c-Abl Kinase Inhibitors Overcome CD40-Mediated Drug Resistance in CLL.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 3078-3078 ◽  
Author(s):  
Delfine Y. Hallaert ◽  
Annelieke Jaspers ◽  
Carel J. van Noesel ◽  
Marinus H.J. Van Oers ◽  
Arnon P. Kater ◽  
...  
Keyword(s):  
Drug Resistance ◽  
Molecular Basis ◽  
Kinase Inhibitors ◽  
Lymphocytic Leukemia ◽  
Recent Analysis ◽  
Combination Strategies ◽  
Cd40 Stimulation ◽  
Erk Inhibition ◽  
Erk Activity

Abstract In chronic lymphocytic leukemia (CLL), proliferation centers reside in lymph node (LN) and possibly also bone marrow, where the environment protects CLL cells from apoptotic and cytotoxic triggers. This protective milieu may well contribute to the lack of curative chemotherapy in CLL, and our recent analysis of the distinct profiles of apoptosis regulators in CLL LN versus peripheral blood supports this notion (Smit et al., Blood 2007, 109: 1660). The aim of the present study was to define the molecular basis for the increased drug resistance and to search for novel strategies to circumvent it. To mimic the situation in CLL LN, we applied prolonged in vitro CD40 stimulation of CLL cells, which results in strong upregulation of anti-apoptotic Bcl-xL and Mcl-1. Moreover, we now also report a gradual reduction of Bim at the protein level, further contributing to the anti-apoptotic profile. Using specific inhibitors (PD98059 and MG132), we found that the decrease in Bim is due to ERK-mediated phosporylation and subsequent proteasomal degradation. ERK inhibition during CD40 triggering abrogated the decrease in Bim levels, but did however not re-establish sensitivity to various drugs (fludarabine, Velcade, Roscovitine). In chronic myeloid leukemia (CML), changes in Bcl-xL, Mcl-1 and Bim levels similar to those observed in our CLL/CD40 system are known to depend on BCR-Abl signaling. Therefore, we next applied c-Abl inhibitors Gleevec or Dasatinib in conjunction with CD40. Both drugs caused a profound reversal of most protective CD40 effects; ERK activity, Bim, Bcl-xL and Mcl-1 levels as well as sensitivity to subsequent drug treatment were restored to pre-CD40 values. These effects also occurred in CLL samples with dysfunctional p53 (n=3). Importantly, in CLL LN samples we also found strong ERK activation together with high Bcl-xL and Mcl-1 but low Bim levels, suggesting that there might be a c-Abl dependent survival pathway in proliferation centers. These data provide a molecular basis for combination strategies that could target refractory niches in CLL, using therapeutics that function independently of p53.

c-Abl kinase inhibitors overcome CD40-mediated drug resistance in CLL: implications for therapeutic targeting of chemoresistant niches

Blood ◽  
2008 ◽  
Vol 112 (13) ◽  
pp. 5141-5149 ◽  
Author(s):  
Delfine Y. H. Hallaert ◽  
Annelieke Jaspers ◽  
Carel J. van Noesel ◽  
Marinus H. J. van Oers ◽  
Arnon P. Kater ◽  
...  
Keyword(s):  
Drug Resistance ◽  
Kinase Inhibitors ◽  
Ex Vivo ◽  
Lymphocytic Leukemia ◽  
Erk Activation ◽  
Abl Kinase ◽  
Cd40 Stimulation ◽  
Sublethal Doses ◽  
Erk Inhibition

Abstract In lymph node (LN) proliferation centers in chronic lymphocytic leukemia (CLL), the environment protects from apoptotic and cytotoxic triggers. Here, we aimed to define the molecular basis for the increased drug resistance and searched for novel strategies to circumvent it. The situation in CLL LN could be mimicked by prolonged in vitro CD40 stimulation, which resulted in up-regulation of antiapoptotic Bcl-xL, A1/Bfl-1, and Mcl-1 proteins, and afforded resistance to various classes of drugs (fludarabine, bortezomib, roscovitine). CD40 stimulation also caused ERK-dependent reduction of Bim-EL protein, but ERK inhibition did not prevent drug resistance. Drugs combined with sublethal doses of the BH3-mimetic ABT-737 displayed partial and variable effects per individual CD40-stimulated CLL. The antiapoptotic profile of CD40-triggered CLL resembled BCR-Abl–dependent changes seen in chronic myeloid leukemia (CML), which prompted application of c-Abl inhibitors imatinib or dasatinib. Both compounds, but especially dasatinib, prevented the entire antiapoptotic CD40 program in CLL cells, and restored drug sensitivity. These effects also occurred in CLL samples with dysfunctional p53. Importantly, ex vivo CLL LN samples also displayed strong ERK activation together with high Bcl-xL and Mcl-1 but low Bim levels. These data indicate that CLL cells in chemoresistant niches may be sensitive to therapeutic strategies that include c-Abl inhibitors.

Possible Mechanisms Of Resistance To The Novel BH3-Mimetic ABT-199 In In Vitro Lymph Node Models Of CLL – The Role Of Abl and Btk

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 4188-4188 ◽  
Author(s):  
Rachel Thijssen ◽  
Christian R Geest ◽  
Martin FM de Rooij ◽  
Nora Liu ◽  
Bogdan I Florea ◽  
...  
Keyword(s):  
Drug Resistance ◽  
Lymph Node ◽  
Chronic Lymphocytic Leukemia ◽  
Clinical Application ◽  
Kinase Inhibitors ◽  
Conflicts Of Interest ◽  
Solid Phase ◽  
Lymphocytic Leukemia ◽  
Bh3 Mimetic

Abstract The new BH3-mimetic ABT-199 antagonizes Bcl-2 and avoids the thrombocytopenia associated with clinical application of its predecessor ABT-263 (navitoclax). Chronic lymphocytic leukemia (CLL) cells are highly sensitive to ABT-199 and the first clinical results show clear reductions in peripheral and bone marrow CLL cells and in lymph node size. In the lymph node, CLL cells receive pro-survival signals that upregulate Bcl-XL, Mcl-1 and Bfl-11. These Bcl-2 family members are not targeted by ABT-199, which poses the potential risk of remaining clones with residual viability. Here, we aimed to define the signals that determine sensitivity for ABT-199 and ABT-737 in an in vitro lymph node model of CLL. We applied CD40 and cytokine stimulation in combination with kinase inhibitors that are known to change microenviroenmental signals and increase drug resistance in CLL. Stimulation via CD40 plus IL-4 or IL-21 differentially affected the expression of Mcl-1, Bcl-XL, Bfl-1 and Noxa and this correlated with strong alterations in sensitivity to ABT-737 and ABT-199 (see table 1 for LC 50 values). As reported before2, in vitro CD40 stimulation reduced sensitivity to ABT-737 by 100-fold, and this was further decreased by IL-4. Strikingly, CD40+IL-4 stimulation in primary CLL cells resulted in full resistance to 10 μM ABT-199, probably due to very high levels of Bcl-XL.Table 1The LC50 of ABT-737 or ABT-199 for CLL cells stimulated with CD40L and IL-21 or IL-4 (averaged values n=8)StimulationLC50 (μM)ABT-737ABT-1993T3 (control)0.0050.0013T40L0.781> 103T40L + IL-210.1950.210 3T40L + IL-46.772> 103T40L + IL-21 + IL-40.4269.121 We next sought ways to circumvent resistance against ABT-199 induced in our in vitro model. We showed previously that the broad spectrum kinase inhibitor dasatinib prevented CD40-mediated resistance to various drugs, including ABT-7373. We therefore first characterized the targets of dasatinib in primary CLL by solid-phase pull-down, mass-spectrometry and competition binding. Abl and Btk were identified as dominant and specific interactors of dasatinib. Importantly, resistance for BH3-mimetics could be overcome by dasatinib (see table 2) and the Abl inhibitor imatinib, but not by the more selective Btk inhibitor ibrutinib. Conversely, BCR- and chemokine-controlled adhesion could be abolished by dasatinib and ibrutinib, but not by imatinib. Thus, Abl and Btk function in two key pro-survival arms; chemoresistance and localization in the protective environment.Table 2The LC50 of ABT-737 or ABT-199 for CLL cells stimulated with CD40L in combination with Dasatinib (averaged values n=4)StimulationLC50 (μM)ABT-737ABT-1993T30.0050.0013T40L0.781> 103T40L + 100 nM Dasatinib0.0810.066 3T40L + 1000 nM Dasatinib0.0370.020 The observed resistance to ABT-199 induced in our in vitro a co-culture system designed to simulate the CLL microenvironment does not reflect the observations from clinical trials in patients. Nevertheless, long-term clinical application of ABT-199 in CLL might select for resistant clones at protective niches. Our data suggest that this may be overcome by combination treatment with kinase inhibitors that either directly abrogate anti-apoptotic signals or cause egress from lymph node sites and prevent the resistance mechanism from coming into play. 1. Smit LA, Hallaert DY, Spijker R et al. Differential Noxa/Mcl-1 balance in peripheral versus lymph node chronic lymphocitic leukemia cells correlates with survival capacity. Blood 2007;109:1660-1668. 2. Vogler M, Butterworth M, Majid A et al. Concurrent up-regulation of BCL-XL and BCL2A1 induces approximately 1000-fold resistance to ABT-737 in chronic lymphocytic leukemia. Blood 2009;113:4403-4413. 3. Hallaert DY, Jaspers A, van Noesel CJ et al. c-Abl kinase inhibitors overcome CD40-mediated drug resistance in CLL; Implications for therapeutic targeting of chemoresistant niches. Blood 2008;112:5141-5149. Disclosures: No relevant conflicts of interest to declare.

Activation of the MAPK pathway mediates resistance to PI3K inhibitors in chronic lymphocytic leukemia (cll)

Blood ◽  
2021 ◽  
Author(s):  
Ishwarya Murali ◽  
Siddha Kasar ◽  
Aishath Naeem ◽  
Svitlana Tyekucheva ◽  
Jasneet Kaur Khalsa ◽  
...  
Keyword(s):  
Chronic Lymphocytic Leukemia ◽  
Mapk Pathway ◽  
Cell Receptor ◽  
Lymphocytic Leukemia ◽  
Erk Activation ◽  
Activating Mutations ◽  
Erk Phosphorylation ◽  
Whole Exome ◽  
Erk Inhibition

Inhibitors of Bruton's tyrosine kinase (BTKi) and phosphatidylinositol 3-kinase delta (PI3Kδi) that target the B cell receptor (BCR) signaling pathway have revolutionized the treatment of chronic lymphocytic leukemia (CLL). While mutations associated with resistance to BTK inhibitors have been identified, limited data are available on mechanisms of resistance to PI3Kδi. Here we present findings from longitudinal whole-exome sequencing of multiply relapsed CLL patients (Ncases=28) enrolled in PI3Ki trials. The non-responder subgroup was characterized by baseline activating mutations in MAP2K1, BRAF and KRAS in 60% of patients. PI3Kδ inhibition failed to inhibit ERK phosphorylation (pERK) in non-responder CLL cells with and without mutations, while treatment with MEKi rescued ERK inhibition. Overexpression of MAP2K1 mutants in vitro led to increased basal and inducible pERK and resistance to idelalisib. These data demonstrate that MAPK/ERK activation plays a key role in resistance to PI3Kδi in CLL and provide rationale for combination therapy with PI3Kδ and ERK inhibitors.

scholarly journals Plasmodium vivax and Drug Resistance

2021 ◽  
Author(s):  
Puji Budi Setia Asih ◽  
Din Syafruddin
Keyword(s):  
Drug Resistance ◽  
Molecular Basis ◽  
Indirect Evidence ◽  
Antimalarial Drugs ◽  
In Vivo Studies ◽  
In Vitro Cultivation ◽  
Radical Cure ◽  
The Status

Resistance to antimalarial drugs is a threat to global efforts to eliminate malaria by 2030. Currently, treatment for vivax malaria uses chloroquine or ACT for uncomplicated P. vivax whereas primaquine is given to eliminate latent liver stage infections (a method known as radical cure). Studies on P. vivax resistance to antimalarials and the molecular basis of resistance lags far behind the P. falciparum as in vitro cultivation of the P. vivax has not yet been established. Therefore, data on the P. vivax resistance to any antimalarial drugs are generated through in vivo studies or through monitoring of antimalarial treatments in mixed species infection. Indirect evidence through drug selective pressure on the parasites genome, as evidenced by the presence of the molecular marker(s) for drug resistance in areas where P. falciparum and P. vivax are distributed in sympatry may reflect, although require validation, the status of P. vivax resistance. This review focuses on the currently available data that may represent the state-of-the art of the P. vivax resistance status to antimalarial to anticipate the challenge for malaria elimination by 2030.

BTK Inhibitors, Irrespective of ITK Inhibition, Increase Efficacy of a CD19/CD3 Bispecific Antibody in CLL

Blood ◽  
2021 ◽  
Author(s):  
Maissa Mhibik ◽  
Erika M. Gaglione ◽  
David Eik ◽  
Ellen K Kendall ◽  
Amy Blackburn ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cytotoxic Activity ◽  
Kinase Inhibitors ◽  
Bispecific Antibody ◽  
Mononuclear Cells ◽  
Cell Function ◽  
Lymphocytic Leukemia ◽  
Peripheral Blood Mononuclear

Bruton Tyrosine Kinase inhibitors (BTKis) are a preferred treatment for patients with chronic lymphocytic leukemia (CLL). Indefinite therapy with BTKis, while effective, presents clinical challenges. Combination therapy can deepen responses, shorten treatment duration, and possibly prevent or overcome drug resistance. We previously reported on a CD19/CD3 bispecific antibody (bsAb) that recruits autologous T cell cytotoxicity against CLL cells in vitro. Compared to observations with samples from treatment-naïve patients, T cells from patients being treated with ibrutinib expanded more rapidly and exerted superior cytotoxic activity in response to the bsAb. In addition to BTK, ibrutinib also inhibits IL2 inducible T cell Kinase (ITK). In contrast, acalabrutinib, does not inhibit ITK. Whether ITK inhibition contributes to the observed immune effects is unknown. To better understand how BTKis modulate T-cell function and cytotoxic activity, we cultured peripheral blood mononuclear cells (PBMCs) from BTKi-naive, and ibrutinib- or acalabrutinib-treated CLL patients with CD19/CD3 bsAb in vitro. T-cell expansion, activation, differentiation, and cytotoxicity were increased in PBMCs from patients on treatment with either BTKi compared to that observed for BKTi-naïve patients. BTKi therapy transcriptionally downregulated immunosuppressive effectors expressed by CLL cells, including CTLA-4 and CD200. CTLA-4 blockade with ipilimumab in vitro increased the cytotoxic activity of the bsAb in BTKi-naïve but not BTKi-treated PBMCS. Taken together, BTKis enhance bsAb induced cytotoxicity by relieving T cells of immunosuppressive restraints imposed by CLL cells. The benefit of combining bsAb immunotherapy with BTKis needs to be confirmed in clinical trials.

scholarly journals CD133 Is Associated with Increased Melanoma Cell Survival after Multikinase Inhibition

2019 ◽  
Vol 2019 ◽  
pp. 1-19 ◽  
Author(s):  
Cynthia M. Simbulan-Rosenthal ◽  
Anirudh Gaur ◽  
Hengbo Zhou ◽  
Maryam AbdusSamad ◽  
Qing Qin ◽  
...  
Keyword(s):  
Drug Resistance ◽  
Kinase Inhibitors ◽  
Braf Inhibitor ◽  
Mek Inhibitor ◽  
Melanoma Cells ◽  
Stem Cell Marker ◽  
High Dose ◽  
Recurrence Rates ◽  
Mapk Inhibitors

FDA-approved kinase inhibitors are now used for melanoma, including combinations of the MEK inhibitor trametinib, and BRAF inhibitor dabrafenib for BRAFV600 mutations. NRAS-mutated cell lines are also sensitive to MEK inhibitionin vitro, and NRAS-mutated tumors have also shown partial response to MEK inhibitors. However, melanoma still has high recurrence rates due to subpopulations, sometimes described as “melanoma initiating cells,” resistant to treatment. Since CD133 is a putative cancer stem cell marker for different cancers, associated with decreased survival, we examined resistance of patient-derived CD133(+) and CD133(-) melanoma cells to MAPK inhibitors. Human melanoma cells were exposed to increasing concentrations of trametinib and/or dabrafenib, either before or after separation into CD133(+) and CD133(-) subpopulations. In parental CD133-mixed lines, the percentages of CD133(+) cells increased significantly (p<0.05) after high-dose drug treatment. Presorted CD133(+) cells also exhibited significantly greater (p<0.05) IC50s for single and combination MAPKI treatment. siRNA knockdown revealed a causal relationship between CD133 and drug resistance. Microarray and qRT-PCR analyses revealed that ten of 18 ABC transporter genes were significantly (P<0.05) upregulated in the CD133(+) subpopulation, while inhibition of ABC activity increased sensitivity, suggesting a mechanism for increased drug resistance of CD133(+) cells.

scholarly journals Use of kinase inhibitors against schistosomes to improve and broaden praziquantel efficacy

Parasitology ◽  
2020 ◽  
Vol 147 (13) ◽  
pp. 1488-1498
Author(s):  
Sujeevi S. K. Nawaratna ◽  
Donald P. McManus ◽  
Robin B. Gasser ◽  
Paul J. Brindley ◽  
Glen M. Boyle ◽  
...  
Keyword(s):  
Drug Resistance ◽  
Kinase Inhibitors ◽  
Lethal Dose ◽  
Alternative Therapies ◽  
Potential Drug ◽  
Dependent Protein Kinase ◽  
Drug Of Choice ◽  
Dependent Protein ◽  
The Impact

AbstractPraziquantel (PZQ) is the drug of choice for schistosomiasis. The potential drug resistance necessitates the search for adjunct or alternative therapies to PZQ. Previous functional genomics has shown that RNAi inhibition of Ca2+/calmodulin-dependent protein kinase II (CaMKII) gene in Schistosoma adult worms significantly improved the effectiveness of PZQ. Here we tested the in vitro efficacy of 15 selective and non-selective CaMK inhibitors against Schistosoma mansoni and showed that PZQ efficacy was improved against refractory juvenile parasites when combined with these CaMK inhibitors. By measuring CaMK activity and the mobility of adult S. mansoni, we identified two non-selective CaMK inhibitors, Staurosporine (STSP) and 1Naphthyl PP1 (1NAPP1), as promising candidates for further study. The impact of STSP and 1NAPP1 was investigated in mice infected with S. mansoni in the presence or absence of a sub-lethal dose of PZQ against 2- and 7-day-old schistosomula and adults. Treatment with STSP/PZQ induced a significant (47–68%) liver egg burden reduction compared with mice treated with PZQ alone. The findings indicate that the combination of STSP and PZQ dosages significantly improved anti-schistosomal activity compared to PZQ alone, demonstrating the potential of selective and non-selective CaMK/kinase inhibitors as a combination therapy with PZQ in treating schistosomiasis.

scholarly journals MEK inhibition enhances the response to tyrosine kinase inhibitors in acute myeloid leukemia

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
María Luz Morales ◽  
Alicia Arenas ◽  
Alejandra Ortiz-Ruiz ◽  
Alejandra Leivas ◽  
Inmaculada Rapado ◽  
...  
Keyword(s):  
Drug Resistance ◽  
Acute Myeloid Leukemia ◽  
Tyrosine Kinase ◽  
Tyrosine Kinase Inhibitors ◽  
Myeloid Leukemia ◽  
Kinase Inhibitors ◽  
Resistance Pathway ◽  
Acute Myeloid

AbstractFMS-like tyrosine kinase 3 (FLT3) is a key driver of acute myeloid leukemia (AML). Several tyrosine kinase inhibitors (TKIs) targeting FLT3 have been evaluated clinically, but their effects are limited when used in monotherapy due to the emergence of drug-resistance. Thus, a better understanding of drug-resistance pathways could be a good strategy to explore and evaluate new combinational therapies for AML. Here, we used phosphoproteomics to identify differentially-phosphorylated proteins in patients with AML and TKI resistance. We then studied resistance mechanisms in vitro and evaluated the efficacy and safety of rational combinational therapy in vitro, ex vivo and in vivo in mice. Proteomic and immunohistochemical studies showed the sustained activation of ERK1/2 in bone marrow samples of patients with AML after developing resistance to FLT3 inhibitors, which was identified as a common resistance pathway. We examined the concomitant inhibition of MEK-ERK1/2 and FLT3 as a strategy to overcome drug-resistance, finding that the MEK inhibitor trametinib remained potent in TKI-resistant cells and exerted strong synergy when combined with the TKI midostaurin in cells with mutated and wild-type FLT3. Importantly, this combination was not toxic to CD34+ cells from healthy donors, but produced survival improvements in vivo when compared with single therapy groups. Thus, our data point to trametinib plus midostaurin as a potentially beneficial therapy in patients with AML.

Bioactive Compounds from Seaweed with Anti-Leukemic Activity: A Mini-Review on Carotenoids and Phlorotannins

2020 ◽  
Vol 20 (1) ◽  
pp. 39-53 ◽  
Author(s):  
Tânia P. Almeida ◽  
Alice A. Ramos ◽  
Joana Ferreira ◽  
Amaya Azqueta ◽  
Eduardo Rocha
Keyword(s):  
Drug Resistance ◽  
Bioactive Compounds ◽  
Myeloid Leukemia ◽  
Molecular Mechanisms ◽  
Kinase Inhibitors ◽  
First Line ◽  
In Vivo Models ◽  
Few Data

: Chronic Myeloid Leukemia (CML) represents 15-20% of all new cases of leukemia and is characterized by an uncontrolled proliferation of abnormal myeloid cells. Currently, the first-line of treatment involves Tyrosine Kinase Inhibitors (TKIs), which specifically inhibits the activity of the fusion protein BCR-ABL. However, resistance, mainly due to mutations, can occur. In the attempt to find more effective and less toxic therapies, several approaches are taken into consideration such as research of new anti-leukemic drugs and “combination chemotherapy” where different drugs that act by different mechanisms are used. Here, we reviewed the molecular mechanisms of CML, the main mechanisms of drug resistance and current strategies to enhance the therapeutic effect of TKIs in CML. Despite major advances in CML treatment, new, more potent anticancer drugs and with fewer side effects are needed. Marine organisms, and particularly seaweed, have a high diversity of bioactive compounds with some of them having anticancer activity in several in vitro and in vivo models. The state-of-art suggests that their use during cancer treatment may improve the outcome. We reviewed here the yet few data supporting anti-leukemic activity of some carotenoids and phlorotannins in some leukemia models. Also, strategies to overcome drug resistance are discussed, particularly the combination of conventional drugs with natural compounds.

Standardizing Co-Culture Conditions Between Chronic Lymphocytic Leukemia Cells and Marrow Stromal Cells: Towards a Reliable and Reproducible System to Assess Cell Adhesion-Mediated Drug Resistance

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 3149-3149
Author(s):  
Antonina Kurtova ◽  
Maite P. Quiroga ◽  
William G. Wierda ◽  
Michael Keating ◽  
Jan A. Burger
Keyword(s):  
Bone Marrow ◽  
Drug Resistance ◽  
Cell Adhesion ◽  
Chronic Lymphocytic Leukemia ◽  
Stromal Cells ◽  
Lymphocytic Leukemia ◽  
Marrow Stromal Cells ◽  
Hpv E6 ◽  
Induced Apoptosis

Abstract Contact between chronic lymphocytic leukemia (CLL) cells and accessory stromal cells in tissue microenvironments is considered to play a major role in regulating CLL cell survival and disease progression. Stromal cells of various origins and species, and variable stromal-CLL cell ratios have been used in the past to study CLL-stromal cell interactions and to assess cell-adhesion mediated drug resistance (CAM-DR). Because of the heterogeneity of the currently used in vitro systems to study CLL-MSC interactions, and the importance of these co-culture systems for development and testing of novel agents, we tested a panel of murine and human MSC lines for their capacities to support CLL cell survival and CAM-DR, using various CLL-MSC ratios and fludarabine (F-ara-A) to induce CLL cell apoptosis. We tested four murine, non-transformed MSC lines derived from bone marrow: M210B4, KUM4, ST-2 and KUSA-H1. Also, we tested three human transformed cell lines: Stroma-NKtert, derived from bone marrow and immortalized by human telomerase reverse transcriptase (hTERT), UE6E7-T2 derived from bone marrow and transformed with human papilloma viruses (HPV) E6, E7 and hTERT, and UCB408E6E7Tert33 derived from umbilical cord blood and transformed with hTERT and HPV E6, E7. CLL cells were isolated from peripheral blood of untreated patients and each cell line was tested with at least three different patients according to the following protocol: viability of CLL was tested after 24, 48 and 72 hours by flow cytometry after staining with DiOC6 and propidium iodide. The following conditions were assayed on each of the MSC lines: CLL cells in suspension culture, CLL cells in suspension culture with 10 mM F-ara-A, CLL cells in co-culture with MSC, and CLL cells in co-culture with MSC and with 10 mM F-ara-A. Firstly, we performed titration experiments in order to identify the most appropriate ratio between stromal and CLL cells, using CLL-MSC ratios of 5:1, 10:1, 20:1, 50:1 and 100:1. We found a decline in MSC-derived CLL cell protection at the highest ratio of 100:1, suggesting that ratios of 50:1 or lower provide optimal conditions for in vitro assays. Results shown in Table 1 were assayed using a 20:1 ratio and represented relative viabilities when compared to untreated controls (mean±SEM). Regarding the protective effect of different MSC, we found that all MSC lines demonstrated remarkable protection of CLL cells from spontaneous and F-ara-A-induced apoptosis. We also found that stromal cells that had round shape morphology and easily formed confluent monolayer (M210B4, KUSA-H1, Stroma-NKTert) showed more prolonged protective effect in comparison to cell lines with more spindle shaped morphology (ST-2, KUM4, UE6E7-T2). The failure of UE6E7-T2 and UCB408E6E7Tert33 to demonstrate long-term protection of CLL cells could be related to their own sensitivity to F-ara-A. In this comparative study we demonstrated that both murine and human MSC provide substantial and comparable levels of protection from spontaneous and drug-induced apoptosis. CLL:MSC ratios of 50:1 or lower can be considered ideal for co-culture experiments. Further experiments have to be done to determine the levels of MSC-derived protection in a larger series of CLL samples and in different laboratories for validation. Collectively, in these co-culture assays we can study CLL-MSC interactions and CLL drugs under more standardized conditions that may allow us to evaluate the efficacy of new treatments that target the CLL microenvironment. Time points 24 hours 48 hours 72 hours +Flu + MSC + MSC +Flu +Flu + MSC + MSC +Flu +Flu +MSC + MSC +Flu M210B4 85.2±2.4 117.2±5.0 110.5±4.9 30.8±12.6 138.1±9.5 113.0±2.2 5.2±3.1 138.1±5.1 120.4±3.4 ST-2 93.6±3.0 99.9±2.6 103.1±0.5 51.6±9.4 111.9±2.6 89.8±8.7 13.9±6.3 112.6±5.7 87.0±16.4 KUM-4 93.6±3.0 106.4±1.8 104.2±1.9 51.6±9.4 112.4±2.6 100.8±2.8 13.9±6.3 111.8±6.7 88.5±11.4 KUSA-H1 79.4±7.4 125.1±3.7 118.2±2.0 33.9±10.9 136.0±3.6 107.2±7.0 11.3±6.1 133.6±5.4 84.9±7.6 Stroma-NKTert 79.3±7.0 118.6±7.0 111.0±7.0 30.5±9.5 130.7±9.5 115.6±8.0 7.1±4.3 133.0±11.5 122.7±9.0 UE6E7-T2 79.3±7.0 113.4±3.9 109.3±3.0 30.5±9.5 118.4±4.8 85.0±7.1 7.1±4.3 119.2±6.9 51.0±10.1 UCB408 E6E7Tert33 81.5±7.2 120.2±5.4 111.8±2.7 36.7±9.4 123.7±6.3 86.7±7.7 8.5±6.7 119.7±6.1 50.8±13.0 Table 1. Flu: fludarabine (10mM/ml), MSC: marrow stromal cells

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