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.     

Progesterone sensitizes breast cancer MCF7 cells to imatinib inhibitory effects

In previous studies, we found that progesterone was able to induce the expression of platelet-derived growth factor (PDGF) in human breast cancer MCF7 cells. Knowing that imatinib mesylate targets PDGF receptor tyrosine kinase activity, the aim of the present study was to examine the effects of imatinib on progesterone-treated MCF7 cells. Expression of phosphorylated (activated) platelet-derived growth factor receptor-alpha (PDGFRalpha) was detected in MCF7 cells. Interestingly, phosphorylated-PDGFRalpha expression was significantly downregulated by imatinib. The effects of imatinib on cell growth, apoptosis and migration were then analyzed. Imatinib effectively inhibited anchorage-dependent colony formation, and cell viability as evaluated by MTT assay. Corroborating these findings, a significant increase in the percentage of apoptotic cells was also observed when cells were treated with imatinib. Surprisingly, these inhibitory effects were all enhanced by the presence of progesterone. Cell migration assays did also show a reduction in the migratory capacity after incubation with imatinib. These findings reveal that imatinib acts by decreasing MCF7 cell viability, growth and migration, with concomitant increase in apoptosis. Furthermore, incubation with progesterone seems to prompt cells to the inhibitory action of imatinib, probably by sustaining PDGFRalpha activity. The current study points out imatinib as a possible therapeutic strategy in progesterone-dependent breast cancer.     

Imatinib Reverses Doxorubicin Resistance by Affecting Activation of STAT3-Dependent NF-κB and HSP27/p38/AKT Pathways and by Inhibiting ABCB1

Despite advances in cancer detection and prevention, a diagnosis of metastatic disease remains a death sentence due to the fact that many cancers are either resistant to chemotherapy (conventional or targeted) or develop resistance during treatment, and residual chemoresistant cells are highly metastatic. Metastatic cancer cells resist the effects of chemotherapeutic agents by upregulating drug transporters, which efflux the drugs, and by activating proliferation and survival signaling pathways. Previously, we found that c-Abl and Arg non-receptor tyrosine kinases are activated in breast cancer, melanoma, and glioblastoma cells, and promote cancer progression. In this report, we demonstrate that the c-Abl/Arg inhibitor, imatinib (imatinib mesylate, STI571, Gleevec), reverses intrinsic and acquired resistance to the anthracycline, doxorubicin, by inducing G2/M arrest and promoting apoptosis in cancer cells expressing highly active c-Abl and Arg. Significantly, imatinib prevents intrinsic resistance by promoting doxorubicin-mediated NF-κB/p65 nuclear localization and repression of NF-κB targets in a STAT3-dependent manner, and by preventing activation of a novel STAT3/HSP27/p38/Akt survival pathway. In contrast, imatinib prevents acquired resistance by inhibiting upregulation of the ABC drug transporter, ABCB1, directly inhibiting ABCB1 function, and abrogating survival signaling. Thus, imatinib inhibits multiple novel chemoresistance pathways, which indicates that it may be effective in reversing intrinsic and acquired resistance in cancers containing highly active c-Abl and Arg, a critical step in effectively treating metastatic disease. Furthermore, since imatinib converts a master survival regulator, NF-κB, from a pro-survival into a pro-apoptotic factor, our data suggest that NF-κB inhibitors may be ineffective in sensitizing tumors containing activated c-Abl/Arg to anthracyclines, and instead might antagonize anthracycline-induced apoptosis.     

Strategic Treatment Interruptions During Imatinib Treatment of Chronic Myelogenous Leukemia

Although imatinib is an effective treatment for chronic myelogenous leukemia (CML), and nearly all patients treated with imatinib attain some form of remission, imatinib does not completely eliminate leukemia. Moreover, if the imatinib treatment is stopped, most patients eventually relapse (Cortes et al. in Clin. Cancer Res. 11:3425–3432, 2005). In Kim et al. (PLoS Comput. Biol. 4(6):e1000095, 2008), the authors presented a mathematical model for the dynamics of CML under imatinib treatment that incorporates the anti-leukemia immune response. We use the mathematical model in Kim et al. (PLoS Comput. Biol. 4(6):e1000095, 2008) to study and numerically simulate strategic treatment interruptions as a potential therapeutic strategy for CML patients. We present the results of numerous simulated treatment programs in which imatinib treatment is temporarily stopped to stimulate and leverage the anti-leukemia immune response to combat CML. The simulations presented in this paper imply that treatment programs that involve strategic treatment interruptions may prevent leukemia from relapsing and may prevent remission for significantly longer than continuous imatinib treatment. Moreover, in many cases, strategic treatment interruptions may completely eliminate leukemic cells from the body. Thus, strategic treatment interruptions may be a feasible clinical approach to enhancing the effects of imatinib treatment for CML. We study the effects of both the timing and the duration of the treatment interruption on the results of the treatment. We also present a sensitivity analysis of the results to the parameters in the mathematical model.     

Proteomic strategy for probing complementary lethality of kinase inhibitors against pancreatic cancer

In the present study, proteomic analysis was performed to discover combinational molecular targets for therapy and chemoresistance by comparing differential protein expression from Panc‐1 cells treated with FDA‐approved drugs such as sunitinib, imatinib mesylate, dasatinib, and PD184352. A total of 4041 proteins were identified in the combined data from all of the treatment groups by nano‐electrospray ultra‐performance LC and MS/MS analysis. Most of the proteins with significant changes are involved in apoptosis, cytoskeletal remodeling, and epithelial‐to‐mesenchymal transition. These processes are associated with increased chemoresistance and progression of pancreatic cancer. Among the differentially expressed proteins, heme oxygenase‐1 (HO‐1) was found in the sunitinib and imatinib mesylate treatment groups, which possibly acts as a specific target for synthetic lethality in combinational treatment. HO‐1 was found to play a key role in sensitizing the chemoresistant Panc‐1 cell line to drug therapy. Viability was significantly decreased in Panc‐1 cells cotreated with sunitinib and imatinib mesylate at low doses, compared to those treated with sunitinib or imatinib mesylate alone. The results suggest that induction of chemosensitization by manipulating specific molecular targets can potentiate synergistic chemotherapeutic effects at lower, better tolerated doses, and in turn reduce the toxicity of multidrug treatment of pancreatic cancer.     

Aclacinomycin A Sensitizes K562 Chronic Myeloid Leukemia Cells to Imatinib through p38MAPK-Mediated Erythroid Differentiation

Expression of oncogenic Bcr-Abl inhibits cell differentiation of hematopoietic stem/progenitor cells in chronic myeloid leukemia (CML). Differentiation therapy is considered to be a new strategy for treating this type of leukemia. Aclacinomycin A (ACM) is an antitumor antibiotic. Previous studies have shown that ACM induced erythroid differentiation of CML cells. In this study, we investigate the effect of ACM on the sensitivity of human CML cell line K562 to Bcr-Abl specific inhibitor imatinib (STI571, Gleevec). We first determined the optimal concentration of ACM for erythroid differentiation but not growth inhibition and apoptosis in K562 cells. Then, pretreatment with this optimal concentration of ACM followed by a minimally toxic concentration of imatinib strongly induced growth inhibition and apoptosis compared to that with simultaneous co-treatment, indicating that ACM-induced erythroid differentiation sensitizes K562 cells to imatinib. Sequential treatment with ACM and imatinib induced Bcr-Abl down-regulation, cytochrome c release into the cytosol, and caspase-3 activation, as well as decreased Mcl-1 and Bcl-xL expressions, but did not affect Fas ligand/Fas death receptor and procaspase-8 expressions. ACM/imatinib sequential treatment-induced apoptosis was suppressed by a caspase-9 inhibitor and a caspase-3 inhibitor, indicating that the caspase cascade is involved in this apoptosis. Furthermore, we demonstrated that ACM induced erythroid differentiation through the p38 mitogen-activated protein kinase (MAPK) pathway. The inhibition of erythroid differentiation by p38MAPK inhibitor SB202190, p38MAPK dominant negative mutant or p38MAPK shRNA knockdown, reduced the ACM/imatinib sequential treatment-mediated growth inhibition and apoptosis. These results suggest that differentiated K562 cells induced by ACM-mediated p38MAPK pathway become more sensitive to imatinib and result in down-regulations of Bcr-Abl and anti-apoptotic proteins, growth inhibition and apoptosis. These results provided a potential management by which ACM might have a crucial impact on increasing sensitivity of CML cells to imatinib in the differentiation therapeutic approaches.     

MYC in chronic myeloid leukemia: induction of aberrant DNA synthesis and association with poor response to imatinib

Untreated chronic myeloid leukemia (CML) progresses from chronic phase to blastic crisis (BC). Increased genomic instability, deregulated proliferation, and loss of differentiation appear associated to BC, but the molecular alterations underlying the progression of CML are poorly characterized. MYC oncogene is frequently deregulated in human cancer, often associated with tumor progression. Genomic instability and induction of aberrant DNA replication are described as effects of MYC. In this report, we studied MYC activities in CML cell lines with conditional MYC expression with and without exposure to imatinib, the front-line drug in CML therapy. In cells with conditional MYC expression, MYC did not rescue the proliferation arrest mediated by imatinib but provoked aberrant DNA synthesis and accumulation of cells with 4C content. We studied MYC mRNA expression in 66 CML patients at different phases of the disease, and we found that MYC expression was higher in CML patients at diagnosis than control bone marrows or in patients responding to imatinib. Further, high MYC levels at diagnosis correlated with a poor response to imatinib. MYC expression did not directly correlate with BCR-ABL levels in patients treated with imatinib. Overall our study suggests that, as in other tumor models, MYC-induced aberrant DNA synthesis in CML cells is consistent with MYC overexpression in untreated CML patients and nonresponding patients and supports a role for MYC in CML progression, possibly through promotion of genomic instability.     

Targeting miR-21 sensitizes Ph+ ALL Sup-b15 cells to imatinib-induced apoptosis through upregulation of PTEN

Philadelphia chromosome positive (Ph+) acute lymphoblastic leukemia (ALL) cells are insensitive to BCR-ABL tyrosine kinase inhibitor imatinib, the underlying mechanisms remain largely unknown. Here, we showed that imatinib treatment induced significant upregulation of miR-21 and downregulation of PTEN in Ph+ ALL cell line Sup-b15. Transient inhibition of miR-21 resulted in increased apoptosis, PTEN upregulation and AKT dephosphorylation, whereas ectopic overexpression of miR-21 further conferred imatinib resistance. Furthermore, knockdown of PTEN protected the cells from imatinib-induced apoptosis achieved by inhibition of miR-21. Additionally, PI3K inhibitors also notably enhanced the effects of imatinib on Sup-b15 cells and primary Ph+ ALL cells similar to miR-21 inhibitor. Therefore, miR-21 contributes to imatinib resistance in Ph+ ALL cells and antagonizing miR-21 demonstrates therapeutic potential by sensitizing the malignancy to imatinib therapy.     

Optimal control of treatment in a mathematical model of chronic myelogenous leukemia

We consider a mathematical model of drug therapy for chronic myelogenous leukemia for an individual patient over a fixed time horizon. The disease dynamics are given by a system of ordinary differential equations that describe the interaction between naive T cells, effector T cells and leukemic cancer cells in a hypothetical patient. We introduce two drug therapies into this model, one a targeted therapy, and the other a broad cytotoxic therapy. Our goal is to find treatment regimens that minimize the cancer cell count and the deleterious effects of the drugs for a given patient. We examine the control setting analytically, and include numerical solutions to illustrate the optimal regimens under various assumptions.     

Role of P-glycoprotein in evolution of populations of chronic myeloid leukemia cells treated with imatinib

Imatinib mesylate (imatinib) is a new generation preparation that is now successfully used for treatment of cancer, particularly for chemotherapy of chronic myeloid leukemia (CML). Imatinib inhibits the activity of chimeric kinase BCR-ABL, which is responsible for the development of CML. The goal of this study was to investigate the role of a multidrug resistance protein, P-glycoprotein (Pgp), in the evolution of CML treated with imatinib. We demonstrate here that although imatinib is a substrate for Pgp, cultured CML cells (strain K562/i-S9), overexpressing active Pgp, do not exhibit imatinib resistance. Studies of CML patients in the accelerated phase have shown variations in the number of Pgp-positive cells (Pgp+) among individual patients treated with imatinib. During treatment of patients with imatinib for 6-12 months, the number of Pgp-positive cells significantly increased in most patients. The high number of Pgp+ cells remained in patients at least for 4.5 years and correlated with active Rhodamine 123 (Rh123) efflux. Such correlation was not found in the group of imatinib-resistant patients examined 35-60 months after onset of imatinib therapy: cells from the imatinib-resistant patients exhibited efficient Rh123 efflux irrespectively of Pgp expression. We also compared the mode of Rh123 efflux by cells from CML patients who underwent imatinib treatment for 6-24 months and the responsiveness of patients to this therapy. There were significant differences in survival of patients depending on the absence or the presence of Rh123 efflux. In addition to Pgp, patients' cells expressed other transport proteins of the ABC family. Our data suggest that treatment with imatinib causes selection of leukemic stem cells characterized by expression of Pgp and other ABC transporters.     

Signaling pathways in chronic myeloid leukemia and leukemic stem cell maintenance: key role of stromal microenvironment

Chronic myeloid leukemia (CML) is caused by the malignant transformation of hematopoietic stem cells in leukemic stem cells. From the introduction of the anti-cancer drug imatinib, the therapy of CML has been positively transformed. However, following treatment most patients display a residual CML disease attributed to the presence of quiescent leukemic stem cells intrinsically resistant to imatinib. Considering that the later cancer cells lose their chemoresistance in vitro, it appears that the stromal microenvironment plays a crucial role in CML-affected cell chemoresistance. In the present review, we summarize and discuss the recent findings on signaling pathways through which stromal cells sustain CML leukemogenesis, as well as leukemic stem cell maintenance and chemoresistance.     

The effect of mesenchymal stem cells and imatinib on macrophage polarization in rat model of liver fibrosis

Liver fibrosis is a disorder in which inflammatory reactions play an important role, and central to the progression and pathogenesis of this disease are the immune-specific cells known as macrophages. Macrophage types are distinguished from each other by the expression of a series of surface markers. STAT6 and Arg1 play an important role in the polarization of macrophages, so these two factors are downstream of interleukin 4 (IL-4) and IL-13 cytokines and cause to differentiate M2. Therefore, this study aimed to compare the independent effects of imatinib and mesenchymal cell treatment on the polarization of macrophages in rat models of liver fibrosis. The liver fibrosis was induced by the injection of CCL4 for 6 weeks in Sprague–Dawley rats. Then, rats were divided into four different groups, and the effects of imatinib and mesenchymal cells on the expression of Arg1, Ly6c, and STAT6 were evaluated. Histopathology experiments considered the amelioration effect of treatments. Our results showed that Arg1 expression was significantly increased in the groups treated with mesenchymal cells and imatinib compared to the control group. On the other hand, expression of STAT6 was significantly increased in the imatinib-treated mice compared to mesenchymal and control groups. Moreover, the expression of LY6C significantly decreased in imatinib and mesenchymal treated groups compared to the control group. Therefore, our data showed that mesenchymal stem cells and imatinib significantly modulate the fibrotic process in rat models of fibrosis, probably by polarizing macrophages towards an anti-inflammatory profile and increasing the frequency of these cells in liver tissue.     

Overexpression of Hes1 is involved in sensitization of K562 cells to Imatinib

Tyrosine kinase inhibitor (TKI)-based therapy has created promising results among much chronic myeloid leukemia (CML) patients. Imatinib as a relatively specific inhibitor of Bcr-Abl is at present one of the undisputed therapeutic agent for newlydiagnosed patients with CML. However, the occurrence of imatinib-resistance enlightens the urgent need to identify other therapeutic agents against CML. Juglone (5-hydroxy-2-methyl-1, 4-naphthoquinone) exerts cytotoxic effects against various human cancer cell lines. However, the mechanisms through which Juglone induces anticancer effects in CML especially in comparison with imatinib treatment remain unknown. Our results revealed that Juglone-inhibited K562 cells growth through inducing apoptosis. Based on our Western blot analyses, Juglone significantly reduced p-Akt levels and increased the expression level of Forkhead box O1 (FoxO1) and FoxO3a proteins. Moreover, hairy/enhancer of split-1 (Hes1) protein, overexpressed under the influence of Juglone, is apparently involved in Juglone-induced apoptosis among K562 cells. Conversely, treatment with imatinib attenuated Hes1 protein expression. Considering the different functional mechanism of Juglone compared with imatinib, it seems that Juglone treatment could be a useful alternative strategy for the treatment of patients with imatinib-resistance.     

Imatinib targets PDGF signaling in melanoma and host smooth muscle neighboring cells

In previous in vitro studies, we showed that imatinib abrogated platelet‐derived growth factor receptor α (PDGFRα) signaling, disrupting both breast cancer and smooth muscle cells (SMC). PDGF is also a powerful mitogen for neural crest origin cells like melanocytes. The purpose of the present study was to evaluate the effect of imatinib on melanoma growth and in angiogenesis, with emphasis to the involvement in PDGF signaling. B16 melanoma cells incubation with 5 µM (IC50) imatinib resulted in a significant reduction in cell proliferation and migration. Apoptosis, however, was not significantly affected. Phosphorylated‐PDGFRα expression was decreased in B16 lysates. In a mouse model of B16 melanoma, intraperitoneal administration of imatinib at early day light significantly decreased tumor growth. These findings were corroborated by a highly significant reduction in cell proliferation and increase in apoptosis in melanoma tumors. This was accompanied by a decrease in microvessel density and in the number of SMC‐presenting vessels. Imatinib further inhibited PDGFRα expression and activity, as confirmed by the down‐regulation of downstream Erk signaling pathway. Altogether, this study demonstrates that besides targeting tumor cells, imatinib also prevents vascular integrity. The current study provides evidence that the paracrine crosstalk between tumor cells and host neighboring cells is crucial for the elucidation of imatinib effects. In addition, the fact that this molecule targets vascular support cells further enlarges its therapeutic purpose to a wide range of vasculoproliferative pathologies. J. Cell. Biochem. 111: 433–441, 2010. © 2010 Wiley‐Liss, Inc.     

Rapid detection of phosphotyrosine proteins by flow cytometric analysis in Bcr-Abl-positive cells.

BACKGROUND: Constitutive tyrosine phosphorylation derived from Bcr-Abl kinase activity is the major characteristic of Bcr-Abl positive cells. In this study, we developed a method to detect the phosphotyrosine proteins by flow cytometry and we asked whether phosphorylation was affected by imatinib mesylate treatment. METHODS: Cells were treated or not with imatinib mesylate, fixed and permeabilized by PFA followed by saponin, then stained with anti-phosphotyrosine (p-tyr) monoclonal antibody and analyzed by flow cytometry. RESULTS: Optimal staining parameters were performed with p-tyr antibody using K562 and LAMA84 lines that displayed high levels of tyrosine phosphorylation as compared to the control line, HL60. Tyrosine phosphorylation was inhibited by imatinib in a dose-dependent manner, but not modified by other inhibitors demonstrating that the staining detected is specific to Bcr-Abl phosphorylation. The staining of imatinib-resistant cell lines such as the mutated BaF/Bcr-AblT315I cell line or resistant CML patient cells, showed that hyperphosphorylation was not affected by imatinib treatment. In one CML patient, our technique permitted us to detect a small hyperphosphorylated population resistant to imatinib that appeared hyperphosphorylated and amplified at the time of relapse. CONCLUSIONS: We have developed a flow cytometric technique presenting several advantages such as rapidity and sensitivity, which requires fewer cells than the Western blot.     

The optimal dose of CAF regimen in adjuvant chemotherapy for breast cancer patients at stage IIB

This paper deals with the idea of balancing drug effects on tumor and normal cell populations based on a variety of criteria, which is evaluated by the oncologist for breast cancer patients at stage IIB. In this paper, the optimal controller represents the optimal drug dosage of CAF (Cyclophosphamide, Adriamycin and Fluorouracil) regimen in adjuvant chemotherapy after surgery for these patients. We determined the doses of CAF regimen by minimizing a cost function with some constraints. The cost function includes the cancer cell and the normal cell growth dynamics with prescribed weighting coefficients for each patient. The physician determines these weighting coefficients based on some individual parameters. The optimal treatment schedules are computed based on a trade-off between the cancer cell reduction and the normal cell preserving. Numerical simulations are given to illustrate the accuracy of the optimal controller.     

The two major imatinib resistance mutations E255K and T315I enhance the activity of BCR/ABL fusion kinase

The resistance to the tyrosine kinase inhibitor imatinib in BCR/ABL-positive leukemias is mostly associated with mutations in the kinase domain of BCR/ABL, which include the most prevalent mutations E255K and T315I. Intriguingly, these mutations have also been identified in some patients before imatinib treatment. Here we examined the effects of these mutations on the kinase activity of a BCR/ABL kinase domain construct that also contained the SH3 and SH2 domains. When expressed in COS7 cells, the BCR/ABL construct with either E255K or T315I exhibited not only the resistance to imatinib but also the increase in activity to induce autophosphorylation as well as tyrosine phosphorylation of various cellular proteins, which included STAT5. The mutant kinases also showed increased activities in in vitro kinase assays. These results raise a possibility that the major imatinib resistance mutations E255K and T315I may confer the growth advantage on leukemic cells to expand in the absence of selective pressure from imatinib treatment.