediators of the pro-fibrotic effects...

An activated JAK/STAT3 pathway and CD45 expression are associated with sensitivity to Hsp90 inhibitors in multiple myeloma

The molecular chaperone Hsp90 is required to maintain the activity of many signaling proteins, including members of the JAK/STAT and the PI3K pathways. Inhibitors of Hsp90 (Hsp90-Is) demonstrated varying activity against multiple myeloma (MM) in clinical trials. We aimed to determine which signaling pathways that account for the differential sensitivity to the Hsp90-I 17DMAG on a panel of MM cell lines and freshly obtained MM cells. Three CD45+ cell lines with an activated JAK/STAT3 pathway were sensitive to 17DMAG and underwent prominent apoptosis upon treatment, while the majority of CD45− cell lines, that were dependent on the activated PI3K pathway, were more resistant to the drug. Culturing the most resistant cell line, LP1, in the presence of IL-6 resulted in up-regulation of CD45 and pSTAT3, and sensitized to 17DMAG-induced apoptosis, primarily in the induced CD45+ sub-population of cells. The high CD45 expressers among primary myeloma cells also expressed significantly higher levels of pSTAT3, as compared to the low CD45 expressers. Ex vivo treatment of primary myeloma cells with 17DMAG resulted in a stronger caspase3 activation in tumor samples with the prevalence of high CD45 expressers. STAT3 activity was efficiently inhibited by Hsp90-Is in both cell lines and primary cells suggesting an importance of STAT3 inactivation for the pro-apoptotic effects of HSP90-Is. Indeed, over-expression of STAT3C, a variant with an increased DNA binding activity, in U266 cells protected them from 17DMAG-induced cell death. The down-regulation of the STAT3 target gene Mcl-1 at both the mRNA and protein levels following 17DMAG treatment was significantly attenuated in STAT3C-expressing cells, and transient over-expression of Mcl-1 protected U266 cells from 17DMAG-induced cell death. The finding that CD45+ MM cells with an IL-6-activated JAK/STAT3 pathway are particularly sensitive to Hsp90-Is as compared to the low CD45 expressers may provide a rational basis for selection of MM patients amenable to Hsp90-I treatment.

A Phase I Trial of the Intravenous (IV) Hsp90 Inhibitor 17-DMAG (alvespimycin) in Patients (pts) with Relapsed Chronic Lymphocytic Leukemia (CLL)/Small Lymphocytic Lymphoma (SLL)

Abstract 180017-DMAG is a geldanamycin derivative that binds the N-terminal domain of the heat shock protein Hsp90, a molecular chaperone protein important in maintaining enzymatic activity of several client proteins involved in cellular signaling pathways. This binding disrupts the interaction between Hsp90 and its client proteins, several important to signaling and survival pathways in CLL including kinases AKT, IKK-α and IKK-β. IKK-α and IKK-β activate the NF-kB pathway, which plays a critical role in promoting proliferation and survival of CLL cells. In vitro data has demonstrated that 17-DMAG is cytotoxic to primary CLL cells, exhibiting minimal toxicity towards normal B-, T- and NK-cells, which may be less immunosuppressive than other available therapies. A prior phase I study of 17-DMAG showed it was safe with clinical activity in pts with relapsed leukemia. This phase I study was designed to determine the maximum tolerated dose (MTD) and dose limiting toxicities (DLTs) in pts with relapsed CLL/SLL as well as to assess preliminary efficacy and determine feasibility of measuring different pharmacodynamic (PD) markers including Hsp90 client proteins AKT, IKK-α and IKK-β.Eligibility included pts with relapsed CLL/SLL requiring therapy by the 2008 International Workshop on CLL guidelines who had received at least one prior therapy including fludarabine (F), equivalent nucleoside analogue (NA) or alternative regimen if contraindication to F existed. Inclusion criteria included normal creatinine (cr) or cr clearance ≥50, AST/ALT ≤ 2.5 × upper limits of normal and QTc 500 msec. Treatment consisted of 17-DMAG IV at escalating dose levels (dl) (16 mg/m2, 20 mg/m2, 24 mg/m2) given on days 1, 4, 8 and 11 of 21 day cycles in a standard cohort-of-3 dose-escalation. All pts were treated for a minimum of 2 cycles in the absence of progressive disease. An expansion cohort of 12 patients was planned at the MTD in order to gain more data on safety and tolerability of 17-DMAG as well as to assess feasibility of measuring PD endpoints in these pts.Fifteen pts (10 males) with a median age of 59 (range 49–76) previously treated with a median of 5 prior therapies (range 1–14) were enrolled. Thirteen pts had prior NA therapy and 1 pt had prior allogeneic stem cell transplant. Five pts were enrolled on dl 1 (16 mg/m2), 3 pts on dl 2 (20 mg/m2) and 6 pts on dl 3 (24 mg/m2). No DLT’s were observed and 24 mg/m2 was determined to be the MTD. One pt was enrolled in the expansion cohort at the MTD. Fourteen of the 15 pts were evaluable for response. Seven pts progressed prior to finishing 1 month of therapy; 4 pts prior to finishing 2 months of therapy and 3 pts prior to finishing 3 months of therapy. Of these 3 pts considered to have stable disease, 2 were treated at dl 2 and 1 was treated at dl 3. Two pts died while on study, both with complications of progressive disease considered unrelated to therapy. Grade 3–4 events included anemia (67%), neutropenia (53%), thrombocytopenia (20%), pain (20%), electrolyte abnormalities (20%), infection (14%), hypertension (14%), dyspnea (14%), hypoxia (7%), hemolysis (7%) and upper gastrointestinal hemorrhage (7%). All were considered unrelated to treatment. Correlative studies included immunoblot analysis of Hsp90 client proteins and real time PCR for NF-kB target genes. We found IKKα and IKKβ levels decreased at cycle 1, day 1 (C1D1) at 24 hours in 4 of 7 pt samples which were evaluable, while AKT levels were not altered to a large extent. Additionally real time PCR was performed to determine any effect on NF-kB target genes Mcl-1, Bcl-2, as these have been previously demonstrated by our group to be decreased by 17-DMAG treatment in vitro. There was a variable effect on these target genes at C1D1 at 24 hours that correlated with the decrease in IKKα and IKKβ. Additionally, SOCS3, a gene that is silenced in CLL and has been demonstrated to increase in vitro with 17-DMAG treatment, was also increased in vivo.IV 17-DMAG given to pts with relapsed CLL/SLL was well tolerated with no DLTs observed. Correlatives suggest that inhibition of Hsp90 may not be complete at the highest dose on this schedule, and higher doses or alternative schedules may be beneficial. Oral administration may provide a more consistent dosing schedule leading to continuous inhibition of Hsp90 and more pronounced effect on downstream signaling pathways. Further mechanistic studies with 17-DMAG as well as alternative Hsp90 inhibitors are being pursued pre-clinically at OSU. Disclosures:No relevant conflicts of interest to declare.

In Vivo Method to Monitor Changes in HER2 Expression Using Near-Infrared Fluorescence Imaging

Human epidermal growth factor receptor type 2 (HER2) is a well-known biomarker that is overexpressed in many breast carcinomas. HER2 expression level is an important factor to optimize the therapeutic strategy and monitor the treatment. We used albumin binding domain-fused HER2-specific Affibody molecules, labeled with Alexa Fluor750 dye, to characterize HER2 expression in vivo. Near-infrared optical imaging studies were carried out using mice with subcutaneous HER2-positive tumors. Animals were divided into groups of five: no treatment and 12 hours and 1 week after treatment of the tumors with the Hsp90 inhibitor 17-dimethylaminoethylamino-17-demethoxygeldanamycin (17-DMAG). The compartmental ligands-receptor model, describing binding kinetics, was used to evaluate HER2 expression from the time sequence of the fluorescence images after the intravenous probe injection. The normalized rate of accumulation of the specific fluorescent biomarkers, estimated from this time sequence, linearly correlates with the conventional ex vivo enzyme-linked immunosorbent assay (ELISA) readings for the same tumor. Such correspondence makes properly arranged fluorescence imaging an excellent candidate for estimating HER2 overexpression in tumors, complementing ELISA and other ex vivo assays. Application of this method to the fluorescence data from HER2-positive xenografts reveals that the 17-DMAG treatment results in downregulation of HER2. Application of the AngioSense 750 probe confirmed the antiangiogenic effect of 17-DMAG found with Affibody-Alexa Fluor 750 conjugate.