Normal ABL1 is a tumor suppressor and therapeutic target in human and mouse leukemias expressing oncogenic ABL1 kinases.

Leukemias expressing constitutively activated mutants of ABL1 tyrosine kinase (BCR-ABL1, TEL-ABL1, NUP214-ABL1) usually contain at least 1 normal ABL1 allele. Because oncogenic and normal ABL1 kinases may exert opposite effects on cell behavior, we examined the role of normal ABL1 in leukemias induced by oncogenic ABL1 kinases. BCR-ABL1-Abl1(-/-) cells generated highly aggressive chronic myeloid leukemia (CML)-blast phase-like disease in mice compared with less malignant CML-chronic phase-like disease from BCR-ABL1-Abl1(+/+) cells. Additionally, loss of ABL1 stimulated proliferation and expansion of BCR-ABL1 murine leukemia stem cells, arrested myeloid differentiation, inhibited genotoxic stress-induced apoptosis, and facilitated accumulation of chromosomal aberrations. Conversely, allosteric stimulation of ABL1 kinase activity enhanced the antileukemia effect of ABL1 tyrosine kinase inhibitors (imatinib and ponatinib) in human and murine leukemias expressing BCR-ABL1, TEL-ABL1, and NUP214-ABL1. Therefore, we postulate that normal ABL1 kinase behaves like a tumor suppressor and therapeutic target in leukemias expressing oncogenic forms of the kinase.

Bosutinib shows low cross intolerance, in chronic myeloid leukemia patients treated in fourth line. Results of the Spanish compassionate use program.

The role of bosutinib as rescue treatment of Philadelphia chromosome-positive chronic myeloid leukemia (CML) patients after failing three previous tyrosine kinase inhibitors (TKIs) is currently unknown. We report here the largest series (to our knowledge) of patients treated with bosutinib in fourth-line, after retrospectively reviewing 30 patients in chronic phase, and pretreated with imatinib, nilotinib, and dasatinib. With a median follow up of 11.1 months, the probability to either maintain or improve their CCyR response was 56.6% (17/30) and 11 patients (36.7%) achieved or maintained their baseline MMR. In patients not having baseline CCyR, the probabilities of obtaining CCyR, MMR, and MR4.5 were 13, 11, and 14%, respectively. The probabilities of obtaining MMR and deep molecular response MR4.5 in patients with baseline CCyR were 40.0% (6/15) and 20.0% (3/15). At 20 months, progression-free survival was 73%. Grade 3-4 hematological toxicities were more frequent in resistant than intolerant patients (45.4 vs. 0.0%). Nonhematological toxicities were also more frequent in resistant patients, being diarrhea the most conspicuous one. Bosutinib seems to be an appropriate treatment option for patients resistant or intolerant to three prior TKI's.

BCR-ABL1 doubling times more reliably assess the dynamics of CML relapse compared with the BCR-ABL1 fold rise: implications for monitoring and management.

Rising BCR-ABL1 transcripts indicate potential loss of imatinib response in CML. We determined whether the BCR-ABL1 doubling time could distinguish nonadherence from resistance as the cause of lost response. Distinct groups were examined: (1) acquired clinical resistance because of blast crisis and/or BCR-ABL1 mutations; and (2) documented imatinib discontinuation/interruption. Short doubling times occurred with blast crisis (median, 9.0 days; range, 6.1-17.6 days; n = 12 patients), relapse after imatinib discontinuation in complete molecular response (median, 9.0 days; range, 6.9-26.5 days; n = 17), and imatinib interruption during an entire measurement interval (median, 9.4 days; range, 4.2-17.6 days; n = 12; P = .72). Whereas these doubling times were consistently short and indicated rapid leukemic expansion, fold rises were highly variable: 71-, 9.5-, and 10.5-fold, respectively. The fold rise depended on the measurement interval, whereas the doubling time was independent of the interval. Longer doubling times occurred for patients with mutations who maintained chronic phase (CP: median, 48 days; range, 17.3-143 days; n = 29; P < .0001). Predicted short and long doubling times were validated on an independent cohort monitored elsewhere. The doubling time revealed major differences in kinetics according to clinical context. Long doubling times observed with mutations in CP allow time for intervention. A short doubling time for a patient in CP should raise the suspicion of nonadherence.

CD117 (c-kit) expression on CD34+ cells participates in the cytogenetic response to imatinib in patients with chronic myeloid leukemia in the first chronic phase.

BACKGROUND: Chronic myeloid leukemia (CML) biology seemed to be perfectly explored especially at the beginning of the tyrosine kinase inhibitors era. Later years with imatinib and second-generation tyrosine kinase inhibitors showed a variety of resistance mechanisms and it became obvious that the bcr-abl chimeric gene is not the only enemy to fight. Some studies assumed the decreased rate of programmed cell death (apoptotic) to be the primary mechanism by which BCR-ABL affects expansion of the leukemic clone in CML. Therefore, the aim of this study was to investigate the role of c-kit inhibition in treatment response. METHODS: Cytogenetic analysis, real-time quantitative reverse-transcriptase polymerase chain reaction, flow-cytometric analysis and imatinib serum level quantification were applied. RESULTS: The percentage of CD34+ cells expressing c-kit (CD117) isolated from bone marrow samples of 54 CML patients treated with standard-dose imatinib was significantly lower among imatinib responders. The fraction of apoptotic CD34+CD117+ cells in this patient group was significantly higher than in nonresponders. CONCLUSION: To achieve optimal treatment response in CML patients, the elimination of CD34+CD117+ may be necessary through an apoptotic pathway.

BCR-ABL1 transcript at 3 months predicts long-term outcomes following second generation tyrosine kinase inhibitor therapy in the patients with chronic myeloid leukaemia in chronic phase who failed Imatinib.

The BCR-ABL1 transcript level at 3 months can predict long-term outcomes following frontline therapy with Imatinib or Dasatinib in chronic myeloid leukaemia (CML) patients. However, data is lacking for second-generation tyrosine kinase inhibitor (2GTKI) therapy after Imatinib failure. A total of 112 patients with CML in chronic phase receiving 2GTKI after Imatinib failure were reviewed. Treatment outcomes including complete cytogenetic (CCyR), major molecular (MMR) and molecular response 4.5 (4.5 log reduction of BCR-ABL1 transcript level, MR(4.5) ), treatment failure, progression-free and overall survival (OS) were compared according to BCR-ABL1 transcript levels at 3 or 6 months, divided into <1%(IS) , 1-10%(IS) and ≥ 10%(IS) . BCR-ABL1 transcript level at 3 months showed better correlation with OS (P < 0.001) than that at 6 months (P = 0.147). Better OS was also observed in the patients achieving <1%(IS) (100%) and 1-10%(IS) (100%) than those with ≥ 10%(IS) at 3 months (70.6%, P < 0.001). Those with <1%(IS) showed the best CCyR, MMR and MR(4.5) rates; 1-10%(IS) , intermediate; and ≥ 10%(IS) , the lowest CCyR, MMR and MR(4.5) rates. The group with <1%(IS) at 3 months maintained significantly lower BCR-ABL1 transcript level compared to other two groups. In conclusion, the BCR-ABL1 transcript level at 3 months is the most relevant surrogate for outcomes following 2GTKI therapy after Imatinib failure.

Impact of BCR-ABL mutations on response to dasatinib after imatinib failure in elderly patients with chronic-phase chronic myeloid leukemia.

To assess the impact of BCR-ABL kinase domain mutations on dasatinib response in elderly chronic myeloid leukemia (CML) patients, we analyzed the outcome of 76 individuals aged >60 affected by imatinib-resistant chronic-phase CML. We found that 36 cases (47 %) displayed mutations before dasatinib. Compared to non-mutated patients, subjects with point mutations had a worse response to dasatinib, with significantly lower rates of complete cytogenetic response (57 vs 32 %), higher percentage of primary resistance (16/36 vs 6/40) and a trend towards a shorter median event-free survival. Our data suggest that, in elderly patients, detection of BCR-ABL mutations negatively affects response to dasatinib.

Blockade of the Bcr-Abl kinase activity induces apoptosis of chronic myelogenous leukemia cells by suppressing signal transducer and activator of transcription 5-dependent expression of Bcl-xL.

Bcr-Abl-expressing leukemic cells are highly resistant to apoptosis induced by chemotherapeutic drugs. Although a number of signaling molecules have been shown to be activated by the Bcr-Abl kinase, the antiapoptotic pathway triggered by this oncogene has not been elucidated. Here, we show that the interleukin 3-independent expression of the antiapoptotic protein, Bcl-xL, is induced by Bcr-Abl through activation of signal transducer and activator of transcription (Stat)5. Inhibition of the Bcr-Abl kinase activity in Bcr-Abl-expressing cell lines and CD34(+) cells from chronic myelogenous leukemia (CML) patients induces apoptosis by suppressing the capacity of Stat5 to interact with the bcl-x promoter. Interestingly, after inhibition of the Bcr-Abl kinase, the expression of Bcl-xL is downregulated more rapidly in chronic phase than in blast crisis CML cells, suggesting an involvement of this protein in disease progression. Overall, we describe a novel antiapoptotic pathway triggered by Bcr-Abl that may contribute to the resistance of CML cells to undergo apoptosis.

[Changes in serum RNase activities in chronic phase and acute crisis of chronic myelocytic leukemia].

We investigated serum acid and alkaline RNase activities in 16 cases of acute crisis of chronic myelocytic leukemia (CML), 21 cases of untreated CML, and 13 cases of treated CML, to clarify clinical significance of the determination of RNase activities in acute crisis of CML. We obtained results as follows; the ratio of acid to alkaline Rnase activities (Ac/Al ratio) of chronic phase of CML was 1.64 +/- 0.47 (mean +/- SD) in the untreated cases, and was 1.32 +/- 0.16 in the treated cases. The Ac/Al ratio always indicated over 1.0 in the chronic phase of CML without any relationship to treatments. On the other hand, in the cases of acute crisis, the Ac/Al ratio was significantly lowered (0.94 +/- 0.22) as compared to the chronic phase of CML (p less than 0.001), and was similar to that of acute leukemia. The acid and alkaline RNase activities of the blast from the patients with acute crisis of CML showed remarkably lower value than those of leukemic cells from patients with chronic phase of CML. Therefore, it was Suggested that the return to normal range of Ac/Al ratio in acute crisis of CML depended on marked decrease of RNase activities of blasts. Thus, serial determinations of the enzyme activities are considered to be one of useful tools for prediction of acute crisis of CML.

Prognostic factors for outcomes of allogeneic stem cell transplantation in chronic phase chronic myeloid leukemia in the era of tyrosine kinase inhibitors.

The aim of this study was to estimate the prognostic factors for the outcomes of chronic myeloid leukemia (CML) patients receiving allogeneic stem cell transplantation (SCT) in chronic phase (CP) in the era of tyrosine kinase inhibitors (TKIs). Ninety-seven patients who underwent allogeneic SCT in CP were analyzed. Forty-seven were TKI-naïve at the time of transplant, and 50 received TKI(s) treatment before transplantation. After a median follow-up of 115.8 months, the 4-year overall survival and event-free survival were 80.4 and 58.8%, respectively. Multivariate analysis showed that there were no differences in survival outcomes based on prior TKI therapy. Older age was a prognostic factor for higher treatment-related mortality (TRM), and the type of graft source and younger age were associated with relapse, but prior TKI therapy and disease status at the time of transplant were not associated with either TRM or relapse. Additionally, a major molecular response at 1 month and an MR(4.5) at 3 months were important predictors of favorable long-term outcomes. This study demonstrates the prognostic factors for the outcomes of allogeneic SCT in CP CML and shows that survival outcomes were not affected by the administration of long-term multi-TKI treatment prior to transplantation.

Current practices in the management of chronic myeloid leukemia.

BACKGROUND: A previous survey of physician self-reported practice patterns in the management of CML was conducted in 2005. The National Comprehensive Cancer Network and European LeukemiaNet guidelines now include nilotinib and dasatinib in their treatment algorithms for CML. To assess these new guidelines, a cross-sectional survey of US hematologists and/or oncologists was conducted in December 2010 through an online survey. MATERIALS AND METHODS: The survey had 43 questions consisting of items updated from the 2005 survey to reflect changes in clinical practice, tyrosine kinase inhibitor therapy, and current guidelines. RESULTS: Analysis of the responses from 507 board certified medical oncologists/hematologists suggests that the use of imatinib 400 mg as an initial treatment option had decreased from 62% in 2005 to 52% in the 2010 survey. Currently, nearly 40% of physicians would choose either nilotinib or dasatinib as first-line treatment. From the surveyed physicians, achievement of at least a major molecular response (MMR) is the predominant treatment goal in chronic phase CML. CONCLUSION: This survey emphasizes the need for continued updates and education regarding optimal therapy, monitoring practices, and therapeutic end points in CML.

Diverse mechanisms of mTOR activation in chronic and blastic phase of chronic myelogenous leukemia.

Chronic myelogenous leukemia (CML) is a stem cell disorder, and leukemia stem cells (LSCs) can contribute to the relapse of the disease. Quiescent LSCs are BCR-ABL independent and resistant to imatinib; therefore, there is an unmet need to identify new therapeutic targets in LSCs. Inhibition of the mammalian target of rapamycin (mTOR) in imatinib-resistant BCR-ABL1-positive cells was effective in vitro, but in a pilot clinical trial, only a few patients responded to the treatment. In this study, we demonstrate that mTOR activation in CML CD34(+) progenitor cells is ERK dependent in chronic phase of the disease and ERK independent in blast crisis. Rapamycin effectively inhibits mTOR in all phases of CML, but does not reduce number of LSC-enriched CD34(+) blast crisis (BC) cells, neither alone nor in combination with imatinib in CML-BC cells. These results show that potential therapeutic benefits of mTOR inhibition may be the result of effects on differentiated leukemic cells and may be potentially achieved only in the chronic phase of the disease.

Evolution of therapies for chronic myelogenous leukemia.

The clinical outcome for patients with chronic myelogenous leukemia (CML) has changed dramatically in the past 15 years. This has been due to the development of tyrosine kinase inhibitors (TKIs), compounds that inhibit the activity of the oncogenic BCR-ABL1 protein. Imatinib was the first TKI developed for CML, and it led to high rates of complete cytogenetic responses and improved survival for patients with this disease. However, approximately 35% of patients in chronic phase treated with imatinib will develop resistance or intolerance to this drug. The recognition of the problem of imatinib failure led to the design of second-generation TKI (dasatinib, nilotinib, and bosutinib). These drugs are highly active in the scenario of imatinib resistance or intolerance. More recently, both nilotinib and dasatinib were approved for frontline use in patients with chronic phase CML. Ponatinib represents the last generation of TKI, and this drug has been developed with the aim of targeting a specific BCR-ABL1 mutation (T315I), which arises in the setting of prolonged TKI therapy and leads to resistance to all commercially available TKI. Parallel to the development of specific drugs for treating CML, major advances were made in the field of disease monitoring and standardization of response criteria. In this review, we summarize how therapy with TKI for CML has evolved during the last decade.

The MEK inhibitor PD184352 enhances BMS-214662-induced apoptosis in CD34+ CML stem/progenitor cells.

The cytotoxic farnesyl transferase inhibitor BMS-214662 has been shown to potently induce mitochondrial apoptosis in primitive CD34+ chronic myeloid leukaemia (CML) stem/progenitor cells. Here, to enhance the BMS-214662 apoptotic effect, we further targeted the extracellular signal-regulated kinase (ERK) pathway, downstream of BCR-ABL, by treating CD34+ CML stem/progenitor cells with a highly selective adenosine triphosphate (ATP) non-competitive MEK inhibitor, PD184352. PD184352 increased the apoptotic effect of BMS-214662 in a CML blast crisis cell line, K562, and in primary chronic phase CD34+ CML cells. Compared with BMS-214662, after combination treatment we observed inhibition of ERK phosphorylation, increased Annexin-V levels, caspase-3, -8 and -9 activation and potentiated mitochondrial damage, associated with decreased levels of anti-apoptotic BCL-2 family protein MCL-1. Inhibition of K-RAS function by a dominant-negative mutant resulted in CML cell death and this process was further enhanced by the addition of BMS-214662 and PD184352. Together, these findings suggest that the addition of a MEK inhibitor improves the ability of BMS-214662 to selectively target CML stem/progenitor cells, notoriously insensitive to tyrosine kinase inhibitor treatment and presumed to be responsible for the persistence and relapse of the disease.

Second-generation tyrosine kinase inhibitors as therapy for chronic myeloid leukemia.

Chronic myeloid leukemia (CML) was the first human malignancy to be associated with a single genetic abnormality, characterized by a reciprocal translocation involving chromosomes 9 and 22 (the Philadelphia chromosome). The fusion gene that results (BCR-ABL) produces a constitutively activated tyrosine kinase that exists in different isoforms depending on BCR break-points. Imatinib mesylate is a highly selective inhibitor of this kinase, producing normal blood-counts in 98% of patients in chronic phase CML and disappearance of the Philadelphia chromosome in 86%. However, 17% of patients in the chronic phase will either relapse or develop resistance resulting mainly from one or more point mutations affecting at least 30 amino acids within the Abl kinase protein. This review focuses on the relevant biology of CML, imatinib mesylate resistance mechanisms, and the current status of the next generation of Bcr-Abl inhibitors.

High expression of UDP-N-acetylglucosamine: beta-D mannoside beta-1,4-N-acetylglucosaminyltransferase III (GnT-III) in chronic myelogenous leukemia in blast crisis.

The activity and mRNA expression of UDP-N-acetylglucosamine: beta-D mannoside beta-1,4-N-acetylglucosaminyl transferase III (GnT-III: EC 2.4.1.144) were investigated in hematological malignancies. GnT-III activity was elevated in patients with chronic myelogenous leukemia (CML) in blast crisis and patients with multiple myeloma (MM), as compared to normal healthy subjects and patients with other hematological malignancies including CML in chronic phase. The GnT-III transcript was the same size in leukemic cells from various hematological diseases and cell lines, while expression of the transcript was not found to correlate significantly with enzyme activity, implying that post-translational modification might regulate the activity of GnT-III. Southern-blot analysis showed no significant variation in the structure and position of the GnT-III genome, indicating that the gene is present as a single copy without isoforms. Furthermore, analyses by immunoprecipitation and Western blot revealed that high GnT-III activity in KU812 cell, a CML cell line, resulted in an increase in E4-PHA binding to CD45, a major surface glycoprotein of the leukocyte, indicating that more bisecting GlcNAc was added to CD45 catalyzed by elevated GnT-III.

Induction of protein-tyrosine-phosphatase activity by interleukin 6 in M1 myeloblastic cells and analysis of possible counteractions by the BCR-ABL oncogene.

Interleukin 6 (IL-6) induces in M1 myeloblastic cells growth arrest and terminal differentiation toward monocytes. It is reported here that IL-6 reduced by 5- to 20-fold the tyrosine phosphorylation of cellular proteins in these cells. The same-fold reduction was also observed in M1 cells that were transfected with the BCR-ABL deregulated protein kinase. In these stable clones, the levels of tyrosine phosphorylation of cellular proteins were 30- to 100-fold higher than in the parental cells. IL-6 did not reduce the expression levels or the inherent tyrosine kinase activity of BCR-ABL p210. By measuring the protein-tyrosine-phosphatase (PTPase; protein-tyrosine-phosphate phosphohydrolase, EC 3.1.3.48) activity in crude cell lysates, we found that protein dephosphorylation resulted, at least partially, from induction of PTPase activity by IL-6. The induction of PTPase in the BCR-ABL-transfected clones was not sufficient to confer the minimal protein phosphorylation levels characteristic of IL-6-treated cells. Yet, the transfected M1 clones showed normal growth and differentiation responses to IL-6. None of the gene responses to IL-6 including suppression in the levels of c-myc, c-myb, and cyclin A mRNA; junB and c-jun mRNA induction; and dephosphorylation of retinoblastoma protein were rescued by the BCR-ABL oncogene. The functional relevance of PTPase induction by IL-6 is discussed.

Terminal deoxynucleotidyl-transferase levels and membrane phenotypes in diagnosis of acute leukaemia.

Terminal deoxynucleotidyl transferase (T.D.T.) has been measured in the cells of 112 leukaemia patients whose cells were characterised by membrane markers as well as by standard haematological and cytochemical criteria. The concentration of enzyme ranged from 0.1 to 1.4 units/10(8) cells in the bone-marrows of 20 patients with normal marrow or benign marrow hyperplasia. The enzyme level was raised in 30 of 31 patients with untreated acute lymphoblastic leukaemia (A.L.L.) whose cells reacted positively with an anti-A.L.L. serum (range 0.9-197, mean 59, units/10(8) cells) and in all 9 patients with thymic A.L.L. (Thy-A.L.L.) (range 1.5-95, mean 36, units/10(8) cells). All but 1 of 17 patients with acute myeloblastic leukaemia gave negative results (range 0-1.5, mean 0.60, units/10(8) cells), and T.D.T. was also normal in all 7 bone-marrow samples from patients with chronic granulocytic leukaemia (C.G.L.) in the chronic phase. The T.D.T. assay gave a clear distinction between 11 patients with C.G.L. in lymphoid transformation, anti-A.L.L.-serum positive (range 15-226, mean 83, units/10(8) cells), and 12 patients with C.G.L. in myeloblastic transformation, anti-A.L.L.-serum negative (range 0-1.9, mean 0.7, units/10(8) cells). Among 17 patients with otherwise unclassifiable acute leukaemia, 10 gave raised values (range 1.6-113 units/10(8) cells) and 7 gave normal values. It is concluded that the assay of T.D.T. in the peripheral blood or bone-marrow of patients with acute leukaemia is of value in differentiating lymphoid (including non-T non-B and Thy-A.L.L.) from myeloid leukaemia.