Clinical resistance to the kinase inhibitor PKC412 in acute myeloid leukemia by mutation of Asn-676 in the FLT3 tyrosine kinase domain

Activating mutations in the FLT3 tyrosine kinase (TK) occur in approximately 35% of patients with acute myeloid leukemia (AML). Therefore, targeting mutated FLT3 is an attractive therapeutic strategy, and early clinical trials testing FLT3 TK inhibitors (TKI) showed measurable clinical responses. Most of these responses were transient; however, in a subset of patients blast recurrence was preceded by an interval of prolonged remission. The etiology of clinical resistance to FLT3-TKI in AML is unclear but is of major significance for the development of future therapeutic strategies. We searched for mechanisms of resistance in 6 patients with AML who had relapses upon PKC412 treatment. In an index AML patient, an algorithm of analyses was applied using clinical material. In vivo and in vitro investigation of primary blasts at relapse revealed persistent TK phosphorylation of FLT3 despite sufficient PKC412 serum levels. Through additional molecular analyses, we identified a single amino acid substitution at position 676 (N676K) within the FLT3 kinase domain as the sole cause of resistance to PKC412 in this patient. Reconstitution experiments expressing the N676K mutant in 32D cells demonstrated that FLT3-ITD-N676K was sufficient to confer an intermediate level of resistance to PKC412 in vitro. These studies point out that a genetically complex malignancy such as AML may retain dependence on a single oncogenic signal.     

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.     

Effective and selective inhibition of chronic myeloid leukemia primitive hematopoietic progenitors by the dual Src/Abl kinase inhibitor SKI-606

Imatinib mesylate (imatinib) is highly effective in the treatment of chronic myeloid leukemia (CML) but is less effective in eliminating CML stem cells. We investigated whether SKI-606, a potent Bcr-Abl and Src kinase inhibitor without anti-PDGF or c-Kit activity, could effectively target primitive CML progenitors. CML and normal progenitors were cultured with SKI-606 or imatinib. SKI-606 effectively inhibited Bcr-Abl kinase activity in CML CD34(+) cells and inhibited Src phosphorylation more potently than imatinib. However, SKI-606 and imatinib resulted in similar suppression of CML primitive and committed progenitor proliferation and growth in CFC and LTC-IC assays. Exposure to either agent alone or in combination resulted in only modest increase in apoptosis. Evaluation of downstream signaling pathways indicated that Akt and STAT5 activity was not changed, but a delayed increase in MAPK activity was seen at high concentrations of SKI-606. SKI-606 inhibited normal progenitor proliferation to a lesser extent than imatinib. SKI-606 effectively inhibits Bcr-Abl and Src kinase activity and inhibits CML progenitor growth with relatively little effect on normal progenitors. However, SKI-606 does not demonstrate increased ability to eliminate primitive CML progenitors by apoptosis compared with imatinib, emphasizing the need for additional strategies besides Bcr-Abl kinase inhibition for curative therapy of CML.     

Patient adherence to tyrosine kinase inhibitor therapy in chronic myeloid leukemia

Dramatically improved survival associated with tyrosine kinase inhibitor (TKI) therapy has transformed the disease model for chronic myeloid leukemia (CML) to one of long-term management, but treatment success is challenged with poor medication adherence. Many risk factors associated with poor adherence can be ameliorated by close monitoring, dose modification, and supportive care. Controlling risk factors for poor adherence in combination with patient education that includes direct communication between the health care team and the patient are essential components for maximizing the benefits of TKI therapy.     

Ultra-deep sequencing leads to earlier and more sensitive detection of the tyrosine kinase inhibitor resistance mutation T315I in chronic myeloid leukemia

Chronic myeloid leukemia cells acquire resistance to tyrosine kinase inhibitors through mutations in the ABL1 kinase domain. The T315I mutation mediates resistance to imatinib, dasatinib, nilotinib and bosutinib, whereas sensitivity to ponatinib remains. Mutation detection by conventional Sanger sequencing requires 10%–20% expansion of the mutated subclone. We studied the T315I mutation development by ultra-deep sequencing on the 454 XL+ platform (Roche) in comparison to Sanger sequencing. By ultra-deep sequencing, mutations were detected at loads of 1%–2%. We selected 40 patients who had failed first-line to third-line treatment (imatinib, dasatinib, nilotinib) and had high loads of the T315I mutation detected by Sanger sequencing. We confirmed T315I mutations by ultra-deep sequencing and investigated the mutation dynamics by backtracking earlier samples. In 20 of 40 patients, we identified the T315I three months (median) before Sanger sequencing detection limits were reached. To exclude sporadic low percentage mutation development without subsequent mutation outgrowth, we selected 42 patients without resistance mutations detected by Sanger sequencing but loss of major molecular response. Here, no mutation was detected by ultradeep sequencing. Additional non-T315I resistance mutations were found in 20 of 40 patients. Only 15% had two mutations per cell; the other cases showed multiple independently mutated clones and the T315I clone demonstrated a rapid outgrowth. In conclusion, T315I mutations could be detected earlier by ultra-deep sequencing compared to Sanger sequencing in a selected group of cases. Earlier mutation detection by ultra-deep sequencing might allow treatment to be changed before clonal increase of cells with the T315I mutation.     

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.     

Bcr-Abl kinase domain mutations, drug resistance, and the road to a cure for chronic myeloid leukemia

Mutations in the kinase domain (KD) of BCR-ABL are the most prevalent mechanism of acquired imatinib resistance in patients with chronic myeloid leukemia (CML). Here we examine predisposing factors underlying acquisition of KD mutations, evidence for acquisition of mutations before and during therapy, and whether the detection of a KD mutation universally implies resistance. We also provide a perspective on how the second-line Abl inhibitors dasatinib and nilotinib are faring in the treatment of imatinib-resistant CML, especially in relation to specific KD mutations. We discuss the growing importance of the multi-inhibitor-resistant 315T>I mutant and the therapeutic potential that a 315T>I inhibitor would have. Last, we assess the potential of Abl kinase inhibitor combinations to induce stable responses even in advanced CML and interpret the emerging data in the context of CML pathogenesis.     

A cell-based screen for resistance of Bcr-Abl-positive leukemia identifies the mutation pattern for PD166326, an alternative Abl kinase inhibitor

In Philadelphia-positive (Ph(+)) leukemia, point mutations within the Bcr-Abl kinase domain emerged as a major mechanism of resistance to imatinib mesylate. We established a cell-based screening strategy for detection of clinically relevant point mutations using Bcr-Abl-transformed Ba/F3 cells. We identified 32 different single-point mutations within the kinase domain of Bcr-Abl. The pattern and frequency of mutations in this cell culture-based screen resembled the pattern and frequency observed in resistant patients. We then applied this screen to an alternative Abl kinase inhibitor. Using PD166326, the frequency of resistant colonies emerging at 5 to 10 times the median growth inhibition (IC50) of PD166326 was significantly lower than with imatinib. In addition, PD166326 produced a distinct pattern of Bcr-Abl mutations. The majority of mutations that came up with both imatinib and PD166326 could effectively be suppressed by increasing the dose of PD166326 to 50 to 500 nM. In contrast, only a few mutations could be suppressed by increasing the imatinib dose to 5 to 10 microM. However, 3 mutations affecting F317 displayed complete resistance to PD166326, but could be effectively inhibited by standard concentrations of imatinib. Thus, this robust and simple screening system provides a rational basis for combinatorial and sequential treatment strategies in targeted cancer therapy.     

Outcome of patients with chronic myeloid leukemia with multiple ABL1 kinase domain mutations receiving tyrosine kinase inhibitor therapy

We investigated the impact of carrying more than one BCR-ABL1 mutation in 207 patients with chronic myeloid leukemia (102 chronic, 61 accelerated, and 44 blast phase) post-imatinib failure. Seven (8%) of 92 patients carrying mutations had more than one mutation: 4 (4%) in chronic phase, 2 (2%) in accelerated phase, and one (1 %) in blast phase. The cytogenetic response rate to second generation TKIs for patients with no, one, or more than one mutation was 88%, 69%, 50% (P=0.03) in chronic phase, 54%, 42%, 50% in accelerated phase (P=0.67), and 35%, 25%, 0% (P=0.63) in blast phase, respectively. No differences were observed in event free survival or overall survival in accelerated or blast phase according to their mutational status. However, the 4-year event free survival rates among patients in chronic phase with no, one, or more than one BCR-ABL1 mutation were 56%, 49%, and 0%, respectively (P=0.02), with overall survival rates of 91%, 69%, and 75%, respectively (P=0.13). In conclusion, patients with more than one BCR-ABL1 mutation fare worse than those with no or one mutation.     

Hepatitis B reactivation in chronic myeloid leukemia patients receiving tyrosine kinase inhibitor

Hepatitis B virus(HBV) reactivation is a well-recognized complication in patients with chronic HBV infection receiving cytotoxic or immunosuppressive chemotherapy.Imatinib mesylate and nilotinib are selective Bcr/Abl tyrosine kinase inhibitors,which are now widely used in the treatment of patients with chronic myeloid leukemia.Although HBV reactivation induced by imatinib mesylate has been reported,nilotinib-related HBV reactivation has not been reported in the English literature.We report here 2 cases of HBV reactivation in chronic myeloid leukemia patients receiving imatinib mesylate and a novel case of nilotinib related HBV reactivation.     

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.     

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.     

Characteristics and outcome of chronic myeloid leukemia patients with F317L BCR-ABL kinase domain mutation after therapy with tyrosine kinase inhibitors

Mutations in codon 317 after treatment with imatinib and dasatinib have been reported. We reviewed patients with chronic myeloid leukemia and mutations after tyrosine kinase inhibitor (TKI) therapy. F317L was detected in 20, including 12/99 (12%) with mutation after imatinib failure, and 8/16 (50%) after dasatinib (P = .001). Median follow-up from mutation detection was 25 months. At the time of F317L, 8 patients were in chronic phase (CP), 6 in accelerated phase, and 6 in blast phase. There was no difference in characteristics between patients with or without F317L mutations, or with no mutations. A complete cytogenetic response was achieved in 3 of 6 patients treated with nilotinib, 2 of 2 with imatinib, and 0 of 3 with dasatinib. Survival of patients with F317L was similar to those with other mutations (P = .45). Patients in CP had better outcome, with a 2-year survival of 75%. F317L mutation is resistant to dasatinib but sensitive to other TKIs. The prognosis is dependent mostly on the disease stage.     

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.