A BCR-ABL Mutant Lacking Direct Binding Sites for the GRB2, CBL and CRKL Adapter Proteins Fails to Induce Leukemia in Mice

The BCR-ABL tyrosine kinase is the defining feature of chronic myeloid leukemia (CML) and its kinase activity is required for induction of this disease. Current thinking holds that BCR-ABL forms a multi-protein complex that incorporates several substrates and adaptor proteins and is stabilized by multiple direct and indirect interactions. Signaling output from this highly redundant network leads to cellular transformation. Proteins known to be associated with BCR-ABL in this complex include: GRB2, c-CBL, p62^DOK, and CRKL. These proteins in turn, link BCR-ABL to various signaling pathways indicated in cellular transformation. In this study we show that a triple mutant of BCR-ABL with mutations of the direct binding sites for GRB2, CBL, p62^DOK and CRKL, is defective for transformation of primary hematopoietic cells in vitro and in a murine CML model, while it retains the capacity to induce IL-3 independence in 32D cells. Compared to BCR-ABL, the triple mutant''s ability to activate the MAP kinase and PI3-kinase pathways is severely compromised, while STAT5 phosphorylation is maintained, suggesting that the former are crucial for the transformation of primary cells, but dispensable for transformation of factor dependent cell lines. Our data suggest that inhibition of BCR-ABL-induced leukemia by disrupting protein interactions could be possible, but would require blocking of multiple sites.     

TAT-CC fusion protein depresses the oncogenicity of BCR-ABL in vitro and in vivo through interrupting its oligomerization

Chronic myeloid leukemia (CML) is a clonal hematologic malignancy characterized by the BCR-ABL protein. BCR-ABL is a constitutively active tyrosine kinase and plays a critical role in the pathogenesis of CML. Imatinib mesylate, a selective tyrosine kinase inhibitor, is effective in CML, but drug resistance and relapse occur. The coiled-coil (CC) domain located in BCR_1–72 mediates BCR-ABL tetramerization, which is essential for the activation of tyrosine kinase and transformation potential of BCR-ABL. CC domain is supposed to be a therapeutic target for CML. We purified a TAT-CC protein competively binding with the endogenous CC domain to reduce BCR-ABL kinase activity. We found that TAT-CC co-located and interacted with BCR-ABL in Ba/F3-p210 and K562 cells. It induced apoptosis and inhibited proliferation in these cells. It increased the sensitivity of these cells to imatinib and reduced the phosphorylation of BCR-ABL, CRKL and STAT5. We confirmed that TAT-CC could attenuate the oncogenicity of Ba/F3-p210 cells and diminish the volume of K562 solid tumor in mice. We conclude targeting the CC may provide a complementary therapy to inhibit BCR-ABL oncogenicity.     

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.     

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     

An overview of chronic myeloid leukemia and its animal models

Chronic myeloid leukemia(CML) is a form of leukemia characterized by the presence of clonal bone marrow stem cells with the proliferation of mature granulocytes(neutrophils, eosinophils, and basophils) and their precursors. CML is a type of myeloproliferative disease associated with a characteristic chromosomal translocation called the Philadelphia(Ph) chromosome or t(9;22) translocation(BCR-ABL). CML is now usually treated with targeted drugs called tyrosine kinase inhibitors(TKIs). The mechanism and natural history of CML is still unclear. Here, we summarize the present CML animal disease models and compare them with each other. Meanwhile, we propose that it is a very wise choice to establish zebrafish(Danio rerio) CML model mimics clinical CML. This model could be used to learn more about the mechanism of CML, and to aid in the development of new drugs to treat CML.     

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.     

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.     

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.     

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.     

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.     

Dihydroorotate dehydrogenase inhibition reveals metabolic vulnerability in chronic myeloid leukemia

The development of different generations of BCR-ABL1 tyrosine kinase inhibitors (TKIs) has led to the high overall survival of chronic myeloid leukemia (CML) patients. However, there are CML patients who show resistance to TKI therapy and are prone to progress to more advanced phases of the disease. So, implementing an alternative approach for targeting TKIs insensitive cells would be of the essence. Dihydroorotate dehydrogenase (DHODH) is an enzyme in the de novo pyrimidine biosynthesis pathway that is located in the inner membrane of mitochondria. Here, we found that CML cells are vulnerable to DHODH inhibition mediated by Meds433, a new and potent DHODH inhibitor recently developed by our group. Meds433 significantly activates the apoptotic pathway and leads to the reduction of amino acids and induction of huge metabolic stress in CML CD34+ cells. Altogether, our study shows that DHODH inhibition is a promising approach for targeting CML stem/progenitor cells and may help more patients discontinue the therapy.Subject terms: Cancer metabolism, Apoptosis