Molecular mechanisms for survival regulation of chronic myeloid leukemia stem cells

Studies on chronic myeloid leukemia (CML) have served as a paradigm for cancer research and therapy. These studies involve the identifi cation of the fi rst cancer-associated chromosomal abnormality and the subsequent development of tyrosine kinase inhibitors (TKIs) that inhibit BCR-ABL kinase activity in CML. It becomes clear that leukemia stem cells (LSCs) in CML which are resistant to TKIs, and eradication of LSCs appears to be extremely diffi cult. Therefore, one of the major issues in current CML biology is to understand the biology of LSCs and to investigate why LSCs are insensitive to TKI monotherapy for developing curative therapeutic strategies. Studies from our group and others have revealed that CML LSCs form a hierarchy similar to that seen in normal hematopoiesis, in which a rare stem cell population with limitless selfrenewal potential gives rise to progenies that lack such potential. LSCs also possess biological features that are different from those of normal hematopoietic stem cells (HSCs) and are critical for their malignant characteristics. In this review, we summarize the latest progress in CML field, and attempt to understand the molecular mechanisms of survival regulation of LSCs.     

Hedgehog pathway activation in chronic myeloid leukemia: A promise for a novel combination therapeutic approach?

Chronic Myeloid Leukemia (CML) is a hematopoietic stem cell malignancy that is driven by the oncogenic BCR-ABL fusion protein, and for which treatment with ABL tyrosine kinase inhibitors (TKI) has yielded great success. While this is the case, BCR-ABL leukemic stem cells can persist despite TKI therapy, and efforts have intensified towards determining the molecular pathways that are critical for the maintenance of such cells. Recent studies indicate that aberrant Hedgehog (Hh) signaling plays a crucial role in the survival of the leukemic stem cell population. The Hh pathway displays crucial roles during embryonic development, tissue regeneration and repair in adults. Several mechanisms that lead to the aberrant activation of the Hh pathway have been identified in various cancers. Here we review in detail the discovery that Hh signaling governs the maintenance of the critical leukemia initiating cells or leukemic stem cells (LSCs) in BCR-ABL-induced CML as well as discuss investigations on the role of Hh signaling in normal hematopoeisis. As inhibitors that directly target the positive Hh signal transducer Smoothened (SMO) have entered clinical trials, these findings offer a unique opportunity to potentially target the LSC population that is not eliminated with ABL tyrosine kinase inhibition therapy in CML.     

ATG7 regulates energy metabolism,differentiation and survival of Philadelphia-chromosome-positive cells

A major drawback of tyrosine kinase inhibitor (TKI) treatment in chronic myeloid leukemia (CML) is that primitive CML cells are able to survive TKI-mediated BCR-ABL inhibition, leading to disease persistence in patients. Investigation of strategies aiming to inhibit alternative survival pathways in CML is therefore critical. We have previously shown that a nonspecific pharmacological inhibition of autophagy potentiates TKI-induced death in Philadelphia chromosome-positive cells. Here we provide further understanding of how specific and pharmacological autophagy inhibition affects nonmitochondrial and mitochondrial energy metabolism and reactive oxygen species (ROS)-mediated differentiation of CML cells and highlight ATG7 (a critical component of the LC3 conjugation system) as a potential specific therapeutic target. By combining extra- and intracellular steady state metabolite measurements by liquid chromatography-mass spectrometry with metabolic flux assays using labeled glucose and functional assays, we demonstrate that knockdown of ATG7 results in decreased glycolysis and increased flux of labeled carbons through the mitochondrial tricarboxylic acid cycle. This leads to increased oxidative phosphorylation and mitochondrial ROS accumulation. Furthermore, following ROS accumulation, CML cells, including primary CML CD34⁺ progenitor cells, differentiate toward the erythroid lineage. Finally, ATG7 knockdown sensitizes CML progenitor cells to TKI-induced death, without affecting survival of normal cells, suggesting that specific inhibitors of ATG7 in combination with TKI would provide a novel therapeutic approach for CML patients exhibiting persistent disease.     

Molecular and cellular bases of chronic myeloid leukemia

Chronic myeloid leukemia (CML) is a myeloproliferative disease characterized by the overproduction of granulocytes, which leads to high white blood cell counts and splenomegaly in patients. Based on clinical symptoms and laboratory findings, CML is classified into three clinical phases, often starting with a chronic phase, progressing to an accelerated phase and ultimately ending in a terminal phase called blast crisis. Blast crisis phase of CML is clinically similar to an acute leukemia; in particular, B-cell acute lymphoblastic leukemia (B-ALL) is a severe form of acute leukemia in blast crisis, and there is no effective therapy for it yet. CML is induced by the BCR-ABL oncogene, whose gene product is a BCR-ABL tyrosine kinase. Currently, inhibition of BCR-ABL kinase activity by its kinase inhibitor such as imatinib mesylate (Gleevec) is a major therapeutic strategy for CML. However, the inability of BCR-ABL kinase inhibitors to completely kill leukemia stem cells (LSCs) indicates that these kinase inhibitors are unlikely to cure CML. In addition, drug resistance due to the development of BCR-ABL mutations occurs before and during treatment of CML with kinase inhibitors. A critical issue to resolve this problem is to fully understand the biology of LSCs, and to identify key genes that play significant roles in survival and self-renewal of LSCs. In this review, we will focus on LSCs in CML by summarizing and discussing available experimental results, including the original studies from our own laboratory.     

Combination of tyrosine kinase inhibitors and the MCL1 inhibitor S63845 exerts synergistic antitumorigenic effects on CML cells

Tyrosine kinase inhibitor (TKI) treatment has dramatically improved the survival of chronic myeloid leukemia (CML) patients, but measurable residual disease typically persists. To more effectively eradicate leukemia cells, simultaneous targeting of BCR-ABL1 and additional CML-related survival proteins has been proposed. Notably, several highly specific myeloid cell leukemia 1 (MCL1) inhibitors have recently entered clinical trials for various hematologic malignancies, although not for CML, reflecting the insensitivity of CML cell lines to single MCL1 inhibition. Here, we show that combining TKI (imatinib, nilotinib, dasatinib, or asciminib) treatment with the small-molecule MCL1 inhibitor {"type":"entrez-nucleotide","attrs":{"text":"S63845","term_id":"400540","term_text":"S63845"}}S63845 exerted strong synergistic antiviability and proapoptotic effects on CML lines and CD34+ stem/progenitor cells isolated from untreated CML patients in chronic phase. Using wild-type BCR-ABL1-harboring CML lines and their T315I-mutated sublines (generated by CRISPR/Cas9-mediated homologous recombination), we prove that the synergistic proapoptotic effect of the drug combination depended on TKI-mediated BCR-ABL1 inhibition, but not on TKI-related off-target mechanisms. Moreover, we demonstrate that colony formation of CML but not normal hematopoietic stem/progenitor cells became markedly reduced upon combination treatment compared to imatinib monotherapy. Our results suggest that dual targeting of MCL1 and BCR-ABL1 activity may efficiently eradicate residual CML cells without affecting normal hematopoietic stem/progenitors.Subject terms: Cancer stem cells, Targeted therapies, Preclinical research     

Philadelphia chromosome-positive leukemia stem cells in acute lymphoblastic leukemia and tyrosine kinase inhibitor therapy

Leukemia stem cells(LSCs),which constitute a minority of the tumor bulk,are functionally defined on the basis of their ability to transfer leukemia into an immunodeficient recipient animal.The presence of LSCs has been demonstrated in acute lymphoblastic leukemia(ALL),of which ALL with Philadelphia chromosome-positive(Ph+).The use of imatinib,a tyrosine kinase inhibitor(TKI),as part of front-line treatment and in combination with cytotoxic agents,has greatly improved the proportions of complete response and molecular remission and the overall outcome in adults with newly diagnosed Ph+ ALL.New challenges have emerged with respect to induction of resistance to imatinib via Abelson tyrosine kinase mutations.An important recent addition to the arsenal against Ph+ leukemias in general was the development of novel TKIs,such as nilotinib and dasatinib.However,in vitro experiments have suggested that TKIs have an antiproliferative but not an antiapoptotic or cytotoxic effect on the most primitive ALL stem cells.None of the TKIs in clinical use target the LSC.Second generation TKI dasatinib has been shown to have a more profound effect on the stem cell compartment but the drug was still unable to kill the most primitive LSCs.Allogeneic stem cell transplantation(SCT) remains the only curative treatment available for these patients.Several mechanisms were proposed to explain the resistance of LSCs to TKIs in addition to mutations.Hence,TKIs may be used as a bridge to SCT rather than monotherapy or combination with standard chemotherapy.Better understanding the biology of Ph+ ALL will open new avenues for effective management.In this review,we highlight recent findings relating to the question of LSCs in Ph+ ALL.     

The Alox5 gene is a novel therapeutic target in cancer stem cells of chronic myeloid leukemia

Cancer stem cells (CSCs) are believed to be the initiating cells for many types of blood cancer and some solid tumors, and curative therapies of these cancers require eradicating CSCs. Specific targeting of CSCs but not normal stem cell counterparts is a correct strategy for developing new anti-cancer therapies, and the success of this approach relies on identification of specific target genes in CSCs. Using BCR-ABL-induced chronic myeloid leukemia (CML) as a cancer model, we recently identified arachidonate 5-lipoxygenase (5-LO) gene (Alox5) as a critical regulator for leukemia stem cells (LSCs) in CML. Without Alox5, BCR-ABL fails to induce CML in mice due to the impairments of the functions of LSCs. The lack of Alox5 does not significantly affect the functions of normal hematopoietic stem cells. In addition, Zileuton, a specific 5-LO inhibitor, also causes the impairments of the functions of LSCs in a similar manner. Our results prove the principle that CSC-specific genes that play key roles in cancer development can be identified and inhibition of these genes can lead to eradication of these cells for cure. Here, we further discuss the mechanisms of Alox5 in CML, and the use of Zileuton as a potential and promising drug in eradicating LSCs in CML and other myeloproliferative diseases. We believe that our discovery of the role of Alox5 in regulating the function of LSCs in CML reminds us of viewing CSCs at a different angel. We predict that CSCs in other types of cancer also utilize specific regulatory pathways to control their survival and self-renewal, and inhibition of these pathways profoundly suppresses CSCs but not their normal stem cell counterparts. Specific targeting of CSCs without causing significant harm to normal stem cells should be a correct direction to go in developing novel therapeutic strategies in the future.     

MAPK15 mediates BCR-ABL1-induced autophagy and regulates oncogene-dependent cell proliferation and tumor formation

A reciprocal translocation of the ABL1 gene to the BCR gene results in the expression of the oncogenic BCR-ABL1 fusion protein, which characterizes human chronic myeloid leukemia (CML), a myeloproliferative disorder considered invariably fatal until the introduction of the imatinib family of tyrosine kinase inhibitors (TKI). Nonetheless, insensitivity of CML stem cells to TKI treatment and intrinsic or acquired resistance are still frequent causes for disease persistence and blastic phase progression experienced in patients after initial successful therapies. Here, we investigated a possible role for the MAPK15/ERK8 kinase in BCR-ABL1-dependent autophagy, a key process for oncogene-induced leukemogenesis. In this context, we showed the ability of MAPK15 to physically recruit the oncogene to autophagic vesicles, confirming our hypothesis of a biologically relevant role for this MAP kinase in signal transduction by this oncogene. Indeed, by modeling BCR-ABL1 signaling in HeLa cells and taking advantage of a physiologically relevant model for human CML, i.e. K562 cells, we demonstrated that BCR-ABL1-induced autophagy is mediated by MAPK15 through its ability to interact with LC3-family proteins, in a LIR-dependent manner. Interestingly, we were also able to interfere with BCR-ABL1-induced autophagy by a pharmacological approach aimed at inhibiting MAPK15, opening the possibility of acting on this kinase to affect autophagy and diseases depending on this cellular function. Indeed, to support the feasibility of this approach, we demonstrated that depletion of endogenous MAPK15 expression inhibited BCR-ABL1-dependent cell proliferation, in vitro, and tumor formation, in vivo, therefore providing a novel “druggable” link between BCR-ABL1 and human CML.     

Heat Shock Protein 90: A Potential Therapeutic Target in Leukemic Progenitor and Stem Cells Harboring Mutant BCR-ABL Resistant to Kinase Inhibitors

Development of drug resistance has become a major obstacle for tyrosine kinase inhibitors (TKIs) in the treatment of Philadelphia chromosome-positive (Ph+) chronic myelogenous leukemia (CML) and other cancers. The BCR-ABL-T315I mutant does not respond to clinically available TKIs, although some newly developed anti-BCR-ABL-T315I TKIs are now being tested in patients. TKIs transiently inhibit kinase activity of BCR-ABL, but do not reduce the level of the BCR-ABL protein. Elimination of mutant BCR-ABL protein would provide a new therapeutic strategy for treating Ph+ leukemia. We recently showed that inhibition of heat shock protein 90 (Hsp90) by a novel Hsp90 inhibitor, IPI-504, causes BCR-ABL protein degradation, decreased numbers of leukemia stem cells, and prolonged survival of mice with CML induced by BCR-ABL-T315I. Here we discuss further the mechanisms and effectiveness of Hsp90 inhibition in suppression of survival and proliferation of leukemic progenitor and stem cells in CML mice, and the potential of this anti-Hsp90 strategy in treating CML patients, including those who have developed resistance to TKIs.     

ATRA-Induced Cellular Differentiation and CD38 Expression Inhibits Acquisition of BCR-ABL Mutations for CML Acquired Resistance

Acquired resistance through genetic mutations is a major obstacle in targeted cancer therapy, but the underlying mechanisms are poorly understood. Here we studied mechanisms of acquired resistance of chronic myeloid leukemia (CML) to tyrosine kinase inhibitors (TKIs) by examining genome-wide gene expression changes in KCL-22 CML cells versus their resistant KCL-22M cells that acquire T315I BCR-ABL mutation following TKI exposure. Although T315I BCR-ABL is sufficient to confer resistance to TKIs in CML cells, surprisingly we found that multiple drug resistance pathways were activated in KCL-22M cells along with reduced expression of a set of myeloid differentiation genes. Forced myeloid differentiation by all-trans-retinoic acid (ATRA) effectively blocked acquisition of BCR-ABL mutations and resistance to the TKIs imatinib, nilotinib or dasatinib in our previously described in vitro models of acquired TKI resistance. ATRA induced robust expression of CD38, a cell surface marker and cellular NADase. High levels of CD38 reduced intracellular nicotinamide adenine dinucleotide (NAD⁺) levels and blocked acquired resistance by inhibiting the activity of the NAD⁺-dependent SIRT1 deacetylase that we have previously shown to promote resistance in CML cells by facilitating error-prone DNA damage repair. Consequently, ATRA treatment decreased DNA damage repair and suppressed acquisition of BCR-ABL mutations. This study sheds novel insight into mechanisms underlying acquired resistance in CML, and suggests potential benefit of combining ATRA with TKIs in treating CML, particularly in advanced phases.     

Molecular and structural characterization of the SH3 domain of AHI-1 in regulation of cellular resistance of BCR-ABL(+) chronic myeloid leukemia cells to tyrosine kinase inhibitors

ABL tyrosine kinase inhibitor (TKI) therapy induces clinical remission in chronic myeloid leukemia (CML) patients but early relapses and later emergence of TKI-resistant disease remain problematic. We recently demonstrated that the AHI-1 oncogene physically interacts with BCR-ABL and JAK2 and mediates cellular resistance to TKI in CML stem/progenitor cells. We now show that deletion of the SH3 domain of AHI-1 significantly enhances apoptotic response of BCR-ABL(+) cells to TKIs compared to cells expressing full-length AHI-1. We have also discovered a novel interaction between AHI-1 and Dynamin-2, a GTPase, through the AHI-1 SH3 domain. The crystal structure of the AHI-1 SH3 domain at 1.53-? resolution reveals that it adopts canonical SH3 folding, with the exception of an unusual C-terminal α helix. PD1R peptide, known to interact with the PI3K SH3 domain, was used to model the binding pattern between the AHI-1 SH3 domain and its ligands. These studies showed that an "Arg-Arg-Trp" stack may form within the binding interface, providing a potential target site for designing specific drugs. The crystal structure of the AHI-1 SH3 domain thus provides a valuable tool for identification of key interaction sites in regulation of drug resistance and for the development of small molecule inhibitors for CML.     

Targeted CML therapy: controlling drug resistance, seeking cure

Targeted cancer therapy with imatinib (Gleevec) has the capability to drive chronic myeloid leukemia (CML) into clinical remission. Some patients, particularly those with advanced disease, develop resistance to imatinib. To counteract this problem, two new BCR-ABL kinase inhibitors for imatinib-refractory disease are currently in clinical trials: the imatinib derivative AMN107 and the dual-specificity SRC/ABL inhibitor dasatinib. Using imatinib to reduce leukemic burden also facilitates the detailed investigation into how the persistence of CML disease depends on BCR-ABL signaling, particularly within the leukemic stem cell compartment. Mathematical models of drug resistance and disease relapse, in addition to experimental systems that recapitulate crucial aspects of advanced disease have deepened our understanding of CML biology. Together, these advances are contributing to a high level of disease control, and might ultimately lead to disease eradication.     

Inhibition of PI3K/mTOR Overcomes Nilotinib Resistance in BCR-ABL1 Positive Leukemia Cells through Translational Down-Regulation of MDM2

Chronic myeloid leukemia (CML) is a cytogenetic disorder resulting from formation of the Philadelphia chromosome (Ph), that is, the t(9;22) chromosomal translocation and the formation of the BCR-ABL1 fusion protein. Tyrosine kinase inhibitors (TKI), such as imatinib and nilotinib, have emerged as leading compounds with which to treat CML. t(9;22) is not restricted to CML, 20-30% of acute lymphoblastic leukemia (ALL) cases also carry the Ph. However, TKIs are not as effective in the treatment of Ph+ ALL as in CML. In this study, the Ph+ cell lines JURL-MK2 and SUP-B15 were used to investigate TKI resistance mechanisms and the sensitization of Ph+ tumor cells to TKI treatment. The annexin V/PI (propidium iodide) assay revealed that nilotinib induced apoptosis in JURL-MK2 cells, but not in SUP-B15 cells. Since there was no mutation in the tyrosine kinase domain of BCR-ABL1 in cell line SUP-B15, the cells were not generally unresponsive to TKI, as evidenced by dephosphorylation of the BCR-ABL1 downstream targets, Crk-like protein (CrkL) and Grb-associated binder-2 (GAB2). Resistance to apoptosis after nilotinib treatment was accompanied by the constitutive and nilotinib unresponsive activation of the phosphoinositide 3-kinase (PI3K) pathway. Treatment of SUP-B15 cells with the dual PI3K/mammalian target of rapamycin (mTOR) inhibitor BEZ235 alone induced apoptosis in a low percentage of cells, while combining nilotinib and BEZ235 led to a synergistic effect. The main role of PI3K/mTOR inhibitor BEZ235 and the reason for apoptosis in the nilotinib-resistant cells was the block of the translational machinery, leading to the rapid downregulation of the anti-apoptotic protein MDM2 (human homolog of the murine double minute-2). These findings highlight MDM2 as a potential therapeutic target to increase TKI-mediated apoptosis and imply that the combination of PI3K/mTOR inhibitor and TKI might form a novel strategy to combat TKI-resistant BCR-ABL1 positive leukemia.     

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.     

Potential Role of Notch Signalling in CD34+ Chronic Myeloid Leukaemia Cells: Cross-Talk between Notch and BCR-ABL

Notch signalling is critical for haemopoietic stem cell (HSC) self-renewal and survival. The role of Notch signalling has been reported recently in chronic myeloid leukaemia (CML) – a stem cell disease characterized by BCR-ABL tyrosine kinase activation. Therefore, we studied the relationship between BCR-ABL and Notch signalling and assessed the expression patterns of Notch and its downstream target Hes1 in CD34⁺ stem and progenitor cells from chronic-phase CML patients and bone marrow (BM) from normal subjects (NBM). We found significant upregulation (p<0.05) of Notch1, Notch2 and Hes1 on the most primitive CD34⁺Thy⁺ subset of CML CD34⁺ cells suggesting that active Notch signalling in CML primitive progenitors. In addition, Notch1 was also expressed in distinct lymphoid and myeloid progenitors within the CD34⁺ population of primary CML cells. To further delineate the possible role and interactions of Notch with BCR-ABL in CD34⁺ primary cells from chronic-phase CML, we used P-crkl detection as a surrogate assay of BCR-ABL tyrosine kinase activity. Our data revealed that Imatinib (IM) induced BCR-ABL inhibition results in significant (p<0.05) upregulation of Notch activity, assessed by Hes1 expression. Similarly, inhibition of Notch leads to hyperactivation of BCR-ABL. This antagonistic relationship between Notch and BCR-ABL signalling was confirmed in K562 and ALL-SIL cell lines. In K562, we further validated this antagonistic relationship by inhibiting histone deacetylase (HDAC) - an effector pathway of Hes1, using valproic acid (VPA) - a HDAC inhibitor. Finally, we also confirmed the potential antagonism between Notch and BCR/ABL in In Vivo, using publically available GSE-database, by analysing gene expression profile of paired samples from chronic-phase CML patients pre- and post-Imatinib therapy. Thus, we have demonstrated an antagonistic relationship between Notch and BCR-ABL in CML. A combined inhibition of Notch and BCR-ABL may therefore provide superior clinical response over tyrosine-kinase inhibitor monotherapy by targeting both quiescent leukaemic stem cells and differentiated leukaemic cells and hence must be explored.     

Knockout of LASP1 in CXCR4 expressing CML cells promotes cell persistence,proliferation and TKI resistance

Chronic myeloid leukaemia (CML) is a clonal myeloproliferative stem cell disorder characterized by the constitutively active BCR-ABL tyrosine kinase. The LIM and SH3 domain protein 1 (LASP1) has recently been identified as a novel BCR-ABL substrate and is associated with proliferation, migration, tumorigenesis and chemoresistance in several cancers. Furthermore, LASP1 was shown to bind to the chemokine receptor 4 (CXCR4), thought to be involved in mechanisms of relapse. In order to identify potential LASP1-mediated pathways and related factors that may help to further eradicate minimal residual disease (MRD), the effect of LASP1 on processes involved in progression and maintenance of CML was investigated. The present data indicate that not only overexpression of CXCR4, but also knockout of LASP1 contributes to proliferation, reduced apoptosis and migration as well as increased adhesive potential of K562 CML cells. Furthermore, LASP1 depletion in K562 CML cells leads to decreased cytokine release and reduced NK cell-mediated cytotoxicity towards CML cells. Taken together, these results indicate that in CML, reduced levels of LASP1 alone and in combination with high CXCR4 expression may contribute to TKI resistance.     

Targeted therapies and autophagy: new insights from chronic myeloid leukemia

Patients who develop chronic myeloid leukemia (CML) are currently treated with tyrosine kinase inhibitors (TKIs), which inhibit the function of the oncogene BCR/Abl. Most CML cells undergo apoptosis when BCR/Abl tyrosine kinase activity is suppressed by TKIs. Cells surviving drug treatment are either stem cells (CML in early phase) or cells with BCR/Abl-dependent or -independent mechanisms of drug resistance (CML in advanced phase). Since survival of these cells is thought to be responsible for disease recurrence, it is critical to find ways to fully eradicate CML stem cells. We have recently shown that when CML cells, including stem cells, are exposed to TKI they activate an autophagic program, which relies on intracellular calcium and is not inhibited by Bcl-2. Pharmacological or RNAi-mediated inhibition of autophagy potentiates the effect of TKI in inducing death of CML cells, including the stem cells. These data strongly suggest that inhibition of autophagy may improve the therapeutic effects of TKIs in the treatment of CML. In addition, they give credence to the idea that in cancer cells autophagy is part of a stereotypic response to stress and specifically to abrogation of their main oncogenic signal(s).     

p53 restoration kills primitive leukemia cells in vivo and increases survival of leukemic mice

Loss of p53 function is a common feature of human cancers and it is required for differentiated tumor cell maintenance; however, it is not known whether sustained inactivation of the p53 pathway is needed for cancer stem cell persistence. Chronic myeloid leukemia (CML) is caused by a chromosome translocation that generates the BCRABL oncogene encoding a constitutively active protein tyrosine kinase. The disease originates in a hematopoietic stem cell and is maintained by leukemic stem cells (LSCs). Treatment with specific tyrosine kinase inhibitors does not eliminate LSCs because they do not depend on the oncogene for survival. We have combined a switchable p53 knock-in mouse model, p53^KI/KI, with the well-characterized Sca1-BCRABLp210 CML transgenic model, to show that transient restoration of p53 slows disease progression and significantly extends the survival of leukemic animals, being the mechanism responsible for this effect, apoptotic death of primitive leukemia cells. In agreement with these in vivo findings, in vitro assays show that restoring p53 reduces hematopoietic colony formation by cells of leukemic animals. These results suggest that reestablishing p53 function may be a therapeutic strategy for the eradication of leukemic stem cells and to prevent disease progression.