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.     

Isolation of specific and biologically active peptides that bind cells from patients with acute myeloid leukemia (AML)

Imatinib was the first BCR-ABL-targeted agent approved for the treatment of patients with chronic myeloid leukemia (CML) and confers significant benefit for most patients; however, a substantial number of patients are either initially refractory or develop resistance. Point mutations within the ABL kinase domain of the BCR-ABL fusion protein are a major underlying cause of resistance. Of the known imatinib-resistant mutations, the most frequently occurring involve the ATP-binding loop (P-loop). In vitro evidence has suggested that these mutations are more oncogenic with respect to other mutations and wild type BCR-ABL. Dasatinib and nilotinib have been approved for second-line treatment of patients with CML who demonstrate resistance (or intolerance) to imatinib. Both agents have marked activity in patients resistant to imatinib; however, they have differential activity against certain mutations, including those of the P-loop. Data from clinical trials suggest that dasatinib may be more effective vs. nilotinib for treating patients harboring P-loop mutations. Other mutations that are differentially sensitive to the second-line tyrosine kinase inhibitors (TKIs) include F317L and F359I/V, which are more sensitive to nilotinib and dasatinib, respectively. P-loop status in patients with CML and the potency of TKIs against P-loop mutations are key determinants for prognosis and response to treatment. This communication reviews the clinical importance of P-loop mutations and the efficacy of the currently available TKIs against them.     

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.     

Intracellular retention of ABL kinase inhibitors determines commitment to apoptosis in CML cells

Clinical development of imatinib in CML established continuous target inhibition as a paradigm for successful tyrosine kinase inhibitor (TKI) therapy. However, recent reports suggested that transient potent target inhibition of BCR-ABL by high-dose TKI (HD-TKI) pulse-exposure is sufficient to irreversibly commit cells to apoptosis. Here, we report a novel mechanism of prolonged intracellular TKI activity upon HD-TKI pulse-exposure (imatinib, dasatinib) in BCR-ABL-positive cells. Comprehensive mechanistic exploration revealed dramatic intracellular accumulation of TKIs which closely correlated with induction of apoptosis. Cells were rescued from apoptosis upon HD-TKI pulse either by repetitive drug wash-out or by overexpression of ABC-family drug transporters. Inhibition of ABCB1 restored sensitivity to HD-TKI pulse-exposure. Thus, our data provide evidence that intracellular drug retention crucially determines biological activity of imatinib and dasatinib. These studies may refine our current thinking on critical requirements of TKI dose and duration of target inhibition for biological activity of TKIs.     

Stem cell and kinase activity-independent pathway in resistance of leukaemia to BCR-ABL kinase inhibitors

BCR-ABL tyrosine kinase inhibitors, such as imatinib (Gleevec) are highly effective in treating human Philadelphia chromosome-positive (Ph+) chronic myeloid leukaemia (CML) in chronic phase but not in terminal acute phase; acquired drug resistance caused mainly by the development of BCR-ABL kinase domain mutations prevents cure of the leukaemia. In addition, imatinib is ineffective in treating Ph+ B-cell acute lymphoblastic leukaemia (B-ALL) and CML blast crisis, even in the absence of the kinase domain mutations. This type of drug resistance that is unrelated to BCR-ABL kinase domain mutations is caused by the insensitivity of leukaemic stem cells to kinase inhibitors such as imatinib and dasatinib, and by activation of a newly-identified signalling pathway involving SRC kinases that are independent of BCR-ABL kinase activity for activation. This SRC pathway is essential for leukaemic cells to survive imatinib treatment and for CML transition to lymphoid blast crisis. Apart from BCR-ABL and SRC kinases, stem cell pathways must also be targeted for curative therapy of Ph+ leukaemia.     

A Target-Disease Network Model of Second-Generation BCR-ABL Inhibitor Action in Ph+ ALL

Philadelphia chromosome-positive acute lymphoblastic leukemia (Ph+ ALL) is in part driven by the tyrosine kinase bcr-abl, but imatinib does not produce long-term remission. Therefore, second-generation ABL inhibitors are currently in clinical investigation. Considering different target specificities and the pronounced genetic heterogeneity of Ph+ ALL, which contributes to the aggressiveness of the disease, drug candidates should be evaluated with regard to their effects on the entire Ph+ ALL-specific signaling network. Here, we applied an integrated experimental and computational approach that allowed us to estimate the differential impact of the bcr-abl inhibitors nilotinib, dasatinib, Bosutinib and Bafetinib. First, we determined drug-protein interactions in Ph+ ALL cell lines by chemical proteomics. We then mapped those interactions along with known genetic lesions onto public protein-protein interactions. Computation of global scores through correlation of target affinity, network topology, and distance to disease-relevant nodes assigned the highest impact to dasatinib, which was subsequently confirmed by proliferation assays. In future, combination of patient-specific genomic information with detailed drug target knowledge and network-based computational analysis should allow for an accurate and individualized prediction of therapy.     

Variable Behavior of iPSCs Derived from CML Patients for Response to TKI and Hematopoietic Differentiation

Chronic myeloid leukemia disease (CML) found effective therapy by treating patients with tyrosine kinase inhibitors (TKI), which suppress the BCR-ABL1 oncogene activity. However, the majority of patients achieving remission with TKI still have molecular evidences of disease persistence. Various mechanisms have been proposed to explain the disease persistence and recurrence. One of the hypotheses is that the primitive leukemic stem cells (LSCs) can survive in the presence of TKI. Understanding the mechanisms leading to TKI resistance of the LSCs in CML is a critical issue but is limited by availability of cells from patients. We generated induced pluripotent stem cells (iPSCs) derived from CD34⁺ blood cells isolated from CML patients (CML-iPSCs) as a model for studying LSCs survival in the presence of TKI and the mechanisms supporting TKI resistance. Interestingly, CML-iPSCs resisted to TKI treatment and their survival did not depend on BCR-ABL1, as for primitive LSCs. Induction of hematopoietic differentiation of CML-iPSC clones was reduced compared to normal clones. Hematopoietic progenitors obtained from iPSCs partially recovered TKI sensitivity. Notably, different CML-iPSCs obtained from the same CML patients were heterogeneous, in terms of BCR-ABL1 level and proliferation. Thus, several clones of CML-iPSCs are a powerful model to decipher all the mechanisms leading to LSC survival following TKI therapy and are a promising tool for testing new therapeutic agents.     

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.     

Leukemia stem cells: the root of chronic myeloid leukemia

Chronic myeloid leukemia (CML) is a clonal myeloproliferative disorder characterized by a chromosome translocation that generates the Bcr-Abl oncogene encoding a constitutive kinase activity. Despite remarkable success in controlling CML at chronic phase by Bcr-Abl tyrosine kinase inhibitors (TKIs), a significant proportion of CML patients treated with TKIs develop drug resistance due to the inability of TKIs to kill leukemia stem cells (LSCs) that are responsible for initiation, drug resistance, and relapse of CML. Therefore, there is an urgent need for more potent and safer therapies against leukemia stem cells for curing CML. A number of LSCassociated targets and corresponding signaling pathways, including CaMKII-γ, a critical molecular switch for co-activating multiple LSC-associated signaling pathways, have been identified over the past decades and various small inhibitors targeting LSC are also under development. Increasing evidence shows that leukemia stem cells are the root of CML and targeting LSC may offer a curable treatment option for CML patients. This review summarizes the molecular biology of LSC and itsassociated targets, and the potential clinical application in chronic myeloid leukemia.     

Curcumin potentiates the anti-leukemia effects of imatinib by downregulation of the AKT/mTOR pathway and BCR/ABL gene expression in Ph+ acute lymphoblastic leukemia

Philadelphia chromosome-positive acute lymphoblastic leukemia (Ph+ ALL) is triggered by BCR/ABL and SRC family tyrosine kinases. They interact with each other and subsequently activate downstream growth-signaling pathways, including Raf/MEK/ERK, Akt/mTOR, and STAT5 pathways. Although imatinib is the standard treatment for Ph+ leukemia, response rate of Ph+ ALL to imatinib is low, relapse is frequent and quick. Studies have documented the potential anti-tumor activities of curcumin. However, whether curcumin can be used in the therapy for Ph+ ALL remains obscure. Here, we reported that curcumin induced apoptosis by inhibition of AKT/mTOR and ABL/STAT5 signaling, down-regulation of BCR/ABL expression, and induction of the BCL2/BAX imbalance. Curcumin exerted synergetic anti-leukemia effects with imatinib by inhibition of the imatinib-mediated overactivation of AKT/mTOR signaling and down-regulation of BCR/ABL gene expression. In primary samples from Ph+ ALL patients, curcumin inhibited cellular proliferation and down-regulated constitutive activation of growth-signaling pathways not only in newly diagnosed patients but also in imatinib-resistant patients. In Ph+ ALL mouse models, curcumin exhibited synergetic anti-leukemia effects with imatinib. These results demonstrated that curcumin might be a promising agent for Ph+ ALL patients.     

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.     

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.     

How does the novel T315L mutation of breakpoint cluster region-abelson (BCR-ABL) kinase confer resistance to ponatinib: a comparative molecular dynamics simulation study

Abstract

Acute lymphocytic leukemia (ALL) is one of the most dangerous types of leukemia, and about 40% of them is Philadelphia chromosome-positive acute lymphocytic leukemia (Ph?+?ALL). Ph?+?ALL is caused by the fusion of the breakpoint cluster region (BCR) and the Ableson (ABL) genes, named the BCR-ABL fused gene that codes for an autonomously active tyrosine kinase. Tyrosine kinase inhibitors (TKIs) are among the first-line therapeutic agents for the treatment of Ph?+?ALL. Drug resistance are the major obstacle, limiting their clinical utility. The latest third-generation TKIs, ponatinib, can tackle most abnormal BCR-ABL kinases, including the T315I mutant that is resistant to first- and second-generations TKIs such as imatinib. However, drug resistance still emerges with the novel T315L mutation and the underlying mechanisms remain elusive. Here, using molecular dynamics (MD) simulations, we explored into the detailed interactions between ponatinib and BCR-ABL in the wild-type (WT), T315I, and T315L systems. The simulations revealed the significant conformational changes of ponatinib in its binding site due to the T315L mutation and the underlying structural mechanisms. Binding free energy analysis unveiled that the affinity of ponatinib to BCR-ABL decreased upon T315L mutation, which resulted in its unfavorable binding and drug resistance. Key residues responsible for the unfavored unbinding were also identified. This study elucidates the detailed mechanisms for the resistance of ponatinib in Ph?+?ALL triggered by the T315L mutation and will provide insights for future drug development and optimization.     

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).     

Regulatory Effects of Sestrin 3 (SESN3) in BCR-ABL Expressing Cells

Chronic myeloid leukemia (CML) and Ph+ acute lymphoblastic leukemia (ALL) are characterized by the presence of the BCR-ABL oncoprotein, which leads to activation of a plethora of pro-mitogenic and pro-survival pathways, including the mTOR signaling cascade. We provide evidence that in BCR-ABL expressing cells, treatment with tyrosine kinase inhibitors (TKIs) results in upregulation of mRNA levels and protein expression of sestrin3 (SESN3), a unique cellular inhibitor of mTOR complex 1 (mTORC1). Such upregulation appears to be mediated by regulatory effects on mTOR, as catalytic inhibition of the mTOR kinase also induces SESN3. Catalytic mTOR inhibition also results in upregulation of SESN3 expression in cells harboring the TKI-insensitive T315I-BCR-ABL mutant, which is resistant to imatinib mesylate. Overexpression of SESN3 results in inhibitory effects on different Ph+ leukemic cell lines including KT-1-derived leukemic precursors, indicating that SESN3 mediates anti-leukemic responses in Ph+ cells. Altogether, our findings suggest the existence of a novel mechanism for the generation of antileukemic responses in CML cells, involving upregulation of SESN3 expression.     

Imatinib and Dasatinib Inhibit Hemangiosarcoma and Implicate PDGFR-β and Src in Tumor Growth

Hemangiosarcoma, a natural model of human angiosarcoma, is an aggressive vascular tumor diagnosed commonly in dogs. The documented expression of several receptor tyrosine kinases (RTKs) by these tumors makes them attractive targets for therapeutic intervention using tyrosine kinase inhibitors (TKIs). However, we possess limited knowledge of the effects of TKIs on hemangiosarcoma as well as other soft tissue sarcomas. We report here on the use of the TKIs imatinib and dasatinib in canine hemangiosarcoma and their effects on platelet-derived growth factor receptor β (PDGFR-β) and Src inhibition. Both TKIs reduced cell viability, but dasatinib was markedly more potent in this regard, mediating cytotoxic effects orders of magnitude greater than imatinib. Dasatinib also inhibited the phosphorylation of the shared PDGFR-β target at a concentration approximately 1000 times less than that needed by imatinib and effectively blocked Src phosphorylation. Both inhibitors augmented the response to doxorubicin, suggesting that clinical responses likely will be improved using both drugs in combination; however, dasatinib was significantly (P < .05) more effective in this context. Despite the higher concentrations needed in cell-based assays, imatinib significantly inhibited tumor growth (P < .05) in a tumor xenograft model, highlighting that disruption of PDGFR-β/PDGF signaling may be important in targeting the angiogenic nature of these tumors. Treatment of a dog with spontaneously occurring hemangiosarcoma established that clinically achievable doses of dasatinib may be realized in dogs and provides a means to investigate the effect of TKIs on soft tissue sarcomas in a large animal model.     

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.     

BCL2 inhibitor ABT-199 and JNK inhibitor SP600125 exhibit synergistic cytotoxicity against imatinib-resistant Ph+ ALL cells

Imatinib (IMT), a specific tyrosine kinase inhibitor (TKI), has drastically changed the treatment strategy for Ph+ ALL (Philadelphia chromosome-positive acute lymphoblastic leukemia). However, TKI resistance remains a serious problem for patient prognosis. Here, a Ph+ ALL cell line NphA2 and the IMT-resistant subline NphA2/STIR were analyzed to identify a potential novel treatment strategy. We also examined other Ph+ ALL cells, MR87 and its IMT-resistant subline, MR87/STIR. IMT induced apoptosis of NphA2 and MR87 but had no effect on resistant sublines. Increased phosphorylated ERK and BCL2, but not BCL-XL, were observed in NphA2/STIR compared with NphA2. NphA2/STIR but not NphA2 was moderately sensitive to U0126, an ERK inhibitor. Interestingly, SP600125, a JNK inhibitor, was potent in cell growth inhibition and apoptosis induction of both parental and IMT-resistant NphA2 and MR87 cells. Moreover, NphA2 and MR87 and their IMT-resistant sublines were sensitive to ABT-199, a specific BCL2 inhibitor. The combination of SP600125 and ABT-199 synergistically suppressed both parental and IMT-resistant cells, including one with T315I mutation, suggesting that Ph+ ALL exhibits high sensitivity to ABT-199 and SP600125 regardless of TKI resistance. This combination might be a possible therapeutic strategy for Ph+ ALL in the future.     

Small Molecule ErbB Inhibitors Decrease Proliferative Signaling and Promote Apoptosis in Philadelphia Chromosome–Positive Acute Lymphoblastic Leukemia

The presence of the Philadelphia chromosome in patients with acute lymphoblastic leukemia (Ph⁺ALL) is a negative prognostic indicator. Tyrosine kinase inhibitors (TKI) that target BCR/ABL, such as imatinib, have improved treatment of Ph⁺ALL and are generally incorporated into induction regimens. This approach has improved clinical responses, but molecular remissions are seen in less than 50% of patients leaving few treatment options in the event of relapse. Thus, identification of additional targets for therapeutic intervention has potential to improve outcomes for Ph+ALL. The human epidermal growth factor receptor 2 (ErbB2) is expressed in ∼30% of B-ALLs, and numerous small molecule inhibitors are available to prevent its activation. We analyzed a cohort of 129 ALL patient samples using reverse phase protein array (RPPA) with ErbB2 and phospho-ErbB2 antibodies and found that activity of ErbB2 was elevated in 56% of Ph⁺ALL as compared to just 4.8% of Ph⁻ALL. In two human Ph+ALL cell lines, inhibition of ErbB kinase activity with canertinib resulted in a dose-dependent decrease in the phosphorylation of an ErbB kinase signaling target p70S6-kinase T389 (by 60% in Z119 and 39% in Z181 cells at 3 µM). Downstream, phosphorylation of S6-kinase was also diminished in both cell lines in a dose-dependent manner (by 91% in both cell lines at 3 µM). Canertinib treatment increased expression of the pro-apoptotic protein Bim by as much as 144% in Z119 cells and 49% in Z181 cells, and further produced caspase-3 activation and consequent apoptotic cell death. Both canertinib and the FDA-approved ErbB1/2-directed TKI lapatinib abrogated proliferation and increased sensitivity to BCR/ABL-directed TKIs at clinically relevant doses. Our results suggest that ErbB signaling is an additional molecular target in Ph⁺ALL and encourage the development of clinical strategies combining ErbB and BCR/ABL kinase inhibitors for this subset of ALL patients.     

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.