Establishment of a novel mesenchymal stem cell-based regimen for chronic myeloid leukemia differentiation therapy

Chronic myeloid leukemia (CML) is characterized by the accumulation of malignant and immature white blood cells which spread to the peripheral blood and other tissues/organs. Despite the fact that current tyrosine kinase inhibitors (TKIs) are capable of achieving the complete remission by reducing the tumor burden, severe adverse effects often occur in CML patients treated with TKIs. The differentiation therapy exhibits therapeutic potential to improve cure rates in leukemia, as evidenced by the striking success of all-trans-retinoic acid in acute promyelocytic leukemia treatment. However, there is still a lack of efficient differentiation therapy strategy in CML. Here we showed that MPL, which encodes the thrombopoietin receptor driving the development of hematopoietic stem/progenitor cells, decreased along with the progression of CML. We first elucidated that MPL signaling blockade impeded the megakaryocytic differentiation and contributed to the progression of CML. While allogeneic human umbilical cord-derived mesenchymal stem cells (UC-MSCs) treatment efficiently promoted megakaryocytic lineage differentiation of CML cells through restoring the MPL expression and activating MPL signaling. UC-MSCs in combination with eltrombopag, a non-peptide MPL agonist, further activated JAK/STAT and MAPK signaling pathways through MPL and exerted a synergetic effect on enhancing CML cell differentiation. The established combinational treatment not only markedly reduced the CML burden but also significantly eliminated CML cells in a xenograft CML model. We provided a new molecular insight of thrombopoietin (TPO) and MPL signaling in MSCs-mediated megakaryocytic differentiation of CML cells. Furthermore, a novel anti-CML treatment regimen that uses the combination of UC-MSCs and eltrombopag shows therapeutic potential to overcome the differentiation blockade in CML.Subject terms: Chronic myeloid leukaemia, Mesenchymal stem cells     

Dynamics of prostate cancer stem cells with diffusion and organism response

We develop a systems based model for prostate cancer, as a sub-system of the organism. We accomplish this in two stages. We first start with a general ODE that includes organism response terms. Then, to account for normally observed spatial diffusion of cell populations, the ODE is extended to a PDE that includes spatial terms. Numerical solutions of the full PDE are provided, and are indicative of traveling wave fronts. This motivates the use of a well known transformation to derive a canonically related (non-linear) system of ODEs for traveling wave solutions. For biological feasibility, we show that the non-negative cone for the traveling wave system is time invariant. We also prove that the traveling waves have a unique global attractor. Biologically, the global attractor would be the limit for the avascular tumor growth. We conclude with comments on clinical implications of the model.     

Synergy between Proteasome Inhibitors and Imatinib Mesylate in Chronic Myeloid Leukemia

Background

Resistance developed by leukemic cells, unsatisfactory efficacy on patients with chronic myeloid leukemia (CML) at accelerated and blastic phases, and potential cardiotoxity, have been limitations for imatinib mesylate (IM) in treating CML. Whether low dose IM in combination with agents of distinct but related mechanisms could be one of the strategies to overcome these concerns warrants careful investigation.

Methods and Findings

We tested the therapeutic efficacies as well as adverse effects of low dose IM in combination with proteasome inhibitor, Bortezomib (BOR) or proteasome inhibitor I (PSI), in two CML murine models, and investigated possible mechanisms of action on CML cells. Our results demonstrated that low dose IM in combination with BOR exerted satisfactory efficacy in prolongation of life span and inhibition of tumor growth in mice, and did not cause cardiotoxicity or body weight loss. Consistently, BOR and PSI enhanced IM-induced inhibition of long-term clonogenic activity and short-term cell growth of CML stem/progenitor cells, and potentiated IM-caused inhibition of proliferation and induction of apoptosis of BCR-ABL+ cells. IM/BOR and IM/PSI inhibited Bcl-2, increased cytoplasmic cytochrome C, and activated caspases. While exerting suppressive effects on BCR-ABL, E2F1, and β-catenin, IM/BOR and IM/PSI inhibited proteasomal degradation of protein phosphatase 2A (PP2A), leading to a re-activation of this important negative regulator of BCR-ABL. In addition, both combination therapties inhibited Bruton''s tyrosine kinase via suppression of NFκB.

Conclusion

These data suggest that combined use of tyrosine kinase inhibitor and proteasome inhibitor might be helpful for optimizing CML treatment.     

Dynamics and potential impact of the immune response to chronic myelogenous leukemia

Recent mathematical models have been developed to study the dynamics of chronic myelogenous leukemia (CML) under imatinib treatment. None of these models incorporates the anti-leukemia immune response. Recent experimental data show that imatinib treatment may promote the development of anti-leukemia immune responses as patients enter remission. Using these experimental data we develop a mathematical model to gain insights into the dynamics and potential impact of the resulting anti-leukemia immune response on CML. We model the immune response using a system of delay differential equations, where the delay term accounts for the duration of cell division. The mathematical model suggests that anti-leukemia T cell responses may play a critical role in maintaining CML patients in remission under imatinib therapy. Furthermore, it proposes a novel concept of an “optimal load zone” for leukemic cells in which the anti-leukemia immune response is most effective. Imatinib therapy may drive leukemic cell populations to enter and fall below this optimal load zone too rapidly to sustain the anti-leukemia T cell response. As a potential therapeutic strategy, the model shows that vaccination approaches in combination with imatinib therapy may optimally sustain the anti-leukemia T cell response to potentially eradicate residual leukemic cells for a durable cure of CML. The approach presented in this paper accounts for the role of the anti-leukemia specific immune response in the dynamics of CML. By combining experimental data and mathematical models, we demonstrate that persistence of anti-leukemia T cells even at low levels seems to prevent the leukemia from relapsing (for at least 50 months). As a consequence, we hypothesize that anti-leukemia T cell responses may help maintain remission under imatinib therapy. The mathematical model together with the new experimental data imply that there may be a feasible, low-risk, clinical approach to enhancing the effects of imatinib treatment.     

Expression of interferon consensus sequence binding protein (ICSBP) is downregulated in Bcr-Abl-induced murine chronic myelogenous leukemia-like disease, and forced coexpression of ICSBP inhibits Bcr-Abl-induced myeloproliferative disorder

Chronic myelogenous leukemia (CML) is a clonal myeloproliferative disorder resulting from the neoplastic transformation of a hematopoietic stem cell. The majority of cases of CML are associated with the (9;22) chromosome translocation that generates the bcr-abl chimeric gene. Alpha interferon (IFN-alpha) treatment induces hematological remission and prolongs life in 75% of CML patients in the chronic phase. It has been shown that mice deficient in interferon consensus sequence binding protein (ICSBP), a member of the interferon regulatory factor family, manifest a CML-like syndrome. We have shown that expression of Bcr-Abl in bone marrow (BM) cells from 5-fluorouracil (5-FU)-treated mice by retroviral transduction efficiently induces a myeloproliferative disease in mice resembling human CML. To directly test whether icsbp can function as a tumor suppressor gene, we examined the effect of ICSBP on Bcr-Abl-induced CML-like disease using this murine model for CML. We found that expression of the ICSBP protein was significantly decreased in Bcr-Abl-induced CML-like disease. Forced coexpression of ICSBP inhibited the Bcr-Abl-induced colony formation of BM cells from 5-FU-treated mice in vitro and Bcr-Abl-induced CML-like disease in vivo. Interestingly, coexpression of ICSBP and Bcr-Abl induced a transient B-lymphoproliferative disorder in the murine model of Bcr-Abl-induced CML-like disease. Overexpression of ICSBP consistently promotes rather than inhibits Bcr-Abl-induced B lymphoproliferation in a murine model where BM cells from non-5-FU-treated donors were used, indicating that ICSBP has a specific antitumor activity toward myeloid neoplasms. We also found that overexpression of ICSBP negatively regulated normal hematopoiesis. These data provide direct evidence that ICSBP can act as a tumor suppressor that regulates normal and neoplastic proliferation of hematopoietic cells.     

A PDE Model for Imatinib-Treated Chronic Myelogenous Leukemia

We derive a model for describing the dynamics of imatinib-treated chronic myelogenous leukemia (CML). This model is a continuous extension of the agent-based CML model of Roeder et al. (Nat. Med. 12(10), 1181–1184, 2006) and of its recent formulation as a system of difference equations (Kim et al. in Bull. Math. Biol. 70(3), 728–744, 2008). The new model is formulated as a system of partial differential equations that describe various stages of differentiation and maturation of normal hematopoietic cells and of leukemic cells. An imatinib treatment is also incorporated into the model. The simulations of the new PDE model are shown to qualitatively agree with the results that were obtained with the discrete-time (difference equation and agent-based) models. At the same time, for a quantitative agreement, it is necessary to adjust the values of certain parameters, such as the rates of imatinib-induced inhibition and degradation.     

Dasatinib preferentially induces apoptosis by inhibiting Lyn kinase in nilotinib-resistant chronic myeloid leukemia cell line

Nilotinib is approved for treatment of newly diagnosed chronic myeloid leukemia (CML) and it is shown superiority over imatinib in first-line treatment for patients of CML. In this study, we established a nilotinib-resistant cell line, K562NR, and evaluated the resistance to nilotinib and efficacy of dasatinib. We found activation of Lyn plays a dominant role in survival of the nilotinib-resistant cell line. We found dasatinib induces the apoptosis of nilotinib-resistant cells and inhibits Lyn kinase activity. This novel nilotinib-resistant CML cell line may help to explore novel therapy for CML.     

Optimal scheduling of radiotherapy and angiogenic inhibitors

We incorporate a previously validated mathematical model of a vascularized tumor into an optimal control problem to determine the temporal scheduling of radiotherapy and angiogenic inhibitors that maximizes the control of a primary tumor. Our results reveal that optimal antiangiogenic monotherapy gives a large initial injection to attain a 20: 1 ratio of tumor cell volume to supporting vasculature volume. It thereafter maintains this 20: 1 ratio via a continuous dose rate that is intensified over time. The optimal radiation monotherapy schedule is characterized by amodest dose intensification over time. The best performance is achieved by our optimal combination regimen, where the antiangiogenic treatment again maintains a constant tumor-to-vasculature ratio, but is administered in a dose-intensified manner only during the latter portion of the radiation fractionation schedule.     

A mathematical model of tumor growth and its response to single irradiation

Background

Mathematical modeling of biological processes is widely used to enhance quantitative understanding of bio-medical phenomena. This quantitative knowledge can be applied in both clinical and experimental settings. Recently, many investigators began studying mathematical models of tumor response to radiation therapy. We developed a simple mathematical model to simulate the growth of tumor volume and its response to a single fraction of high dose irradiation. The modelling study may provide clinicians important insights on radiation therapy strategies through identification of biological factors significantly influencing the treatment effectiveness.

Methods

We made several key assumptions of the model. Tumor volume is composed of proliferating (or dividing) cancer cells and non-dividing (or dead) cells. Tumor growth rate (or tumor volume doubling time) is proportional to the ratio of the volumes of tumor vasculature and the tumor. The vascular volume grows slower than the tumor by introducing the vascular growth retardation factor, θ. Upon irradiation, the proliferating cells gradually die over a fixed time period after irradiation. Dead cells are cleared away with cell clearance time. The model was applied to simulate pre-treatment growth and post-treatment radiation response of rat rhabdomyosarcoma tumors and metastatic brain tumors of five patients who were treated with Gamma Knife stereotactic radiosurgery (GKSRS).

Results

By selecting appropriate model parameters, we showed the temporal variation of the tumors for both the rat experiment and the clinical GKSRS cases could be easily replicated by the simple model. Additionally, the application of our model to the GKSRS cases showed that the α-value, which is an indicator of radiation sensitivity in the LQ model, and the value of θ could be predictors of the post-treatment volume change.

Conclusions

The proposed model was successful in representing both the animal experimental data and the clinically observed tumor volume changes. We showed that the model can be used to find the potential biological parameters, which may be able to predict the treatment outcome. However, there is a large statistical uncertainty of the result due to the small sample size. Therefore, a future clinical study with a larger number of patients is needed to confirm the finding.

     

Adenovirus-mediated PDCD5 gene transfer sensitizes K562 cells to apoptosis induced by idarubicin in vitro and in vivo

PDCD5 (programmed cell death 5) accelerates apoptosis of certain tumor cells and is expressed at low levels in marrow-nucleated cells of AML and CML patients. In the present study, we evaluated the effects of PDCD5 overexpression on drug sensitivity of leukemia cells. K562 cells were treated with idarubicin (IDR) alone or in combination with adenoviral vectors expressing PDCD5 (Ad-PDCD5). As shown by annexin-V-FITC/PI dual labeling, apoptosis rates were markedly increased after combined treatment with Ad-PDCD5 compared to IDR treatment alone. We observed that PDCD5 overexpression significantly improves the antitumor effects of low dose IDR treatment in vivo. Tumor sizes were significantly decreased in combined Ad-PDCD5 and low dose IDR treatment groups compared with single IDR treatment groups. Similar results were obtained with combined systemic treatment of Ad-PDCD5 and low dose IDR, and combined treatment with Ad-PDCD5 local injection and low dose IDR i.p. injection. These results indicate that Ad-PDCD5 may be a promising agent for enhancing chemosensitivity. Guo-Rui Ruan and Hong-Shan Zhao contributed equally to this work.     

Inhibition of neoplastic cell growth by quiescent cells is mediated by serum concentration and cAMP phosphodiesterase inhibitors

We have demonstrated that confluent monolayers of the mouse fibroblast cell line C3H/10T1/2 (10T1/2) have the ability to cause reversible growth inhibition of cocultured transformed cells. This was first demonstrated for de novo transformed cells and later extended to established cell lines of proven oncogenicity in vivo. This growth inhibition could be increased by growing the 10T1/2 cells to high density in increasing concentrations of serum or by elevating intracellular concentrations of cAMP using inhibitors of phosphodiesterase (PDE). These manipulations, which in cocultures of nontransformed and transformed cells caused complete inhibition of tumor cell growth, had no effect on growth rate or saturation density of either ceil type when cultured alone, demonstrating the cooperative nature of this phenomenon. This cooperation could not be produced by transfer of culture medium, demonstrating the requirement for intimate cell contact. Inhibition of the formation of transformed foci of cells in these mixed cultures was accompanied by a decrease in the incorporation of labeled thymidine into these cultures; the kinetics of this inhibition and recovery suggested a rapidly reversible effect on cell cycle transit times. The potent inhibitor of cAMP PDE, Ro 20-1724 induced dose dependent increases in intracellular cAMP in both nontransformed and in transformed cells. However, at a concentration of 10^?4 M Ro 20-1724, which inhibited tumor cell growth in mixed cultures, cAMP was elevated 30-fold in nontransformed versus only 3-fold in transformed cells. The inhibitory effects of PDE inhibitors on tumor growth have been extended to an in vivo model system, utilizing Lewis lung carcinoma cells growing as metastases in the lungs of C57B1 mice. In these mice, inoculated intravenously with a single cell suspension of Lewis lung cells, the formation of lung metastases was dramatically decreased by the twice daily administration of either isobutylmethylxanthine or Ro 20-1724; PDE inhibitors were shown to be active in vitro. The latter compound, which showed highest activity in vitro, was also substantially more potent in vivo as an inhibitor of lung tumor colony formation and doubled the life span of the tumor bearing animals. Cell cycle analysis of lung tumor colonies by the labeled mitosis method showed that both phosphodiesterase inhibitors caused a prolonged G₁ phase in the cell cycle but failed to influence other phases. Although detailed analysis of host tissues is not complete, prolonged treatment with these drugs caused no statistically significant weight loss or changes in counts of red or white blood cells indicating a selective growth inhibition of transformed cells at these doses. Studies to determine the mechanism of the cellular communication and the nature of the signal are in progress.     

CDKIs p18INK4c and p57Kip2 are involved in quiescence of CML leukemic stem cells after treatment with TKI

Chronic Myeloid Leukemia (CML) is sustained by a small population of cells with stem cell characteristics known as Leukemic Stem Cells that are positive to BCR-ABL fusion protein, involved with several abnormalities in cell proliferation, expansion, apoptosis and cell cycle regulation. Current treatment options for CML involve the use of Tirosine Kinase Inhibitor (Imatinib, Nilotinib and Dasatinib), that efficiently reduce proliferation proliferative cells but do not kill non proliferating CML primitive cells that remain and contributes to the persistence of the disease.

In order to understand the role of Cyclin Dependent Kinase Inhibitors in CML LSC permanence after TKI treatment, in this study we analyzed cell cycle status, the levels of several CDKIs and the subcellular localization of such molecules in different CML cell lines, as well as primary CD34⁺CD38^?lin^? LSC and HSC.

Our results demonstrate that cellular location of p18^INK4c and p57^Kip2 seems to be implicated in the antiproliferative activity of Imatinib and Dasatinib in CML cells and also suggest that the permanence of quiescent stem cells after TKI treatment could be associated with a decrease in p18^INK4c and p57^Kip2 nuclear location. The differences in p18^INK4cand p57^Kip2activities in CML and normal stem cells suggest a different cell cycle regulation and provide a platform that could be considered in the development of new therapeutic options to eliminate LSC.     

Tadalafil alters energy metabolism in C2C12 skeletal muscle cells

Phosphodiesterases (PDEs) are a family of enzymes that hydrolyze cyclic nucleotides, thereby modulating cell functions. Three highly selective PDE5 inhibitors (PDE5i), sildenafil, vardenafil and tadalafil, have been developed for treatment of erectile dysfunction (ED). Experimental evidence showed that chronic treatment with sildenafil PDE5i in a mouse model of diet-induced obesity and insulin resistance improved insulin action and decreased circulating fatty acid levels. It has recently been shown that healthy athletes use PDE5i as performance enhancers, hence in the present study we investigated whether the long-lasting PDE5i tadalafil influences energy metabolism in C2C12 skeletal muscle cells by evaluating lactate production, glucose consumption, and citrate synthase and 3-OH acyl CoA dehydrogenase activities. Our data demonstrate that tadalafil is able to modulate energy homeostasis in mouse skeletal muscle cells, depending on the treatment length and dose.     

Modulating Autophagy for Therapeutic Benefit

Autophagy is an ancient cell survival pathway that allows cells to recoup ATP and essential building blocks for biosynthesis when they are starved of nutrients or when they are exposed to hypoxia, which are hallmarks of the tumor microenvironment. This pathway involves the formation of double-membraned vesicles, coined autophagosomes, which envelop bulk cellular material and/or organelles and that subsequently fuse with lysosomes that degrade their cargo. Autophagy has been suggested to play important roles in chemoresistance of cancer to some therapeutic agents, which typically induce an apoptotic response. For example, the histone deacetylase inhibitor SAHA induces both apoptosis and autophagy, suggesting that agents that disrupt the autophagy pathway might augment its efficacy as a therapeutic agent. We tested this notion in a model of Imatinib-refractory chronic myelogenous leukemia (CML) and in imatinib-resistant primary CML cells from patients bearing mutations in Bcr-Abl, including the T315I mutation that causes resistance to currently utilized tyrosine kinase inhibitors and translates into a very poor clinical prognosis. Agents that disrupt autophagy were shown to synergize with SAHA in provoking apoptotic death of these refractory tumors. These findings support the use of agents that disrupt the autophagy pathway in settings of chemorefractory malignancies.

Addendum to:

Targeting Autophagy Augments the Anticancer Activity of the Histone Deacetylase Inhibitor SAHA to Overcome Bcr-Abl-Mediated Drug Resistance

J.S. Carew, S.T. Nawrocki, C.N. Kahue, H. Zhang, C. Yang, L. Chung, J.A. Houghton, P. Huang, F.J. Giles and J.L. Cleveland

Blood 2007; In press     

Carboxyamidotriazole-orotate inhibits the growth of imatinib-resistant chronic myeloid leukaemia cells and modulates exosomes-stimulated angiogenesis

The Bcr/Abl kinase has been targeted for the treatment of chronic myelogenous leukaemia (CML) by imatinib mesylate. While imatinib has been extremely effective for chronic phase CML, blast crisis CML are often resistant. New therapeutic options are therefore needed for this fatal disease. Although more common in solid tumors, increased microvessel density was also reported in chronic myelogenous leukaemia and was associated with a significant increase of angiogenic factors, suggesting that vascularity in hematologic malignancies is a controlled process and may play a role in the leukaemogenic process thus representing an alternative therapeutic target. Carboxyamidotriazole-orotate (CTO) is the orotate salt form of carboxyamidotriazole (CAI), an orally bioavailable signal transduction inhibitor that in vitro has been shown to possess antileukaemic activities. CTO, which has a reduced toxicity, increased oral bioavailability and stronger efficacy when compared to the parental compound, was tested in this study for its ability to affect imatinib-resistant CML tumor growth in a xenograft model. The active cross talk between endothelial cells and leukemic cells in the bone marrow involving exosomes plays an important role in modulating the process of neovascularization in CML. We have thus investigated the effects of CTO on exosome-stimulated angiogenesis. Our results indicate that CTO may be effective in targeting both cancer cell growth and the tumor microenvironment, thus suggesting a potential therapeutic utility for CTO in leukaemia patients.     

Adoptive transfer of mammaglobin-a epitope specific CD8 T cells combined with a single low dose of total body irradiation eradicates breast tumors

Adoptive T cell therapy has proven to be beneficial in a number of tumor systems by targeting the relevant tumor antigen. The tumor antigen targeted in our model is Mammaglobin-A, expressed by approximately 80% of human breast tumors. Here we evaluated the use of adoptively transferred Mammaglobin-A specific CD8 T cells in combination with low dose irradiation to induce breast tumor rejection and prevent relapse. We show Mammaglobin-A specific CD8 T cells generated by DNA vaccination with all epitopes (Mammaglobin-A2.1, A2.2, A2.4 and A2.6) and full-length DNA in vivo resulted in heterogeneous T cell populations consisting of both effector and central memory CD8 T cell subsets. Adoptive transfer of spleen cells from all Mammaglobin-A2 immunized mice into tumor-bearing SCID/beige mice induced tumor regression but this anti-tumor response was not sustained long-term. Additionally, we demonstrate that only the adoptive transfer of Mammaglobin-A2 specific CD8 T cells in combination with a single low dose of irradiation prevents tumors from recurring. More importantly we show that this single dose of irradiation results in the down regulation of the macrophage scavenger receptor 1 on dendritic cells within the tumor and reduces lipid uptake by tumor resident dendritic cells potentially enabling the dendritic cells to present tumor antigen more efficiently and aid in tumor clearance. These data reveal the potential for adoptive transfer combined with a single low dose of total body irradiation as a suitable therapy for the treatment of established breast tumors and the prevention of tumor recurrence.     

An “Age” Structured Model of Hematopoietic Stem Cell Organization with Application to Chronic Myeloid Leukemia

Previously, we have modeled hematopoietic stem cell organization by a stochastic, single cell-based approach. Applications to different experimental systems demonstrated that this model consistently explains a broad variety of in vivo and in vitro data. A major advantage of the agent-based model (ABM) is the representation of heterogeneity within the hematopoietic stem cell population. However, this advantage comes at the price of time-consuming simulations if the systems become large. One example in this respect is the modeling of disease and treatment dynamics in patients with chronic myeloid leukemia (CML), where the realistic number of individual cells to be considered exceeds 10⁶. To overcome this deficiency, without losing the representation of the inherent heterogeneity of the stem cell population, we here propose to approximate the ABM by a system of partial differential equations (PDEs). The major benefit of such an approach is its independence from the size of the system. Although this mean field approach includes a number of simplifying assumptions compared to the ABM, it retains the key structure of the model including the “age”-structure of stem cells. We show that the PDE model qualitatively and quantitatively reproduces the results of the agent-based approach.