Chronic myeloid leukaemia as a model of disease evolution in human cancer.

Chronic myeloid leukaemia (CML) can be considered as a paradigm for neoplasias that evolve through a multi-step process. CML is also one of the best examples of a disease that can be targeted by molecular therapy; however, the success of new 'designer drugs' is largely restricted to the chronic phase of the disease. If not cured at this stage, CML invariably progresses and transforms into an acute-type leukaemia undergoing a 'blast crisis'. The causes of this transformation are still poorly understood. What mechanisms underlie this progression, and are they shared by other common cancers?

Safety and efficacy of imatinib cessation for CML patients with stable undetectable minimal residual disease: results from the TWISTER study.

Most patients with chronic myeloid leukemia (CML) treated with imatinib will relapse if treatment is withdrawn. We conducted a prospective clinical trial of imatinib withdrawal in 40 chronic-phase CML patients who had sustained undetectable minimal residual disease (UMRD) by conventional quantitative polymerase chain reaction (PCR) on imatinib for at least 2 years. Patients stopped imatinib and were monitored frequently for molecular relapse. At 24 months, the actuarial estimate of stable treatment-free remission was 47.1%. Most relapses occurred within 4 months of stopping imatinib, and no relapses beyond 27 months were seen. In the 21 patients treated with interferon before imatinib, a shorter duration of interferon treatment before imatinib was significantly associated with relapse risk, as was slower achievement of UMRD after switching to imatinib. Highly sensitive patient-specific BCR-ABL DNA PCR showed persistence of the original CML clone in all patients with stable UMRD, even several years after imatinib withdrawal. No patients with molecular relapse after discontinuation have progressed or developed BCR-ABL mutations (median follow-up, 42 months). All patients who relapsed remained sensitive to imatinib re-treatment. These results confirm the safety and efficacy of a trial of imatinib withdrawal in stable UMRD with frequent, sensitive molecular monitoring and early rescue of molecular relapse.

Natural course and biology of CML.

Chronic myeloid leukaemia (CML) is a myeloproliferative disorder arising in the haemopoietic stem cell (HSC) compartment. This disease is characterised by a reciprocal t(9;22) chromosomal translocation, resulting in the formation of the Philadelphia (Ph) chromosome containing the BCR-ABL1 gene. As such, diagnosis and monitoring of disease involves detection of BCR-ABL1. It is the BCR-ABL1 protein, in particular its constitutively active tyrosine kinase activity, that forges the pathogenesis of CML. This aberrant kinase signalling activates downstream targets that reprogram the cell to cause uncontrolled proliferation and results in myeloid hyperplasia and 'indolent' symptoms of chronic phase (CP) CML. Without successful intervention, the disease will progress into blast crisis (BC), resembling an acute leukaemia. This advanced disease stage takes on an aggressive phenotype and is almost always fatal. The cell biology of CML is also centred on BCR-ABL1. The presence of BCR-ABL1 can explain virtually all the cellular features of the leukaemia (enhanced cell growth, inhibition of apoptosis, altered cell adhesion, growth factor independence, impaired genomic surveillance and differentiation). This article provides an overview of the clinical and cell biology of CML, and highlights key findings and unanswered questions essential for understanding this disease.

SHP-1 expression accounts for resistance to imatinib treatment in Philadelphia chromosome-positive cells derived from patients with chronic myeloid leukemia.

We prove that the SH2-containing tyrosine phosphatase 1 (SHP-1) plays a prominent role as resistance determinant of imatinib (IMA) treatment response in chronic myelogenous leukemia cell lines (sensitive/KCL22-S and resistant/KCL22-R). Indeed, SHP-1 expression is significantly lower in resistant than in sensitive cell line, in which coimmunoprecipitation analysis shows the interaction between SHP-1 and a second tyrosine phosphatase SHP-2, a positive regulator of RAS/MAPK pathway. In KCL22-R SHP-1 ectopic expression restores both SHP-1/SHP-2 interaction and IMA responsiveness; it also decreases SHP-2 activity after IMA treatment. Consistently, SHP-2 knocking-down in KCL22-R reduces either STAT3 activation or cell viability after IMA exposure. Therefore, our data suggest that SHP-1 plays an important role in BCR-ABL-independent IMA resistance modulating the activation signals that SHP-2 receives from both BCR/ABL and membrane receptor tyrosine kinases. The role of SHP-1 as a determinant of IMA sensitivity has been further confirmed in 60 consecutive untreated patients with chronic myelogenous leukemia, whose SHP-1 mRNA levels were significantly lower in case of IMA treatment failure (P < .0001). In conclusion, we suggest that SHP-1 could be a new biologic indicator at baseline of IMA sensitivity in patients with chronic myelogenous leukemia.

BCR-ABL1 tyrosine kinase sustained MECOM expression in chronic myeloid leukaemia.

MECOM oncogene expression correlates with chronic myeloid leukaemia (CML) progression. Here we show that the knockdown of MECOM (E) and MECOM (ME) isoforms reduces cell division at low cell density, inhibits colony-forming cells by 34% and moderately reduces BCR-ABL1 mRNA and protein expression but not tyrosine kinase catalytic activity in K562 cells. We also show that both E and ME are expressed in CD34(+) selected cells of both CML chronic phase (CML-CP), and non-CML (normal) origin. Furthermore, MECOM mRNA and protein expression were repressed by imatinib mesylate treatment of CML-CP CD34(+) cells, K562 and KY01 cell lines whereas imatinib had no effect in non-CML BCR-ABL1 -ve CD34(+) cells. Together these results suggest that BCR-ABL1 tyrosine kinase catalytic activity regulates MECOM gene expression in CML-CP progenitor cells and that the BCR-ABL1 oncoprotein partially mediates its biological activity through MECOM. MECOM gene expression in CML-CP progenitor cells would provide an in vivo selective advantage, contributing to CML pathogenesis.

Wrapping BCR-ABL: it's in the bag.

Leukemia, with its origin in a specific genetic abnormality, will only arise if the cell properly folds and processes the oncogenic protein encoded by the mutant gene. In this issue of Blood, Tsukahara and Maru describe a set of proteins that control the processing of the nascent BCR-ABL oncoprotein, providing new avenues for potential therapeutic intervention in chronic myelogenous leukemia (CML).

In search of the original leukemic clone in chronic myeloid leukemia patients in complete molecular remission after stem cell transplantation or imatinib.

It is not clear if absence of BCR-ABL transcripts--complete molecular response (CMR)--is synonymous with, or required for, cure of chronic myeloid leukemia (CML). Some patients achieve CMR with imatinib (IM), but most relapse shortly after treatment discontinuation. Furthermore, most patients in long-term remission (LTR) post-stem cell transplantation (SCT) are considered functionally cured, although some remain occasionally positive for low-level BCR-ABL mRNA. Interpretation of the latter is complicated because it has been observed in healthy subjects. We designed a patient-specific, highly sensitive, DNA quantitative polymerase chain reaction to test follow-up samples for the original leukemic clone, identified by its unique genomic BCR-ABL fusion (gBCR-ABL). In 5 IM-treated patients in CMR, gBCR-ABL was detected in transcript-negative samples; 4 patients became gBCR-ABL-negative with continuing IM therapy. In contrast, of 9 patients in LTR (13-27 years) post-SCT, gBCR-ABL was detected in only 1, despite occasional transcript-positive samples in 8 of them. In conclusion, in IM-treated patients, absence of transcripts should not be interpreted as absence of the leukemic clone, although continuing IM after achievement of CMR may lead to further reduction of residual disease. Post-SCT, we found little evidence that the transcripts occasionally detected originate from the leukemic clone.

A gene expression signature of CD34+ cells to predict major cytogenetic response in chronic-phase chronic myeloid leukemia patients treated with imatinib.

In chronic-phase chronic myeloid leukemia (CML) patients, the lack of a major cytogenetic response (< 36% Ph(+) metaphases) to imatinib within 12 months indicates failure and mandates a change of therapy. To identify biomarkers predictive of imatinib failure, we performed gene expression array profiling of CD34(+) cells from 2 independent cohorts of imatinib-naive chronic-phase CML patients. The learning set consisted of retrospectively selected patients with a complete cytogenetic response or more than 65% Ph(+) metaphases within 12 months of imatinib therapy. Based on analysis of variance P less than .1 and fold difference 1.5 or more, we identified 885 probe sets with differential expression between responders and nonresponders, from which we extracted a 75-probe set minimal signature (classifier) that separated the 2 groups. On application to a prospectively accrued validation set, the classifier correctly predicted 88% of responders and 83% of nonresponders. Bioinformatics analysis and comparison with published studies revealed overlap of classifier genes with CML progression signatures and implicated beta-catenin in their regulation, suggesting that chronic-phase CML patients destined to fail imatinib have more advanced disease than evident by morphologic criteria. Our classifier may allow directing more aggressive therapy upfront to the patients most likely to benefit while sparing good-risk patients from unnecessary toxicity.

Discovery of a small-molecule type II inhibitor of wild-type and gatekeeper mutants of BCR-ABL, PDGFRalpha, Kit, and Src kinases: novel type II inhibitor of gatekeeper mutants.

Many clinically validated kinases, such as BCR-ABL, c-Kit, PDGFR, and EGFR, become resistant to adenosine triphosphate-competitive inhibitors through mutation of the so-called gatekeeper amino acid from a threonine to a large hydrophobic amino acid, such as an isoleucine or methionine. We have developed a new class of adenosine triphosphate competitive inhibitors, exemplified by HG-7-85-01, which is capable of inhibiting T315I- BCR-ABL (clinically observed in chronic myeloid leukemia), T670I-c-Kit (clinically observed in gastrointestinal stromal tumors), and T674I/M-PDGFRalpha (clinically observed in hypereosinophilic syndrome). HG-7-85-01 is unique among all currently reported kinase inhibitors in having the ability to accommodate either a gatekeeper threonine, present in the wild-type forms of these kinases, or a large hydrophobic amino acid without becoming a promiscuous kinase inhibitor. The distinctive ability of HG-7-85-01 to simultaneously inhibit both wild-type and mutant forms of several kinases of clinical relevance is an important step in the development of the next generation of tyrosine kinase inhibitors.

Gaining insights into the Bcr-Abl activity-independent mechanisms of resistance to imatinib mesylate in KCL22 cells: a comparative proteomic approach.

Imatinib mesylate is a potent inhibitor of Bcr-Abl tyrosine kinase, an oncoprotein that plays a key role in the development of chronic myeloid leukemia. Consequently, imatinib is used as front-line therapy for this disease. A major concern in imatinib treatment is the emergence of resistance to the drug. Here we used the imatinib-resistant KCL22R and imatinib-sensitive KCL22S cells in which none of the known resistance mechanisms has been detected and hence novel Bcr-Abl activity-independent mechanisms could be envisaged. We characterized proteins that were differentially expressed between the KCL22R and KCL22S cells. Using two-dimensional differential gel electrophoresis coupled with mass spectrometry and Western blot analysis we identified 51 differentially expressed proteins: 27 were over-expressed and 24 were under-expressed in KCL22R versus KCL22S cells. Several of these proteins are likely to be involved in such survival mechanisms as modulation of redox balance and activation of anti-apoptotic pathways mediated by NF-kappaB and Ras-MAPK signaling. The data reported may be useful for further studies on mechanisms of imatinib resistance and for the screening of biomarkers to develop new combinatorial therapeutic approaches.

Selecting optimal second-line tyrosine kinase inhibitor therapy for chronic myeloid leukemia patients after imatinib failure: does the BCR-ABL mutation status really matter?

Preclinical studies of BCR-ABL mutation sensitivity to nilotinib or dasatinib suggested that the majority would be sensitive. Correspondingly, the initial clinical trials demonstrated similar response rates for CML patients after imatinib failure, irrespective of the mutation status. However, on closer examination, clinical evidence now indicates that some mutations are less sensitive to nilotinib (Y253H, E255K/V, and F359V/C) or dasatinib (F317L and V299L). T315I is insensitive to both. Novel mutations (F317I/V/C and T315A) are less sensitive/insensitive to dasatinib. We refer to these collectively as second-generation inhibitor (SGI) clinically relevant mutations. By in vitro analysis, other mutations confer a degree of insensitivity; however, clinical evidence is currently insufficient to define them as SGI clinically relevant. Here we examine the mutations that are clearly SGI clinically relevant, those with minimal impact on response, and those for which more data are needed. In our series of patients with mutations at imatinib cessation and/or at nilotinib or dasatinib commencement, 43% had SGI clinically relevant mutations, including 14% with T315I. The frequency of SGI clinically relevant mutations was dependent on the disease phase at imatinib failure. The clinical data suggest that a mutation will often be detectable after imatinib failure for which there is compelling clinical evidence that one SGI should be preferred.

Association between BMI-1 expression, acute graft-versus-host disease, and outcome following allogeneic stem cell transplantation from HLA-identical siblings in chronic myeloid leukemia.

Expression of CD7, ELA-2, PR-3, and the polycomb group gene BMI-1 reflects the intrinsic heterogeneity and predicts prognosis of patients with chronic myeloid leukemia (CML) who were not treated with allogeneic stem cell transplantation (allo-SCT). This study investigated whether expression of these genes determined outcome following allo-SCT in a cohort of 84 patients with chronic-phase (CP) CML. We found that patients expressing BMI-1 at a "high" level before allo-SCT had an improved overall survival (P = .005) related to a reduced transplantation-related mortality. In multivariate analysis, when adjusted for the European Group for Blood and Marrow Transplantation (EBMT)-Gratwohl score and other prog-nostic factors, there was an independent association between BMI-1 expression and grades 2 to 4 acute graft-versus-host disease (relative risk [RR] = 2.85; 95% confidence interval [CI], 1.3-6.4; P = .011), suggesting that BMI-1 measured prior to allo-SCT can serve as a biomarker for predicting outcome in patients with CP-CML receiving allo-SCT, and may thus contribute to better therapeutic decisions.

Abnormalities in glucose uptake and metabolism in imatinib-resistant human BCR-ABL-positive cells.

The development of imatinib resistance has become a significant therapeutic problem in which the etiology seems to be multifactorial and poorly understood. As of today, clinical criteria to predict the development of imatinib resistance in chronic myelogenous leukemia (CML), other than rebound of the myeloproliferation, are under development. However, there is evidence that the control of glucose-substrate flux is an important mechanism of the antiproliferative action of imatinib because imatinib-resistant gastrointestinal stromal KIT-positive tumors reveal highly elevated glucose uptake in radiologic images. We used nuclear magnetic resonance spectroscopy and gas chromatography mass spectrometry to assess (13)C glucose uptake and metabolism (glycolysis, TCA cycle, and nucleic acid ribose synthesis) during imatinib treatment in CML cell lines with different sensitivities to imatinib. Our results show that sensitive K562-s and LAMA84-s BCR-ABL-positive cells have decreased glucose uptake, decreased lactate production, and an improved oxidative TCA cycle following imatinib treatment. The resistant K562-r and LAMA84-r cells maintained a highly glycolytic metabolic phenotype with elevated glucose uptake and lactate production. In addition, oxidative synthesis of RNA ribose from (13)C-glucose via glucose-6-phosphate dehydrogenase was decreased, and RNA synthesis via the nonoxidative transketolase pathway was increased in imatinib-resistant cells. CML cells which exhibited a (oxidative/nonoxidative) flux ratio for nucleic acid ribose synthesis of >1 were sensitive to imatinib. The resistant K562-r and LAMA84-r exhibited a (oxidative/nonoxidative) flux ratio of <0.7. The changes in glucose uptake and metabolism were accompanied by intracellular translocation of GLUT-1 from the plasma membrane into the intracellular fraction in sensitive cells treated with imatinib, whereas GLUT-1 remained located at the plasma membrane in LAMA84-r and K562-r cells. The total protein load of GLUT-1 was unchanged among treated sensitive and resistant cell lines. In summary, elevated glucose uptake and nonoxidative glycolytic metabolic phenotype can be used as sensitive markers for early detection of imatinib resistance in BCR-ABL-positive cells.

A kinase-independent role for CDK8 in BCR-ABL1+ leukemia.

Cyclin-dependent kinases (CDKs) are frequently deregulated in cancer and represent promising drug targets. We provide evidence that CDK8 has a key role in B-ALL. Loss of CDK8 in leukemia mouse models significantly enhances disease latency and prevents disease maintenance. Loss of CDK8 is associated with pronounced transcriptional changes, whereas inhibiting CDK8 kinase activity has minimal effects. Gene set enrichment analysis suggests that the mTOR signaling pathway is deregulated in CDK8-deficient cells and, accordingly, these cells are highly sensitive to mTOR inhibitors. Analysis of large cohorts of human ALL and AML patients reveals a significant correlation between the level of CDK8 and of mTOR pathway members. We have synthesized a small molecule YKL-06-101 that combines mTOR inhibition and degradation of CDK8, and induces cell death in human leukemic cells. We propose that simultaneous CDK8 degradation and mTOR inhibition might represent a potential therapeutic strategy for the treatment of ALL patients.

Production of P-glycoprotein from the MDR1 upstream promoter is insufficient to affect the response to first-line chemotherapy in advanced breast cancer.

Multidrug resistance, the phenomenon by which cells treated with a drug become resistant to the cytotoxic effect of a variety of other structurally and functionally unrelated drugs, is often associated with the expression of P-glycoprotein, an efflux membrane pump coded by the MDR1 (ABCB1) gene. Transcription from MDR1 can start at 2 promoters: a well-characterized downstream promoter and an as yet uncharacterized upstream promoter (USP). We have previously determined that the USP is activated in some drug-resistant cell lines, in primary breast tumors and in metastatic epithelial cells isolated from the lymph nodes of breast cancer patients. In this study, we report the cloning and characterization of the MDR1 USP and studied its association with chemotherapy response in breast cancer patients. Deletion analysis indicated that a nearby endogenous retroviral long terminal repeat is not responsible for promoter activation, and that the region within the first 400 nucleotides upstream from the transcription start point contained all the elements necessary for promoter activity in drug-resistant cells. We identified an element recognized by the transcription factor NF-IL6 (activated upon interleukin-6 exposure) which is necessary for promoter activity in drug-resistant cells and plays a role in the activation of the promoter in response to interleukin-6 in breast cancer MCF-7 cells. Although transcripts from this promoter are associated with translating polyribosomes, their low abundance makes the amount of synthesized P-glycoprotein insufficient to affect the response to first-line chemotherapy in patients with advanced breast cancer.

BCR-ABL in chronic myelogenous leukemia--how does it work?

The discovery of the BCR-ABL fusion gene on the Philadelphia (Ph) chromosome in 1985 was the start of a new era in understanding the molecular basis of hematologic malignancies. It provided the rationale for producing first imatinib and then a series of small molecules designed to inhibit the tyrosine kinase activity of the Bcr-Abl oncoprotein, all of which can induce complete cytogenetic remissions in the majority of patients with chronic myelogenous leukemia (CML) in the chronic phase. However, we still do not know for sure whether the BCR-ABL fusion gene is really the initiating lesion for the chronic phase of CML and we have an incomplete understanding of the so-called genomic instability that underlies the production of the fusion gene and predisposes the Ph-positive clone to acquire further genetic events that lead to advanced-phase disease. Moreover, it is clear that though some of the mutant Ph-positive subclones that develop in patients taking tyrosine kinase inhibitors (TKIs) are the direct cause of the resistance observed, in other cases, its cause is unclear. It is likely that in the next few years we will see (1) improved methods for predicting responses to TKIs, (2) the use of TKIs in combination with other effective molecules such as farnesyl transferase inhibitors, and (3) a gradual reduction in the proportion of chronic-phase patients resistant to therapy.

Inactivation of HOXA genes by hypermethylation in myeloid and lymphoid malignancy is frequent and associated with poor prognosis.

PURPOSE: The HOX genes comprise a large family of homeodomain-containing transcription factors, present in four separate clusters, which are key regulators of embryonic development, hematopoietic differentiation, and leukemogenesis. We aimed to study the role of DNA methylation as an inducer of HOX gene silencing in leukemia. EXPERIMENTAL DESIGN: Three hundred and seventy-eight samples of myeloid and lymphoid leukemia were quantitatively analyzed (by COBRA analysis and pyrosequencing of bisulfite-modified DNA) for methylation of eight HOXA and HOXB cluster genes. The biological significance of the methylation identified was studied by expression analysis and through re-expression of HOXA5 in a chronic myeloid leukemia (CML) blast crisis cell line model. RESULTS: Here, we identify frequent hypermethylation and gene inactivation of HOXA and HOXB cluster genes in leukemia. In particular, hypermethylation of HOXA4 and HOXA5 was frequently observed (26-79%) in all types of leukemias studied. HOXA6 hypermethylation was predominantly restricted to lymphoid malignancies, whereas hypermethylation of other HOXA and HOXB genes was only observed in childhood leukemia. HOX gene methylation exhibited clear correlations with important clinical variables, most notably in CML, in which hypermethylation of both HOXA5 (P = 0.00002) and HOXA4 (P = 0.006) was strongly correlated with progression to blast crisis. Furthermore, re-expression of HOXA5 in CML blast crisis cells resulted in the induction of markers of granulocytic differentiation. CONCLUSION: We propose that in addition to the oncogenic role of some HOX family members, other HOX genes are frequent targets for gene inactivation and normally play suppressor roles in leukemia development.

Novel compounds with antiproliferative activity against imatinib-resistant cell lines.

Chronic myelogenous leukemia is caused by the Bcr-Abl hybrid gene that encodes the p210Bcr-Abl chimeric oncoprotein. Although it reduces the total body burden of leukemia cells, the use of imatinib mesylate as a single agent may be accompanied by the evolution of resistance due mainly to the acquisition of point mutations. Imatinib has been combined with drugs that inhibit both the active and the inactive states of the p210Bcr-Abl kinase. These combinations have reduced but not completely eliminated the rate at which point mutations are acquired in the p210Bcr-Abl kinase. Thus, it is important to identify additional new inhibitors of the p210Bcr-Abl kinase. One possible method to prevent evolution of resistance is to simultaneously use multiple kinase inhibitors each with a different mechanism of action. To identify such a new class of inhibitors that could suppress the growth of chronic myelogenous leukemia cells and prevent the evolution of cells that are resistant to imatinib, we screened two low-complexity libraries of compounds based on planar and linear scaffolds. These libraries were screened using a cell-based assay for molecules that suppress p210Bcr-Abl-dependent cell growth. The application of this method resulted in the isolation of two new classes of drugs, both of which inhibited imatinib-resistant cells in the low micromolar range. Some of these drugs were potent inhibitors not only of Abl tyrosine kinase but also of the Src, Lyn, and Fyn tyrosine kinases.

Phenotyping and Target Expression Profiling of CD34+/CD38- and CD34+/CD38+ Stem- and Progenitor cells in Acute Lymphoblastic Leukemia.

Leukemic stem cells (LSCs) are an emerging target of curative anti-leukemia therapy. In acute lymphoblastic leukemia (ALL), LSCs frequently express CD34 and often lack CD38. However, little is known about markers and targets expressed in ALL LSCs. We have examined marker- and target expression profiles in CD34+/CD38- LSCs in patients with Ph+ ALL (n = 22) and Ph- ALL (n = 27) by multi-color flow cytometry and qPCR. ALL LSCs expressed CD19 (B4), CD44 (Pgp-1), CD123 (IL-3RA), and CD184 (CXCR4) in all patients tested. Moreover, in various subgroups of patients, LSCs also displayed CD20 (MS4A1) (10/41 = 24%), CD22 (12/20 = 60%), CD33 (Siglec-3) (20/48 = 42%), CD52 (CAMPATH-1) (17/40 = 43%), IL-1RAP (13/29 = 45%), and/or CD135 (FLT3) (4/20 = 20%). CD25 (IL-2RA) and CD26 (DPPIV) were expressed on LSCs in Ph+ ALL exhibiting BCR/ABL1p210, whereas in Ph+ ALL with BCR/ABL1p190, LSCs variably expressed CD25 but did not express CD26. In Ph- ALL, CD34+/CD38- LSCs expressed IL-1RAP in 6/18 patients (33%), but did not express CD25 or CD26. Normal stem cells stained negative for CD25, CD26 and IL-1RAP, and expressed only low amounts of CD52. In xenotransplantation experiments, CD34+/CD38- and CD34+/CD38+ cells engrafted NSG mice after 12-20 weeks, and targeting with antibodies against CD33 and CD52 resulted in reduced engraftment. Together, LSCs in Ph+ and Ph- ALL display unique marker- and target expression profiles. In Ph+ ALL with BCR/ABL1p210, the LSC-phenotype closely resembles the marker-profile of CD34+/CD38- LSCs in chronic myeloid leukemia, confirming the close biologic relationship of these neoplasms. Targeting of LSCs with specific antibodies or related immunotherapies may facilitate LSC eradication in ALL.