Differential binding of hyaluronic acid in two CD44+ sublines: Relationship with tumor infiltration.

A partir de un linfoma T murino de origen espontáneo LB se estableció una línea celular LBL que fue caracterizada. Por análisis histopatológicos se demostró que el tumor parental inoculado en ratones singeneicos infiltró bazo, ganglios linfáticos, hígado, timo, médula ósea y pulmón, mientras que la línea celular lo hizo en estos mismos órganos pero no en pulmón. A partir de la línea LBL se originaron dos sublíneas con características diferentes. Una de ellas creció en suspensión formando grumos (LBLc) mientras que la otra lo hizo adhiriéndose al frasco de cultivo (LBLa). Cuando se evaluó la velocidad de crecimiento, respuesta a estímulos mitogénicos e inducción de apoptosis se observaron algunas diferencias entre la línea celular y las sublíneas. Se analizó la expresión de la glucoproteína de superficie CD44 encontrándose que tanto la línea madre LBL como la sublínea LBLa expresaron esta molécula constitutivamente, mientras que LBLc necesitó ser estimulada con PMA para alcanzar estos niveles de expresión. Las células LBLa unieron ácido hialurónico (AH), por el contrario las células LBL y LBLc no lo hicieron ni aun luego de ser activadas con PMA. Se postula que la unión diferencial de AH podría estar relacionada con una diferente capacidad de anclaje e infiltración en los distintos órganos.We have established and characterized a cell line (LBL) from a spontaneous murine T lymphoma LB. Histopatological analysis has demonstrated LB primary tumor infiltration in spleen, lymph nodes, liver, thymus, bone marrow and lung. However LBL cells infiltrated all these organs except lung. Two sublines with different growth behavior were derived from LBL cell line. One of them grew in suspension as clusters (LBLc) while the other one grew as adherent monolayers (LBLa). Growth rate, response to mitogenic stimuli and apoptosis induction were different among the parental cell line and the derived sulblines. CD44 was expressed constitutively in LBL and LBLa cells. In contrast LBLc cells only expressed similar levels of this molecule when stimulated with PMA. LBLa cells showed hyaluronic acid (HA) binding properties, while LBL and LBLc cells were not able to bind HA even when activated with PMA. We postulate that differences in HA binding could be related with different infiltration behaviors.Fil: Ernst, Glenda. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Estudios de la Inmunidad Humoral Prof. Ricardo A. Margni. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Estudios de la Inmunidad Humoral Prof. Ricardo A. Margni; ArgentinaFil: Caldas Lopes, Maria Eloisi. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Estudios de la Inmunidad Humoral Prof. Ricardo A. Margni. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Estudios de la Inmunidad Humoral Prof. Ricardo A. Margni; ArgentinaFil: Cabrera, Paula V.. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica; ArgentinaFil: Alvarez, Elida. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica; ArgentinaFil: Hajos, Silvia Elvira. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Estudios de la Inmunidad Humoral Prof. Ricardo A. Margni. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Estudios de la Inmunidad Humoral Prof. Ricardo A. Margni; Argentin

Measuring the Pharmacodynamic Effects of a Novel Hsp90 Inhibitor on HER2/neu Expression in Mice Using 89Zr-DFO-Trastuzumab

The positron-emitting radionuclide (89)Zr (t(1/2) = 3.17 days) was used to prepare (89)Zr-radiolabeled trastuzumab for use as a radiotracer for characterizing HER2/neu-positive breast tumors. In addition, pharmacodynamic studies on HER2/neu expression levels in response to therapeutic doses of PU-H71 (a specific inhibitor of heat-shock protein 90 [Hsp90]) were conducted.Trastuzumab was functionalized with desferrioxamine B (DFO) and radiolabeled with [(89)Zr]Zr-oxalate at room temperature using modified literature methods. ImmunoPET and biodistribution experiments in female, athymic nu/nu mice bearing sub-cutaneous BT-474 (HER2/neu positive) and/or MDA-MB-468 (HER2/neu negative) tumor xenografts were conducted. The change in (89)Zr-DFO-trastuzumab tissue uptake in response to high- and low-specific-activity formulations and co-administration of PU-H71 was evaluated by biodistribution studies, Western blot analysis and immunoPET. (89)Zr-DFO-trastuzumab radiolabeling proceeded in high radiochemical yield and specific-activity 104.3+/-2.1 MBq/mg (2.82+/-0.05 mCi/mg of mAb). In vitro assays demonstrated >99% radiochemical purity with an immunoreactive fraction of 0.87+/-0.07. In vivo biodistribution experiments revealed high specific BT-474 uptake after 24, 48 and 72 h (64.68+/-13.06%ID/g; 71.71+/-10.35%ID/g and 85.18+/-11.10%ID/g, respectively) with retention of activity for over 120 h. Pre-treatment with PU-H71 was followed by biodistribution studies and immunoPET of (89)Zr-DFO-trastuzumab. Expression levels of HER2/neu were modulated during the first 24 and 48 h post-administration (29.75+/-4.43%ID/g and 41.42+/-3.64%ID/g, respectively). By 72 h radiotracer uptake (73.64+/-12.17%ID/g) and Western blot analysis demonstrated that HER2/neu expression recovered to baseline levels.The results indicate that (89)Zr-DFO-trastuzumab provides quantitative and highly-specific delineation of HER2/neu positive tumors, and has potential to be used to measure the efficacy of long-term treatment with Hsp90 inhibitors, like PU-H71, which display extended pharmacodynamic profiles

Determinants of Apoptotic Sensitivity to HSP90 Inhibition In Acute Myeloid Leukemia

Abstract Abstract 2159 Background: Acute myeloid leukemia (AML) is a heterogeneous and intrinsically resistant disease group of malignant hematopoietic disorders that accounts for approximately 80% of all adult leukemias. Heat shock proteins (HSPs) are often overexpressed in AML are their expression is associated with poor-prognosis and resistance to chemotherapy. Among HSPs, HSP90 is the main chaperone required for the stabilization of multiple oncogenic kinases, which contribute to AML pathogenesis, providing a rationale for the use of HSP90 inhibitors in the treatment of AML. Hypothesis: To identify patients with AML who will benefit from HSP90 inhibitor therapy there is a need to discover molecules and pathways in AML cells that confer sensitivity and lead to significant apoptosis upon HSP90 inhibition. Study design and Results: To evaluate the spectrum of sensitivities of AML cells to HSP90 inhibitors, and to investigate a possible relationship between their genetic background and apoptotic sensitivity to HSP90 inhibition, we investigated the effects of HSP90 inhibitors in a set of genetically characterized human AML cells. Addition of several HSP90 inhibitors to each of these cell lines potently inhibited cell growth, with a potency reflective of their affinity for HSP90. Normal peripheral blood leukocytes were unaffected at similar concentrations. HSP90 inhibition was associated with destabilization and subsequent degradation of Akt and c-Raf in all tested cells, as well as of several cell-specific onco-proteins such as mutant Flt3 in MOLM-13, TEL-TRKC in M0-91, AML1-ETO and mutant cKit in Kasumi-1 and SKNO-1, and mutant Jak2 in HEL cells, respectively. Notably, the proclivity for these cells to undergo apoptosis upon HSP90 inhibition varied considerably. The most sensitive cell lines were MOLM-13, MV-4-11 and M0-91 cells, and for each these cell lines we observed near 100% killing of the initial cell population after 48–72 h of HSP90 inhibitor treatment. In contrast, only 20% death was seen in HEL and HL-60 cells under these conditions. We next made use of specific inhibitors of known oncogenic signaling pathways known to be dysregulated in AML to demonstrate that apoptotic sensitivity of AML cells to HSP90 inhibition correlated with PI3K-Akt and STAT5 activation, but not with activation of the Raf-MAPK pathway. Importantly, similar results were observed in cells lines, xenograft models and isogenic cell line systems. We also found that dual activation of these two pathways, especially in the context of Bcl-xL overexpression, lowers the apoptotic threshold of AML when HSP90 is inhibited. Conclusions: We found that activation of oncogenic signaling pathways and expression of leukemogenic anti-apoptotic molecules, most importantly p-Akt, predicts for AML sensitivity to HSP90 inhibitors. Importantly, 50– 70% of patients with AML display phosphorylation of both Thr308 and Ser4 Akt. This molecule contributes to proliferation, survival and drug resistance in AML, and is associated with adverse outcome. Taken together, our findings suggest that AML patients with activation of Akt and STAT5 signaling are most likely to benefit from HSP90 inhibitor therapy, and clinical trials should aim to enroll patients with specific activation of these important signaling pathways. Disclosures: No relevant conflicts of interest to declare

Extracellular vesicles in DLBCL provide abundant clues to aberrant transcriptional programming and genomic alterations.

The biological role of extracellular vesicles (EVs) in diffuse large B-cell lymphoma (DLBCL) initiation and progression remains largely unknown. We characterized EVs secreted by 5 DLBCL cell lines, a primary DLBCL tumor, and a normal control B-cell sample, optimized their purification, and analyzed their content. We found that DLBCLs secreted large quantities of CD63, Alix, TSG101, and CD81 EVs, which can be extracted using an ultracentrifugation-based method and traced by their cell of origin surface markers. We also showed that tumor-derived EVs can be exchanged between lymphoma cells, normal tonsillar cells, and HK stromal cells. We then examined the content of EVs, focusing on isolation of high-quality total RNA. We sequenced the total RNA and analyzed the nature of RNA species, including coding and noncoding RNAs. We compared whole-cell and EV-derived RNA composition in benign and malignant B cells and discovered that transcripts from EVs were involved in many critical cellular functions. Finally, we performed mutational analysis and found that mutations detected in EVs exquisitely represented mutations in the cell of origin. These results enhance our understanding and enable future studies of the role that EVs may play in the pathogenesis of DLBCL, particularly with regards to the exchange of genomic information. Current findings open a new strategy for liquid biopsy approaches in disease monitoring

BCL6 repression of EP300 in human diffuse large B cell lymphoma cells provides a basis for rational combinatorial therapy

B cell lymphoma 6 (BCL6), which encodes a transcriptional repressor, is a critical oncogene in diffuse large B cell lymphomas (DLBCLs). Although a retro-inverted BCL6 peptide inhibitor (RI-BPI) was recently shown to potently kill DLBCL cells, the underlying mechanisms remain unclear. Here, we show that RI-BPI induces a particular gene expression signature in human DLBCL cell lines that included genes associated with the actions of histone deacetylase (HDAC) and Hsp90 inhibitors. BCL6 directly repressed the expression of p300 lysine acetyltransferase (EP300) and its cofactor HLA-B–associated transcript 3 (BAT3). RI-BPI induced expression of p300 and BAT3, resulting in acetylation of p300 targets including p53 and Hsp90. Induction of p300 and BAT3 was required for the antilymphoma effects of RI-BPI, since specific blockade of either protein rescued human DLBCL cell lines from the BCL6 inhibitor. Consistent with this, combination of RI-BPI with either an HDAC inhibitor (HDI) or an Hsp90 inhibitor potently suppressed or even eradicated established human DLBCL xenografts in mice. Furthermore, HDAC and Hsp90 inhibitors independently enhanced RI-BPI killing of primary human DLBCL cells in vitro. We also show that p300-inactivating mutations occur naturally in human DLBCL patients and may confer resistance to BCL6 inhibitors. Thus, BCL6 repression of EP300 provides a basis for rational targeted combinatorial therapy for patients with DLBCL