Pushing the limits of targeted therapy in chronic myeloid leukaemia

Tyrosine kinase inhibitor (TKI) therapy targeting the BCR-ABL1 kinase is effective against chronic myeloid leukaemia (CML), but is not curative for most patients. Minimal residual disease (MRD) is thought to reside in TKI-insensitive leukaemia stem cells (LSCs) that are not fully addicted to BCR-ABL1. Recent conceptual advances in both CML biology and therapeutic intervention have increased the potential for the elimination of CML cells, including LSCs, through simultaneous inhibition of BCR-ABL1 and other newly identified, crucial targets.     

Chronic myeloid leukemia stem cells in the era of targeted therapies: resistance, persistence and long-term dormancy

Targeted therapies of chronic myeloid leukemia (CML) using tyrosine kinase inhibitors (TKI) have profoundly changed the natural history of the disease with a major impact on survival. Molecular monitoring with BCR-ABL quantification shows that a status of undetectable molecular residual disease (UMRD) is obtained in a significant minority of patients. However, it remains unclear whether these patients are definitively cured of their leukemia. Imatinib mesylate withdrawal trials have demonstrated the rapid appearance of the malignant clone in the majority of the patients whereas some patients remain in a state of UMRD. It has clearly been demonstrated that the most primitive stem cells are refractory to all TKIs used in clinical practice. In addition, long-term dormancy is one of the most fundamental characteristics of hematopoietic stem cells. In this context, we have recently undertaken a systematic analysis of the bone marrow stem cell compartment in several patients in durable UMRD. We have demonstrated the long-term persistence of a considerable amount of BCR-ABL-expressing stem cells, even in the absence of relapse. The phenomenon of long-term leukemic stem cell dormancy is of major importance in CML and one of the key questions in cancer biology in general. We discuss, here, the potential mechanisms, including intrinsic and microenvironmental factors, that control the response of leukemic stem cells (LSCs) to targeted therapies and potential novel strategies currently in progress with a curative intent. Moreover, we propose a molecular evaluation of the residual LSC compartment in selected patients in order to develop rational TKI-cessation strategies in CML.     

Wnt信号通路与慢性粒细胞白血病

 慢性粒细胞白血病作为血液系统的恶性增殖性肿瘤，其主要发病机制为BCR-ABL1融合基因形成，因此针对BCR-ABL1基因的靶向药物TKI为其治疗带来了希望，但TKI无法完全消除CML患者体内的LSCs，CML患者容易出现TKI药物耐药及治疗后复发。随着Wnt信号通路在实体瘤中的应用，大量研究发现Wnt信号通路同样在CML中起到重要作用，我们就Wnt信号通路对CML的影响作一综述。     

IL-6 controls leukemic multipotent progenitor cell fate and contributes to chronic myelogenous leukemia development

Using a mouse model recapitulating the main features of human chronic myelogenous leukemia (CML), we uncover the hierarchy of leukemic stem and progenitor cells contributing to disease pathogenesis. We refine the characterization of CML leukemic stem cells (LSCs) to the most immature long-term hematopoietic stem cells (LT-HSCs) and identify some important molecular deregulations underlying their aberrant behavior. We find that CML multipotent progenitors (MPPs) exhibit an aberrant B-lymphoid potential but are redirected toward the myeloid lineage by the action of the proinflammatory cytokine IL-6. We show that BCR/ABL activity controls Il-6 expression thereby establishing a paracrine feedback loop that sustains CML development. These results describe how proinflammatory tumor environment affects leukemic progenitor cell fate and contributes to CML pathogenesis.     

Novel combination treatments targeting chronic myeloid leukemia stem cells

Chronic myeloid leukemia (CML) is currently considered incurable in most patients. Stem cell transplantation, an accepted curative option for which extensive experience has been gained, is limited by high morbidity and mortality rates, particularly in older patients. Tyrosine kinase inhibitors targeting BCR-ABL are widely used and induce remission in a high proportion of patients, but resistance and incomplete response to these agents portends eventual relapse and disease progression. Although BCR-ABL inhibitors eradicate most CML cells, they are largely ineffective against the reservoir of quiescent leukemic stem cells (LSCs). Thus a strong medical need exists for therapies that effectively eradicate LSCs and is currently a focus of extensive research. To date, evidence obtained from in vitro studies, animal models, and clinical CML specimens suggests that an effective approach may be to partner existing BCR-ABL inhibitors with compounds targeting key stem cell molecular effectors, including Wnt/β-catenin, hedgehog pathway components, histone deacetylase (HDAC), transforming growth factor-β (TGF-β), Janus kinase 2, promyelocytic leukemia protein, and arachidonate 5-lipoxygenase (ALOX5). Novel combinations may sensitize LSCs to BCR-ABL inhibitors, thereby overcoming resistance and creating the possibility of improving disease outcome beyond the current standard of care.     

Growth autonomy and lineage switching in BCR-ABL-transduced human cord blood cells depend on different functional domains of BCR-ABL.

The tyrosine kinase activity of p210BCR-ABL is essential to its leukemogenic potential, but the role of other functional domains in primary human hematopoietic cells has not been previously investigated. Here we show that infection of normal human CD34+ cord blood (CB) cells with a retroviral vector encoding p210BCR-ABL rapidly activates a factor-independent phenotype and autocrine interleukin-3/granulocyte colony-stimulating factor/erythropoietin production in the transduced cells. These changes are characteristic of primitive chronic myeloid leukemic (CML) cells and are important to the leukemogenicity of BCR-ABL-transduced murine hematopoietic stem cells. When BCR-ABL-transduced human CB cells were incubated with imatinib mesylate, an inhibitor of the p210BCR-ABL kinase, or when human CB cells were transduced with a BCR-ABL cDNA lacking the SH2 domain (p210DeltaSH2), factor independence was significantly reduced. In contrast, deletion of the SH2 domain had little impact on the p210BCR-ABL kinase-dependent promotion of erythropoietic differentiation also seen immediately following the BCR-ABL transduction of primitive human CB cells, but not in naturally occurring CML. Thus, p210BCR-ABL has distinct biological effects in primary human hematopoietic cells, which variably mimic features of human CML, and activation of these changes can show different dependencies on the integrity of the SH1 and SH2 domains of p210BCR-ABL.     

Imatinib mesylate and nilotinib (AMN107) exhibit high-affinity interaction with ABCG2 on primitive hematopoietic stem cells.

The majority of chronic phase chronic myeloid leukemia (CML) patients treated with the tyrosine kinase inhibitor (TKI) imatinib mesylate maintain durable responses to the drug. However, most patients relapse after withdrawal of imatinib and advanced stage patients often develop drug resistance. As CML is considered a hematopoietic stem cell cancer, it has been postulated that inherent protective mechanisms lead to relapse in patients. The ATP binding-cassette transporters ABCB1 (MDR-1; P-glycoprotein) and ABCG2 are highly expressed on primitive hematopoietic stem cells (HSCs) and have been shown to interact with TKIs. Herein we demonstrate a dose-dependent, reversible inhibition of ABCG2-mediated Hoechst 33342 dye efflux in primary human and murine HSC by both imatinib and nilotinib (AMN107), a novel aminopyrimidine inhibitor of BCR-ABL. ABCG2-transduced K562 cells were protected from imatinib and nilotinib-mediated cell death and from downregulation of P-CRKL. Moreover, photoaffinity labeling revealed interaction of both TKIs with ABCG2 at the substrate binding sites as they compete with the binding of [(125)I] IAAP and also stimulate the transporter's ATPase activity. Therefore, our evidence suggests for the role of ABC transporters in resistance to TKI on primitive HSCs and CML stem cells and provides a rationale how TKI resistance can be overcome in vivo.     

Role of Pten in leukemia stem cells

Chronic myeloid leukemia (CML) is initiated from the BCR-ABL-expressing leukemia stem cells (LSCs). These LSCs are highly resistant to BCR-ABL kinase inhibitors, imatinib, dasantinib and nilotinib, and methods for eradication of LSCs are still not available. It is critical to identify genes that play roles in survival and proliferation of LSCs. We recently discovered that the tumor suppressor gene Pten is downregulated in LSCs of CML mice. By genetic deletion or overexpression of Pten, we confirmed that Pten functions as a tumor suppressor in LSCs of CML, consistent with the role of Pten in LSCs of acute myeloid leukemia (AML) and progenitor cells of T-ALL progenitors. Functional enhancement of the Pten pathway provides a therapeutic strategy for targeting LSCs.     

Role of SPA-1 in phenotypes of chronic myelogenous leukemia induced by BCR-ABL-expressing hematopoietic progenitors in a mouse model

SPA-1 is a negative regulator of Rap1 signal in hematopoietic cells, and SPA-1-deficient mice develop myeloproliferative disorders (MPD) of long latency. In the present study, we showed that the MPDs in SPA-1(-/-) mice were associated with the increased hematopoietic stem cells expressing LFA-1 in bone marrow and their premature mobilization to spleen with extensive extramedullary hematopoiesis, resembling human chronic myelogenous leukemia (CML). We further showed that human BCR-ABL oncogene caused a partial down-regulation of endogenous SPA-1 gene expression in mouse hematopoietic progenitor cells (HPC) and immature hematopoietic cell lines. Although both BCR-ABL-transduced wild-type (wt) and SPA-1(-/-) HPC rapidly developed CML-like MPD when transferred to severe combined immunodeficient mice, the latter recipients showed significantly increased proportions of BCR-ABL(+) Lin(-) c-Kit(+) cells compared with the former ones. Serial transfer experiments revealed that spleen cells of secondary recipients of BCR-ABL(+) wt HPC failed to transfer MPD to tertiary recipients due to a progressive reduction of BCR-ABL(+) Lin(-) c-Kit(+) cells. In contrast, SPA-1(-/-) BCR-ABL(+) Lin(-) c-Kit(+) cells were sustained at high level in secondary recipients, and their spleen cells could transfer MPD to tertiary recipients, a part of which rapidly developed blast crisis. Present results suggest that endogenous SPA-1 plays a significant role in regulating expansion and/or survival of BCR-ABL(+) leukemic progenitors albeit partial repression by BCR-ABL and that Rap1 signal may represent a new molecular target for controlling leukemic progenitors in CML.     

细胞因子在慢性髓系白血病中的研究进展

慢性髓系白血病（chronic myeloid leukemia CML）是一类起源于造血干细胞的恶性克隆性疾病,酪氨酸激酶抑制剂(TKI)对CML慢性期有很好的治疗效果,但治疗过程中会出现耐药、发生急变。CML急变和TKI的耐药是目前影响该病治疗和预后的主要障碍。近年来研究发现细胞因子网络紊乱及其活动与CML是相关的,造血微环境中许多造血和趋化相关的细胞因子在CML中异常表达。细胞因子在CML的进展和耐药中起到重要作用。本文将对细胞因子在CML进展机制中的作用,细胞因子与临床耐药的相关性及当前针对细胞因子的新的治疗方法等方面作一综述。     

Essential role for telomerase in chronic myeloid leukemia induced by BCR-ABL in mice

The telomerase protein is constitutively activated in malignant cells from many patients with cancer, including the chronic myeloid leukemia (CML), but whether telomerase is essential for the pathogenesis of this disease is not known. Here, we used telomerase deficient mice to determine the requirement for telomerase in CML induced by BCR-ABL in mouse models of CML. Loss of one telomerase allele or complete deletion of telomerase prevented the development of leukemia induced by BCR-ABL. However, BCR-ABL was expressed and active in telomerase heterozygous and null leukemic hematopoietic stem cells. These results demonstrate that telomerase is essential for oncogene-induced reprogramming of hematopoietic stem cells in CML development and validate telomerase and the genes it regulates as targets for therapy in CML.     

Potentiation of Nilotinib-mediated cell death in the context of the bone marrow microenvironment requires a promiscuous JAK inhibitor in CML

In this study, we show that conditioned media (CM) generated from bone marrow (BM)-derived mesenchymal stromal cells lead to BCR-ABL independent STAT3 activation. Activation of STAT3 is important not only for survival of CML cells but also for its protection against Nilotinib (NI), within the BM microenvironment. Reducing the expression of both JAK2 and TYK2 or utilizing a pan-JAK inhibitor blocked CM-mediated STAT3 activation and sensitized CML cells to NI-mediated cell death. Finally, we demonstrate that in patient-derived primitive leukemic cells, co-cultured with BM stromal cells, inhibition of BCR-ABL and JAK activity was a successful strategy to potentiate their elimination.     

Getting to the stem of chronic myeloid leukaemia

Tyrosine kinase inhibitor (TKI) therapy for chronic myeloid leukaemia (CML) is the consummate success story for targeted therapy, yet relapse is a nearly inevitable consequence of cessation or interruption of therapy. Primitive TKI-refractory CML stem cells are the likely source of these relapses, as they provide sanctuary for the Philadelphia chromosome. In advanced disease, their progressively anaplastic progeny ultimately maintain CML independently of the CML haematopoietic stem cell (HSC). Interestingly, there are at least two distinct cell types capable of self-renewal in different phases of CML: first, a primitive HSC with BCR-ABL mutation, which maintains the more indolent chronic-phase disease and, second, a coexisting mutated progenitor cell which acquires stem cell characteristics responsible for rapid cell expansion in advanced disease.     

Imatinib (ST1571) provides only limited selectivity for CML cells and treatment might be complicated by silent BCR-ABL genes

Very promising results have been obtained in clinical trials on chronic-phase chronic myeloid leukemia (CP-CML) patients treated with imatinib mesylate (IM; Gleevecr, STI571), a BCR-ABL tyrosine kinase inhibitor. However, we found that IM caused considerable inhibition of normal hematopoietic progenitor cells upon treating control bone marrow (BM) cultures. In vitro IM treatment gave a decrease in the yield and size of colonies from BM of untreated CP-CML patients that was only two to three times that from the normal samples. Moreover, about 30% of myeloid progenitors (CFU-GM) from CML BM still formed colonies in the presence of IM, most of which had BCR-ABL RNA. About half of these treated colonies also displayed methylation of the internal ABL Pa promoter, a CML-specific epigenetic alteration, which was used in this study as a marker for BCR-ABL translocation-containing cells. However, ~5-8% of the treated or the untreated CML BM-derived colonies had no detectable BCR-ABL RNA by two or three rounds of RT-PCR despite being positive for the internal standard RNA and displaying hallmarks of CML, either t(9;22)(q34;ql 1) or ABL Pa methylation. Our results indicate that IM is only partially specific for CML progenitor cells compared to normal hematopoietic progenitor cells and suggest that some CML cells may have a silent BCR-ABL oncogene that could interfere with therapy.     

HOXA10 expression induced by Abl kinase inhibitors enhanced apoptosis through PI3K pathway in CML cells

Chronic myelogenous leukemia is characterized by the reciprocal chromosomal translocation (9;22), which generates a novel fusion gene, BCR-ABL. Bcr-Abl-expressing leukemia cells are highly resistant to apoptosis. Imatinib an Abl kinase inhibitor, is a highly effective agent for patients with CML. However, a small percentage of these patients and most advanced-phase patients relapse on imatinib therapy. It is poorly understood whether the Abl kinase inhibitors are able to eradicate CML progenitor or stem cells. In this study, we investigated the role of HOXA10 in CML cell lines and the hematopoietic progenitor cells derived from CML patients, and whether the regulation of HOXA10 eradicates Bcr-Abl(+) hematopoietic stem/progenitor cells. The Abl kinase inhibitors and PI3K inhibitor, LY294002, induced the expression of HOXA10, and it enhanced apoptosis in CML cells. Moreover, the reduction of HOXA10 expression by siRNA in CML cells inhibited apoptosis by treatment with the Abl kinase inhibitors and LY294002. These results revealed that HOXA10 had an important role in induction of apoptosis by the Abl kinase inhibitors in CML cells. Finally, we showed that the inhibition of HOXA10 expression by siRNA increased the numbers of CFU-GEMM, BFU-E, and CFU-GM when the cells were treated with the combination of BMS354825 and LY294002 compared to control cells, and HOXA10 played a critical role in the committed colony-formation in CML. This study shows for the first time that the Abl kinase inhibitor and LY294002 induced HOXA10, and HOXA10 had an important role in apoptosis or cell growth inhibition in CML cells in vitro.     

The PIM inhibitor AZD1208 synergizes with ruxolitinib to induce apoptosis of ruxolitinib sensitive and resistant JAK2-V617F-driven cells and inhibit colony formation of primary MPN cells

Classical myeloproliferative neoplasms (MPNs) are hematopoietic stem cell disorders that exhibit excess mature myeloid cells, bone marrow fibrosis, and risk of leukemic transformation. Aberrant JAK2 signaling plays an etiological role in MPN formation. Because neoplastic cells in patients are largely insensitive to current anti-JAK2 therapies, effective therapies remain needed. Members of the PIM family of serine/threonine kinases are induced by JAK/STAT signaling, regulate hematopoietic stem cell growth, protect hematopoietic cells from apoptosis, and exhibit hematopoietic cell transforming properties. We hypothesized that PIM kinases may offer a therapeutic target for MPNs. We treated JAK2-V617F-dependent MPN model cells as well as primary MPN patient cells with the PIM kinase inhibitors SGI-1776 and AZD1208 and the JAK2 inhibitor ruxolitinib. While MPN model cells were rather insensitive to PIM inhibitors, combination of PIM inhibitors with ruxolitinib led to a synergistic effect on MPN cell growth due to enhanced apoptosis. Importantly, PIM inhibitor mono-therapy inhibited, and AZD1208/ruxolitinib combination therapy synergistically suppressed, colony formation of primary MPN cells. Enhanced apoptosis by combination therapy was associated with activation of BAD, inhibition of downstream components of the mTOR pathway, including p70S6K and S6 protein, and activation of 4EBP1. Importantly, PIM inhibitors re-sensitized ruxolitinib-resistant MPN cells to ruxolitinib by inducing apoptosis. Finally, exogenous expression of PIM1 induced ruxolitinib resistance in MPN model cells. These data indicate that PIMs may play a role in MPNs and that combining PIM and JAK2 kinase inhibitors may offer a more efficacious therapeutic approach for MPNs over JAK2 inhibitor mono-therapy.     

Maintaining Low BCR-ABL Signaling Output to Restrict CML Progression and Enable Persistence

Deregulated BCR-ABL oncogenic activity leads to transformation, oncogene addiction and drives disease progression in chronic myeloid leukemia (CML). Inhibition of BCR-ABL using Abl-specific kinase inhibitors (TKI) such as imatinib induces remarkable clinical responses. However, approximately only less than 15 % of all chronic-phase CML patients will remain relapse-free after discontinuation of imatinib in deep molecular remission. It is not well understood why persisting CML cells survive under TKI therapy without developing clonal evolution and frank TKI resistance. BCR-ABL expression level may be critically involved. Whereas higher BCR-ABL expression has been described as a pre-requisite for malignant CML stem cell transformation and CML progression to blast crisis, recent evidence suggests that during persistence TKI select for CML precursors with low BCR-ABL expression. Genetic, translational and clinical evidence is discussed to suggest that TKI-induced maintenance of low BCR-ABL signaling output may be potently tumor suppressive, because it abrogates oncogenic addiction.     

BCR-ABL alters the proliferation and differentiation response of multipotent hematopoietic cells to stem cell factor

Chronic myeloid leukaemia (CML), a hematopoietic stem cell disorder is characterized by the expression of BCR-ABL. To investigate the effects of BCR-ABL on multipotent hematopoietic cells, a temperature sensitive BCR-ABL tyrosine kinase was expressed in the cell line, FDCP-Mix. BCR-ABL mediated an increase in c-kit expression that correlated with an enhanced mitogenic response to SCF. This was not observed in the absence of Bcr-Abl kinase activity or presence of the BCR-ABL inhibitor STI571, which also inhibits c-kit. When cultured in a combination of SCF plus G-CSF the FDCP-Mix cells undergo neutrophilic differentiation over a 7-10 day period. When BCR-ABL was active there was a marked inhibition of cell maturation compared to control cells in which BCR-ABL was either inactive or not present. However, BCR-ABL did not block differentiation as the cells eventually undergo terminal maturation. These data argue that BCR-ABL is directly responsible for the enhanced response to SCF reported in CML progenitor cells. Furthermore, although the primary effect of STI571 is via direct inhibition of BCR-ABL, STI571 additionally reduces the enhanced response to SCF. Thus there are two sites of STI571 action of potential importance in Bcr-Abl expressing cells.