Restoration of sensitivity to STI571 in STI571-resistant chronic myeloid leukemia cells.

STI571 induces sustained hematologic remission in patients with chronic myeloid leukemia (CML) in chronic phase. However, in advanced phases, especially blast crisis, the leukemia usually becomes resistant within months. It has been investigated whether resistance to STI571 is stable and immutable or whether it can be reversed in selected CML cell lines. Withdrawal of STI571 for varying lengths of time from cultures of 3 resistant lines (K562-r, KCL22-r, and Baf/BCR-ABL-r1) did not restore sensitivity to the inhibitor. In contrast, LAMA84-resistant cells experienced a sharp reduction in survival and proliferation during the first week of STI571 withdrawal but recovered thereafter. Moreover, when left off the inhibitor for 2 months or longer, this cell line reacquired sensitivity to STI571. It is hypothesized, therefore, that patients who have become resistant to the drug may respond again if STI571 therapy is temporarily interrupted.     

Molecular mechanisms of resistance to STI571 in chronic myeloid leukemia

Therapeutic use of the recently FDA-approved drug STI571 has been successful in the treatment of Philadelphia chromosome-positive leukemias. STI571 is a small molecule inhibitor with activity against BCR-ABL, the deregulated tyrosine kinase responsible for initiation and maintenance of the disease in the chronic phase of chronic myeloid leukemia (CML). Clinical trials demonstrated the ability of STI571 to induce remissions in patients with chronic phase CML with only rare relapses after 18 months of follow-up. However, in patients with more advanced stages of disease, responses to STI571 were less common and often transient. Studies investigating the molecular mechanisms of resistance to this novel compound have progressed rapidly and point to the continued importance of BCR-ABL in disease maintenance even at its latest stages. Here the authors review recent work aimed at elucidating the nature of STI51 resistance.     

Primitive, quiescent, Philadelphia-positive stem cells from patients with chronic myeloid leukemia are insensitive to STI571 in vitro.

In clinical trials, the tyrosine kinase inhibitor STI571 has proven highly effective in reducing leukemic cell burden in chronic myeloid leukemia (CML). The overall sensitivity of CML CD34(+) progenitor cells to STI571 and the degree to which cell death was dependent on cell cycle status were determined. Stem cells (Lin(-)CD34(+)) from the peripheral blood of patients with CML in chronic phase and from granulocyte-colony-stimulating factor-mobilized healthy donors were labeled with carboxy-fluorescein diacetate succinimidyl diester dye to enable high-resolution tracking of cell division. Then they were cultured for 3 days with and without growth factors +/- STI571. After culture, the cells were separated by fluorescence-activated cell sorting into populations of viable quiescent versus cycling cells for genotyping. For healthy controls, in the presence of growth factors, STI571 affected neither cell cycle kinetics nor recovery of viable cells. In the absence of growth factors, normal cells were unable to divide. For CML samples, in the presence or absence of growth factors, the response to STI571 was variable. In the most sensitive cases, STI571 killed almost all dividing cells; however, a significant population of viable CD34(+) cells was recovered in the undivided peak and confirmed to be part of the leukemic clone. STI571 also appeared to exhibit antiproliferative activity on the quiescent population. These studies confirm that CML stem cells remain viable in a quiescent state even in the presence of growth factors and STI571. Despite dramatic short-term responses in vivo, such in vitro insensitivity to STI571, in combination with its demonstrated antiproliferative activity, could translate into disease relapse after prolonged therapy.     

Reversal of bone marrow angiogenesis in chronic myeloid leukemia following imatinib mesylate (STI571) therapy

The effect of imatinib mesylate (imatinib) therapy on angiogenesis and myelofibrosis was investigated and compared with interferon (IFN) and hydroxyurea (HU) in 98 patients with newly diagnosed Philadelphia chromosome-positive/BCR-ABL(+) (Ph(+)/BCR-ABL(+)) chronic myeloid leukemia in first chronic phase and no other pretreatment. By means of immunostaining (CD34) and morphometry, a relationship between microvessel frequency and fiber density was detectable in initial bone marrow (BM) biopsies and sequential examinations after at least 8 months of therapy. First-line monotherapy with imatinib induced a significant reduction (normalization in comparison with controls) of microvessels and reticulin fibers. In most patients, decrease in BM vascularity was associated with a complete cytogenetic response. A significant anti-angiogenic effect was also observed after HU treatment, contrasting with IFN administration or combination regimens (IFN plus HU). In conclusion, our data support the anti-angiogenic capacity of imatinib by normalization of vascularity. In contrast, hematologic response following IFN treatment is independent from BM angiogenesis.     

Alpha1-acid glycoprotein expressed in the plasma of chronic myeloid leukemia patients does not mediate significant in vitro resistance to STI571.

Despite the efficacy of STI571 (Glivec, Novartis, Basle, Switzerland) in treating chronic myeloid leukemia (CML), drug resistance has already been noted both in vitro and in vivo. As plasma proteins, including alpha-1-acid glycoprotein (AGP), may reduce drug efficacy through binding, AGP was investigated for its ability to interact with STI571. At all stages of CML, AGP plasma level was significantly higher than in normal controls (P <.05). The glycoprotein was purified from normal plasma and individual chronic myeloid leukemia (CML) patients' plasma by low-pressure chromatography. The influence of alpha1-acid glycoprotein (AGP), in the presence of STI571, on the proliferation of Philadelphia chromosome-positive (Ph+) cells was examined. Normal AGP, even at supraphysiological concentrations, did not block the effect of STI571 on K562-cell proliferation in vitro. Moreover, CML-derived AGP failed to block the effect of STI571 on Ph+ cells in vitro. Thus, these in vitro findings suggest that AGP will not abrogate the antileukemic activity of STI571.     

Ph(+) acute lymphoblastic leukemia resistant to the tyrosine kinase inhibitor STI571 has a unique BCR-ABL gene mutation

The tyrosine kinase inhibitor STI571 is a promising agent for the treatment of advanced Philadelphia chromosome positive (Ph(+)) acute lymphoblastic leukemia (ALL), but resistance develops rapidly in most patients after an initial response. To identify mechanisms of resistance to STI571, 30 complementary DNAs (including 9 matched samples) obtained from the bone marrow of individuals with Ph(+) ALL were analyzed by direct sequencing of a 714-base pair region of ABL encoding for the adenosine triphosphate (ATP)-binding site and the kinase activation loop. A single point mutation was found at nucleotide 1127 (GI6382056) resulting in Glu255Lys. This mutation occurred in 6 of 9 patients (67%) following their treatment with STI571 but not in the samples from patients before beginning treatment with STI571. Glu255Lys is within the motif important for forming the pocket of the ATP-binding site in ABL and it is highly conserved across species. In conclusion, Ph(+) ALL samples resistant to STI571 have a unique mutation Glu255Lys of BCR-ABL.     

The tyrosine kinase inhibitor STI571, like interferon-alpha, preferentially reduces the capacity for amplification of granulocyte-macrophage progenitors from patients with chronic myeloid leukemia

OBJECTIVE: To determine whether the compound STI571 (formerly known as CGP571418B), a selective inhibitor of the protein tyrosine kinase (PTK) activity of ABL and BCR-ABL proteins, preferentially reduces the capacity for amplification of granulocyte-macrophage progenitors (CFU-GM) from patients with chronic myeloid leukemia while sparing normal CFU-GM and to compare responses of CML and normal cells with STI571 and IFN-alpha. MATERIALS AND METHODS: Chronic phase CML and normal CFU-GM were grown with and without STI571, IFN-alpha, or the two agents in combination. Colonies were plucked and replated in 96-well microtiter plates. Secondary colonies were scored, and the results were expressed as the area-under-the-curve (AUC) of the distribution of secondary colony numbers per primary CFU-GM. This value gives an overall measure of the replating ability or amplification of the original CFU-GM population. RESULTS: STI571 selectively inhibits the formation of granulocyte-macrophage colony-forming cells (CFU-GM) from CML patients. It also significantly inhibits the amplification of CML CFU-GM (p = 0.002) as measured by secondary colony formation after replating primary CFU-GM colonies. In contrast, amplification of normal CFU-GM was enhanced (p = 0.001) at low concentrations (0.1 microM) of STI571 with a return to baseline at 10 microM STI571. Addition of interferon (IFN)-alpha to STI571 abolished the increase in normal CFU-GM amplification seen with either agent alone. There was a highly significant correlation between the in vitro response to STI571 and the in vitro response to IFN-alpha (r = 0.74 for CML cells, and 0.77 for normal cells). CONCLUSION: We conclude that STI571, like IFN-alpha, preferentially suppresses amplification of CML CFU-GM while sparing normal CFU-GM.     

Clinical resistance to the kinase inhibitor STI-571 in chronic myeloid leukemia by mutation of Tyr-253 in the Abl kinase domain P-loop

The Abl tyrosine kinase inhibitor STI-571 is effective therapy for stable phase chronic myeloid leukemia (CML) patients, but the majority of CML blast-crisis patients that respond to STI-571 relapse because of reactivation of Bcr-Abl signaling. Mutations of Thr-315 in the Abl kinase domain to Ile (T315I) were previously described in STI-571-resistant patients and likely cause resistance from steric interference with drug binding. Here we identify mutations of Tyr-253 in the nucleotide-binding (P) loop of the Abl kinase domain to Phe or His in patients with advanced CML and acquired STI-571 resistance. Bcr-Abl Y253F demonstrated intermediate resistance to STI-571 in vitro and in vivo when compared with Bcr-Abl T315I. The response of Abl proteins to STI-571 was influenced by the regulatory state of the kinase and by tyrosine phosphorylation. The sensitivity of purified c-Abl to STI-571 was increased by a dysregulating mutation (P112L) in the Src homology 3 domain of Abl but decreased by phosphorylation at the regulatory Tyr-393. In contrast, the Y253F mutation dysregulated c-Abl and conferred intrinsic but not absolute resistance to STI-571 that was independent of Tyr-393 phosphorylation. The Abl P-loop is a second target for mutations that confer resistance to STI-571 in advanced CML, and the Y253F mutation may impair the induced-fit interaction of STI-571 with the Abl catalytic domain rather than sterically blocking binding of the drug. Because clinical resistance induced by the Y253F mutation might be overcome by dose escalation of STI-571, molecular genotyping of STI-571-resistant patients may provide information useful for rational therapeutic management.     

Determination of alpha-1 acid glycoprotein in patients with Ph+ chronic myeloid leukemia during the first 13 weeks of therapy with STI571

The tyrosine kinase activity of the BCR/ABL fusion protein is required for the transformation in patients with chronic myeloid leukemia. The tyrosine kinase inhibitor STI571 inhibits the BCR/ABL and ABL kinase activity and consequently inhibits growth of BCR/ABL-positive cells. However, resistance to STI571 has been demonstrated in Ph+ cell lines and in CML patients and can be explained in some cases by point mutations within the ATP-binding pocket or amplification of the bcr/abl gene. In previous investigations using a nu/nu mouse model, the binding of STI571 to elevated levels of the plasmaprotein -1 acid glycoprotein (AGP) was identified as an additional mechanism of resistance to this therapeutic approach. Here we provide data on the expression of AGP in CML patients under therapy with STI571. Patients received 400 or 600 mg STI571 daily and apart from clinical parameters we determined AGP and C-reactive protein (CRP) plasma levels as well as the quantitative expression of both BCR/ABL and AGP mRNA in peripheral blood cells. Our data suggest that despite elevated AGP levels in 52% of our patients, no upfront resistance against STI571 was present. In conclusion, we demonstrated that during the first 13 weeks of STI571 therapy (i) plasma AGP levels in CML patients correlate with white blood cell count and stage of disease; (ii) patients with elevated AGP responded less rapidly to STI571; (iii) elevated AGP and CRP levels normalized in patients during treatment with STI571, although mRNA levels of AGP remained stable; (iv) initially normal levels of AGP remained in the normal range during treatment with STI571, indicating that STI571 does not trigger AGP expression in humans; and (v) in relapsed patients, elevation of AGP levels is present prior to hematological progress.     

High frequency of point mutations clustered within the adenosine triphosphate-binding region of BCR/ABL in patients with chronic myeloid leukemia or Ph-positive acute lymphoblastic leukemia who develop imatinib (STI571) resistance

Point mutations were found in the adenosine triphosphate (ATP) binding region of BCR/ABL in 12 of 18 patients with chronic myeloid leukemia (CML) or Ph-positive acute lymphoblastic leukemia (Ph(+) ALL) and imatinib resistance (defined as loss of established hematologic response), but they were found in only 1 of 10 patients with CML with imatinib refractoriness (failure to achieve cytogenetic response). In 10 of 10 patients for whom samples were available, the mutation was not detected before the initiation of imatinib therapy. Three mutations (T315I, Y253H, and F317L present in 3, 1, and 1 patients, respectively) have a predicted role in abrogating imatinib binding to BCR/ABL, whereas 3 other mutations (E255K, G250E, and M351T, present in 4, 2, and 2 patients, respectively) do not. Thus we confirm a high frequency of mutations clustered within the ATP-binding region of BCR/ABL in resistant patients. Screening may allow intervention before relapse by identifying emerging mutations with defined impacts on imatinib binding. Certain mutations may respond to higher doses of imatinib, whereas other mutations may mandate switching to another therapeutic strategy.     

Involvement of Akt kinase in the action of STI571 on chronic myelogenous leukemia cells

To elucidate the role of mitogen-activated protein kinases (MAPKs) and Akt kinase in leukemogenesis caused by the breakpoint cluster region (BCR)-Abelson (ABL) tyrosine kinase oncoprotein, we examined the activities of MAPKs and Akt kinase and their roles in the action of STI571, a specific inhibitor of BCR-ABL tyrosine kinase, in chronic myelogenous leukemia (CML) cells. We found that extracellular signal-regulated kinase (ERK) 1/2 and Akt kinase are constitutively active in the chronic phase of CML, blast crisis of CML, and the CML-derived K562 cell line. Both interferon-alpha and STI571 suppressed ERK1/2 activity in K562 cells. In contrast, Akt kinase activity was inhibited only by STI571. K562 cell proliferation was markedly suppressed by LY294002, a specific inhibitor of PI3K/Akt kinase, and STI571 but not by PD98059, a specific inhibitor of MEK1/2. In addition, caspase-3 was activated by treatment of cells with STI571 and LY294002 but not with PD98059. These data indicate that Akt kinase may play a role in the proliferation of CML leukemia cells and the action of STI571. Primary leukemia cells from patients with CML blast crisis did not show inhibition of ERK1/2 or Akt kinase activity and were resistant to caspase-3-associated apoptosis after treatment with STI571. These findings suggest that STI571 does not effectively block signaling molecules downstream of the BCR-ABL tyrosine kinase in some cases of CML blast crisis.     

Comparative gene expression profile of chronic myeloid leukemia cells innately resistant to imatinib mesylate

OBJECTIVE: Resistance to imatinib mesylate monotherapy is clearly a barrier to successful treatment of chronic myeloid leukemia (CML) patients. In some patients, resistance arises due to powerful selective pressure on rare cells that carry amplified copies of the BCR-ABL fusion oncogene or point mutations in the Bcr-Abl tyrosine kinase domain that affect binding of the drug to the oncoprotein. However, in a proportion of patients neither mechanism operates, and resistance appears to be a priori, existing prior to exposure to the drug. These mechanisms of imatinib resistance are poorly understood and may be heterogeneous. MATERIALS AND METHODS: We have previously described such innate resistance to imatinib in subclones of a myeloid leukemia cell line, KCL22, in which imatinib exposure inhibits the activity of Bcr-Abl and yet fails to induce apoptosis. We describe here whole-genome expression analysis of imatinib-sensitive and -resistant cells derived from the original KCL22 line, using Affymetrix microarray analysis. RESULTS: We detected differential expression of 39 genes that correlate with the imatinib-resistant phenotype. The resistant cells overexpress several genes associated with the suppression of apoptosis or with conferral of a transformed phenotype. CONCLUSION: Amongst the differentially-expressed genes correlating with imatinib resistance, several suggest the activation of alternative pathway(s) that maintain viability and growth independently of Bcr-Abl kinase activity. Given the high rate of primary imatinib resistance in blast crisis, the potential of activating such alternative pathways appears to correlate with disease progression.     

BCR-ABL independence and LYN kinase overexpression in chronic myelogenous leukemia cells selected for resistance to STI571

Clinical studies have shown that the tyrosine kinase inhibitor STI571 effectively controls BCR-ABL-positive chronic myelogenous leukemia (CML). However, disease progression while on STI571 therapy has been reported, suggesting de novo or intrinsic resistance to BCR-ABL-targeted therapy. To investigate possible mediators of acquired STI571 resistance, K562 cells resistant to 5 microM STI571 (K562-R) were cloned and compared to the parental cell population. K562-R cells had reduced BCR-ABL expression and limited activation of BCR-ABL signaling cascades (Stat 5, CrkL, MAPK). STI571 failed to activate caspase cascades or to suppress expression of survival genes (bcl-xL) in resistant cells. Gene sequencing and tyrosine kinase activity measurements demonstrated that K562-R cells retained wild-type and active BCR-ABL tyrosine kinase that was inhibitable by in vitro incubation with STI571, suggesting that BCR-ABL was not coupled to proliferation or survival of K562-R cells. The src-related kinase LYN was highly overexpressed and activated in K562-R cells, and its inhibition reduced proliferation and survival of K562-R cells while having limited effects of K562 cells. Specimens taken from patients with advanced CML that progressed on STI571 therapy also were analyzed for LYN kinase expression, and they were found to be elevated to a level similar to that of K562-R cells. Comparison of samples from patients taken prior to and following STI571 failure suggested that expression and/or activation of LYN/HCK occurs during disease progression. Together, these results suggest that acquired STI571 resistance may be associated with BCR-ABL independence and mediated in part through overexpression of other tyrosine kinases.     

The proteasome inhibitor bortezomib interacts synergistically with histone deacetylase inhibitors to induce apoptosis in Bcr/Abl+ cells sensitive and resistant to STI571

Interactions between the proteasome inhibitor bortezomib and histone deacetylase inhibitors (HDIs) have been examined in Bcr/Abl+ human leukemia cells (K562 and LAMA 84). Coexposure of cells (24-48 hours) to minimally toxic concentrations of bortezomib + either suberoylanilide hydroxamic acid (SAHA) or sodium butyrate (SB) resulted in a striking increase in mitochondrial injury, caspase activation, and apoptosis, reflected by caspases-3 and -8 cleavage and poly(adenosine diphosphate-ribose) polymerase (PARP) degradation. These events were accompanied by down-regulation of the Raf-1/mitogen-induced extracellular kinase (MEK)/extracellular signal-related kinase (ERK) pathway as well as diminished expression of Bcr/Abl and cyclin D1, cleavage of p21CIP1 and phosphorylation of the retinoblastoma protein (pRb), and induction of the stress-related kinases Jun kinase (JNK) and p38 mitogen-activated protein kinase (MAPK). Transient transfection of cells with a constitutively active MEK construct significantly protected them from bortezomib/SAHA-mediated lethality. Coadministration of bortezomib and SAHA resulted in increased reactive oxygen species (ROS) generation and diminished nuclear factor kappa B (NF-kappa B) activation; moreover, the free radical scavenger L-N-acetylcyteine (LNAC) blocked bortezomib/SAHA-related ROS generation, induction of JNK and p21CIP1, and apoptosis. Lastly, this regimen potently induced apoptosis in STI571 (imatinib mesylate)-resistant K562 cells and CD34+ mononuclear cells obtained from a patient with STI571-resistant disease, as well as in Bcr/Abl- leukemia cells (eg, HL-60, U937, Jurkat). Together, these findings raise the possibility that combined proteasome/histone deacetylase inhibition may represent a novel strategy in leukemia, including apoptosis-resistant Bcr/Abl+ hematologic malignancies.