FGF2 Mediates Resistance In CML Patients In The Absence Of Kinase Domain Mutations, and Resistance Is Overcome By Ponatinib

Abstract Background Development of resistance to kinase inhibitors remains a challenge in chronic myeloid leukemia (CML). Kinase domain mutations are a common mechanism of resistance, yet the mechanism of resistance in the absence of mutations remains less clear. Recent evidence suggests that the bone marrow microenvironment provides a sanctuary for leukemia cells, and may be involved in mediating resistance to imatinib – particularly in the absence of BCR-ABL kinase domain mutations. We tested selected cytokines, growth factors, and extracellular matrix proteins expressed by cells in the bone marrow microenvironment for their ability to protect CML cells from imatinib. Results We found that fibroblast growth factor 2 (FGF2) was the most protective protein for the K562 CML cell line when exposed to imatinib. FGF2 was not only capable of promoting growth in short-term culture, but uniquely able to promote long-term resistance in vitro (p<0.0001 by 2-way ANOVA analysis). To analyze the mechanism of resistance, we used siRNA to target the FGF receptors 1-4 and found that only siRNA targeting FGFR3 was able to abrogate the protective effect of FGF2. Phospho-chip and Western blot analysis revealed that FGF2 binds FGFR3, which then signals the downstream kinases Ras, c-RAF, MEK1, and ERK1/2 to promote survival in the presence of imatinib. Inhibition of FGFR3 with the specific FGFR inhibitor PD173074 led to dephosphorylation of this signaling cascade, and restored sensitivity to imatinib of FGF2-mediated resistant K562 cells. Resistance could also be overcome with ponatinib, a multi-kinase inhibitor that targets both BCR-ABL and FGFR, whereas imatinib, nilotinib and dasatinib were all ineffective against FGF2-mediated resistant K562 cells. Although ponatinib was rationally designed to circumvent the BCR-ABL T315I gatekeeper mutation, it was also able to achieve major cytogenetic responses in 62% of patients without detectable kinase domain mutations in the recent PACE trial. We theorized that increased FGF2 may drive resistance in the subset of patients without kinase domain mutations who respond to ponatinib, similar to our in vitro findings. To evaluate this possibility, we identified patients without kinase domain mutations who were responsive to ponatinib and quantified bone marrow FGF2 by immunohistochemistry. In comparison to ponatinib-responsive patients with kinase domain mutations, patients without kinase domain mutations had increased FGF2 in their bone marrow (50.5% versus 36.6%, p=0.033). Moreover, FGF2 in the marrow decreased concurrently with response to ponatinib, further suggesting that FGF2-mediated resistance is interrupted by FGFR inhibition (-15.9% versus 0.8%, when compared to the change in FGF2 of patients with kinase domain mutations, p=0.012). Qualitatively, FGF2 was predominantly localized in supportive stromal cells (consistent with previous reports), supporting a paracrine mechanism of resistance. Furthermore, we also evaluated a single patient without kinase domain mutations who was resistant to ponatinib. In this patient’s marrow, there was no elevation in FGF2 or change in FGF2 with ponatinib treatment. Taken together, inhibition of FGFR appears to be critical for the clinical activity of ponatinib in patients without kinase domain mutations. Conclusions In summary, our data supports a model of resistance in which FGF2 production by the marrow stromal cells promotes resistance to multiple ABL kinase inhibitors without the need for mutation of the ABL kinase domain. Resistance occurs via FGF2 ligand-induced activation of the FGFR3/Ras/MAPK pathway, and can be overcome by concomitant inhibition of ABL and FGFR. In combination with recent clinical data with ponatinib, our data suggest that FGF2-mediated resistance is a major mechanism of resistance in CML patients without kinase domain mutations. These results illustrate the clinical importance of ligand-induced resistance to kinase inhibitors and support an approach of developing rational inhibitor combinations to circumvent resistance, particularly in other kinase-driven malignancies that routinely develop resistance to kinase inhibitors. Disclosures: Tyner: InCyte Corporation: Research Funding. Druker:Novartis, Bristol-Myers Squibb, & ARIAD: Novartis, BMS & ARIAD clin trial funding. OHSU holds contracts; no salary/lab research funds. OHSU & Druker have financial interest in MolecularMD; technology used in some studies licensed to MolecularMD. This conflict reviewed and managed by OHSU. Other.

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Dynamics of BCR-ABL Kinase Domain Mutations in Patients with Chronic Myeloid Leukemia (CML) after Treatment with One, Two or Three Tyrosine Kinase Inhibitors (TKI).

Blood ◽

10.1182/blood.v108.11.750.750 ◽

2006 ◽

Vol 108 (11) ◽

pp. 750-750 ◽

Cited By ~ 8

Author(s):

Elias Jabbour ◽

Dan Jones ◽

Hagop Kantarjian ◽

Susan O’Brien ◽

Guillermo Garcia-Manero ◽

...

Keyword(s):

Tyrosine Kinase ◽

Tyrosine Kinase Inhibitors ◽

Kinase Inhibitors ◽

Kinase Domain ◽

Imatinib Resistance ◽

Mutation Status ◽

Abl Kinase ◽

Kinase Domain Mutations ◽

New Mutations

Abstract Dasatinib (D) and nilotinib (N) are potent tyrosine kinase inhibitors (TKIs) with activity against many imatinib (IM) resistant BCR-ABL kinase domain mutants, except T315I. In vitro mutant models have selected specific mutations occurring after incubation with IM, D and N. Therapy with these new TKI may select for patients with T315I or other mutations relatively insensitive to them. We assessed the change in mutation status of the bcr-abl kinase domain (codons 220 to 500) in 113 patients (pts) with CML who received therapy with D and/or N after imatinib failure. Median age was 60 years (range, 21 to 82 years). Seventy-one (63%) pts received prior interferon (IFN). Median time on imatinib was 28 months (range, 2 to 78 months). At the time of imatinib failure, mutations were detected in 46 of 85 (54%) pts who had DNA sequencing. The evolution of mutations after a second TKI was as follows (Table 1). Twenty pts received a third TKI after failing IM and a second TKI. The evolution of mutations in this cohort was as follows (Table 2). Overall, 19 of 101 evaluable pts (19%), cases had new mutations emerge following TKI switch 17 after a 2nd TKI (12 nilotinib, 5 dasatinib), and 2 after a 3rd TKI (2 dasatinib). We analyzed whether these N- and D-associated new mutations were at sites that have been detected following D and N treatment in vitro (Burgess et al, PNAS 2005; Bradeen et al, Blood 2006; Von Bubnoff et al, Blood 2006). Only 14/46 (30%) kinase domain mutations that developed after D (7) or N (7) corresponded with an in vitro-identified site. Only 5 of 134 (4%) mutations identified were T315I (3 after dasatinib, 2 after nilotinib), but the mutation status of these patients was unknown after IM. We conclude that the spectrum of mutations that develops in vivo after TKI switch is broader and includes common imatinib-resistance sites as well. There appears to no marked increase in the incidence of T315I mutation after TKI switch. Table 1. Dynamics of mutations after 2nd TKI Post IM mutation No. Post-2nd TKI Mutation (New + Same + Lost) *1 pt acquired new mutation with persistence of pre-existing mutation, 1 lost 3 mutations and acquired 1, and 1 pt lost 2 mutations. Nilotinib Dasatinib Absent 39 8+NA+NA/21 3+NA+NA/18 Present 46 3+20+3/26 2+16+2*/20 Unknown 28 8/9 13/19 Table 2. Dynamics of mutations after 3rd TKI Post IM mutation No. Post-3nd TKI Mutation (compared to status after 2nd TKI) (New + Same + Lost) Nilotinib Dasatinib Absent 5 0/1 1+NA+NA/4 Present 12 0+1+0/1 2+6+3/11 Unknown 3 1/3 NA

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De Novo ABL Kinase Domain Mutations in Murine Bone Marrow Cells Retrovirally Transduced with Wild-Type p210 BCR-ABL and Amplified in Culture with Imatinib Mesylate.

Blood ◽

10.1182/blood.v104.11.1990.1990 ◽

2004 ◽

Vol 104 (11) ◽

pp. 1990-1990

Author(s):

Stephane Flamant ◽

Martine Guillier ◽

Marie-Laure Bonnet ◽

Jean-Pierre Lecouedic ◽

William Vainchenker ◽

...

Keyword(s):

Bone Marrow ◽

Imatinib Mesylate ◽

Cell Lines ◽

De Novo ◽

Bone Marrow Cells ◽

Kinase Domain ◽

Abl Kinase ◽

Kinase Domain Mutations ◽

Marrow Cells

Abstract Imatinib mesylate (IM) is a tyrosine kinase inhibitor which is highly efficient in chronic myelogenous leukemia (CML), especially in the firts chronic phase of the disease. Recent data showed, however, that resistance to IM can develop in patients in more aggressive phases of their disease, which mainly occurs through mutations within the ABL kinase domain that interfere with IM binding, leading to IM-resistant relapses. The mechanisms of the occurrence of ABL kinase domain mutations in patients on IM therapy are not well understood, and in some of them, a mutation pre-existing to the introduction of IM was described, suggesting the possibility of a clonal selection under IM therapy. To determine if ABL kinase domain mutations could be induced de novo in primary marrow cells, we used an ecotropic BCR-ABL retrovirus (MIGR-p210 vector, 5.105 viral particles / ml) and infected 5-FU-treated bone marrow cells from C57BL/6 mice. Retrovirally transduced cells (30% GFP+) were transplanted in lethally irradiated animals in which they induced lethal leukemia in 3 weeks. Both BCR-ABL-transduced and control C57BL/6 bone marrow cells were seeded in liquid cultures (104 cells/well) in the presence of 0.25 μM IM with weekly half medium changes during which the concentration of IM was increased gradually from 0.25 to 2 μM over 2 months. In these conditions, no growth could be obtained from normal bone marrow cells (0/192 wells) whereas in 10/192 wells containing BCR-ABL-transduced cells, we observed significant growth on IM. These cells were then amplified in the presence of murine stromal MS-5 cells and 2 μM IM for over 6 months and 2 clones (C3 and C10) exhibiting persistent growth were further characterized. At cytological analysis both cell lines had a typical mast cell morphology. Flow cytomery analyses demonsrated the presence of CD41 marker on both cell lines, with absence of myeloid (Gr1), erythroid (Ter119) and B-cell (B220) markers. Cells were not polyploid and interestingly, they exhibited higher growth rates in the presence of IM, with reduced growth upon IM deprivation. Both cell lines had evidence of BCR-ABL vector integration by PCR analysis and were highly GFP+. To explore the mechanisms of IM-resistance in these cells, we extracted high molecular weight genomic DNA and amplified a BCR-ABL fragment of 1236 bp encompassing the ABL kinase domain of the integrated construct. We then sequenced the ABL kinase domain using internal primers in both 5′-3′ directions. Plasmid DNA from the original MIGR-p210 BCR-ABL vector served as control. In both clones (C3 and C10) ABL kinase point mutations were readily detectable which were not found in the BCR-ABL retroviral vector. C3 carried two mutations interesting the C helix (E300K) and the SH2 contact region (E371K) of ABL kinase domain, whereas C10 carried a single mutation in the C helix (D295N). These mutations were previously detected in a random in vitro mutagenesis assay of BCR-ABL in bacterial systems. Thus, our model is the first demonstration of the occurrence of ABL kinase domain mutations and the concomittant generation of an IM-resistant phenotype in primary marrow stem cells transduced with BCR-ABL vector DNA and selected in the presence of IM. The mechanisms of the occurrence of these mutations in vitro are currently under study but these results suggest that de novo ABL kinase mutations could also occur in vivo in CML patients treated with IM.

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Leukemic Stem Cells of Chronic Phase CML Patients Possess Uniquely Elevated BCR-ABL Kinase Activity and Acquire Spontaneous BCR-ABL Kinase Domain Mutations at a High Frequency.

Blood ◽

10.1182/blood.v106.11.438.438 ◽

2005 ◽

Vol 106 (11) ◽

pp. 438-438 ◽

Cited By ~ 1

Author(s):

Xiaoyan Jiang ◽

Kyi Min Saw ◽

Allen Eaves ◽

Connie Eaves

Keyword(s):

Stem Cells ◽

Tyrosine Kinase ◽

Kinase Activity ◽

Point Mutations ◽

Chronic Phase ◽

Kinase Domain ◽

Tyrosine Kinase Activity ◽

Abl Kinase ◽

Kinase Domain Mutations

Abstract Growing evidence indicates that the therapeutic potential of imatinib mesylate (IM) for the treatment of CML may be limited initially by a relative innate resistance of the leukemic stem cells and eventually by an accumulation of cells with BCR-ABL tyrosine kinase domain mutations. We now show that the amount and tyrosine kinase activity of p210-BCR-ABL in the most primitive and relatively IM-unresponsive lin−CD34+CD38− CML cells is 3 to 10-fold higher than in the majority of the lin−CD34+CD38+ CML progenitors (n=3). These results confirm previous BCR-ABL transcript data and identify elevated p210-BCR-ABL expression to be a likely important factor in the characteristic IM-insensitivity of very primitive CML cells. To determine whether in vivo, CML stem cells also accumulate gene mutations affecting the BCR-ABL kinase domain, cDNAs were prepared from RNA extracts of purified lin−CD34+CD38− cells isolated from 3 chronic phase patients that had not received IM therapy. Bidirectional sequencing of individually cloned cDNAs from these samples revealed BCR-ABL kinase domain mutations in 2 of the 3 patients at frequencies of 10% (1/10), 20% (2*/10,*identical mutations). Incubation of these lin−CD34+CD38− cells in vitro for 2–3 wk ± a high concentration of IM (up to 10 μM, which was sufficient to reduce the tyrosine kinase activity in the input cells by 70±12% and in their 2 wk progeny by 10±5%) selected a subpopulation of more differentiated and completely IM-resistant cells. This was shown in Western blots by the inability of 10 μM IM to reduce either their p210-BCR-ABL tyrosine kinase activity or CrkL phosphorylation and in methylcellulose assays ±5 μM IM. As predicted, IM-selected cells showed a higher frequency of kinase domain mutations (13–20% vs 0–20% of cDNA clones analyzed from 3 wk cells cultured ±IM). Analysis of individual colonies produced from CFCs in the cultured cells showed all (21/21) colonies from IM-selected cells had mutations vs 50% (5/10) in those cultured without IM. The total frequency of mutant cDNAs detected was also increased in the IM-resistant cells (35–55% vs 10–25% mutant cDNAs in selected vs control cells). Interestingly, in most cases, both wild-type and mutant cDNAs were identified in the same colony, indicating de novo generation of mutations in vitro. Overall, >50 different mutations were identified. These included 10 point mutations previously associated with clinical IM resistance (including G250 and T315), another 13 point mutations previously identified in a comprehensive mutational screen, and >20 previously undescribed mutations. Several of the latter affect the critical region of the P loop, the c-helix and the activation loop and would be predicted to confer significant IM resistance. To investigate the possibility that the observed genomic instability of very primitive CML cells might be related to their elevated innate p210-BCR-ABL activity, BCR-ABL transcript levels in individual IM-selected, fully resistant and control (similarly treated but no IM exposure) colonies were compared. This showed that BCR-ABL transcripts were ~20-fold higher (P<0.05) in the resistant colonies (30 assessed from 3 patients). These findings suggest that the increased BCR-ABL expression and activity that uniquely characterizes the most primitive CML cells may contribute not only to their innate insensitivity to IM but also to a deregulation of genomic stability leading to the emergence of IM-resistant mutants and other subclones associated with disease progression.

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Correlation of Clinical Response to Nilotinib with BCR-ABL Mutation Status in Advanced Phase Chronic Myelogenous Leukemia (CML-AP) Patients with Imatinib-Resistance or Intolerance.

Blood ◽

10.1182/blood.v110.11.1940.1940 ◽

2007 ◽

Vol 110 (11) ◽

pp. 1940-1940 ◽

Cited By ~ 2

Author(s):

Giuseppe Saglio ◽

Dong-Wook Kim ◽

Andreas Hochhaus ◽

Simona Soverini ◽

P. Erben ◽

...

Keyword(s):

Disease Progression ◽

Cytogenetic Response ◽

Kinase Domain ◽

Mutant Clone ◽

Imatinib Resistance ◽

Mutation Status ◽

Abl Kinase ◽

Imatinib Resistant ◽

Kinase Domain Mutations

Abstract The 2nd-generation bcr-abl inhibitor nilotinib is more potent than imatinib (IC50 <30 nM) against unmutated bcr-abl and active against 32/33 imatinib-resistant BCR-ABL mutants in vitro. We investigated the in vivo activity of nilotinib stratified by the baseline BCR-ABL mutation status in 127 imatinib-resistant or -intolerant CML-AP patients (pts) enrolled in an open-label phase II trial of nilotinib. Eighty-five pts (85/127, 67%) were screened prior to nilotinib therapy for BCR-ABL kinase domain mutations by direct sequencing. Of the 85 pts, 75 (88%) were resistant to imatinib and 10 (12%) were intolerant using standard published criteria. Twenty-two different baseline mutations involving 19 amino acids were identified in 50 (59%) pts analyzed. Other 35 (41%) pts did not have a baseline mutation. The most frequent mutation types identified included M351T (8 pts), G250E (7 pts), Y253H (6 pts), M244V (5 pts), F359V (5 pts) and T315I (5 pts). Twenty-two percent of pts with baseline mutations (11/50) showed more than one mutation (9 with two, 1 with three, and 1 with four mutations). All baseline mutations occurred in imatinib-resistant pts but none in intolerant pts. After 12 months of therapy, confirmed (confirmed in two consecutive analyses 4 week apart) hematologic response (HR) was achieved in 48% (21/50), major cytogenetic response (MCR) in 20% (10/50), and complete cytogenetic response (CCR) in 16% (8/50) of imatinib-resistant pts with baseline mutation versus 44% (12/25), 40% (10/25), and 20% (2/25) of imatinib-resistant pts without baseline mutation, respectively. Responses appeared to be affected by the in vitro sensitivity of the mutant clone against nilotinib. Pts with less sensitive mutation (cellular IC50 of >200nM: Y253H, E255K, E255V, F359C) representing 13% (11/85) of all patients assessed for baseline mutation, showed 13% (1/11) HR and 13% (1/11) MCyR compared to 74% (17/28) and 18% (5/28) respectively in the mutant group with IC50 of ≤200 nM. The nilotinib resistant T315I mutation occurred in 5 pts. Only one of these 5 pts who had T315I and G250E dual mutation achieved HR conceivably reflecting the sensitivity of G250E or non-mutant clone to nilotinib. At the time of data analyses, 50% of pts with baseline mutation were free of disease progression versus 62% of pts without baseline mutation. Rate of progression was 64% (7/11) in the group with less sensitive mutations and 60% (3/5) in pts. with T315I. However, the mutants most frequently associated with progression were F359V and M244V both having 4/5 pts (80%) progressed. In summary, BCR-ABL kinase domain mutations were identified at baseline in 59% of all pts in this cohort and in 67% of pts with imatinib resistance. Responses were observed across a broad spectrum of mutant genotypes. The rate of responses and disease progression may be affected by the baseline mutation types, although a larger data set with longer follow up is needed to further establish the correlation.

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Properties of CD34+ CML Cells That Predict Clinical Response to Tyrosine Kinase Inhibitors.

Blood ◽

10.1182/blood.v110.11.324.324 ◽

2007 ◽

Vol 110 (11) ◽

pp. 324-324

Author(s):

Xiaoyan Jiang ◽

Donna Forrest ◽

Franck Nicolini ◽

Karen Lambie ◽

Kyi Min Saw ◽

...

Keyword(s):

Stem Cells ◽

Tyrosine Kinase ◽

Blast Crisis ◽

Chronic Phase ◽

Kinase Domain ◽

Abl Kinase ◽

Cd34 Cells ◽

Clinical Responses ◽

Kinase Domain Mutations

Abstract Imatinib (IM) treatment causes remission in a majority of patients with chronic myeloid leukemia (CML) but relapses remain a problem. The frequent presence in relapsing cells of BCR-ABL kinase domain mutations suggests that their prior but undetected acquisition by rare CML stem cells may be a major contributor to IM treatment failures. We have recently demonstrated that enriched populations of CML stem cells (lin−CD34+CD38− cells) are relatively insensitive to IM and possess multiple unique features that would be expected to promote both innate and acquired mechanisms of resistance to BCR-ABL-targeted therapeutics. These include elevated BCR-ABL expression and tyrosine kinase activity, increased expression of ABCB1/MDR1 and ABCG2, decreased expression of OCT1, and a high degree of genetic instability, as demonstrated by a rapid accumulation of BCR-ABL mutations in vitro. To determine whether these parameters may be predictive of clinical responses to IM, immunomagnetically selected CD34+ stem/progenitor cells from 18 chronic phase CML patients’ samples obtained prior to IM therapy were evaluated and the results compared with subsequent clinical responses. Direct sequencing of transcripts cloned from extracts of freshly isolated CD34+ cells (10 clones/sample) detected a high frequency of pre-existing BCR-ABL kinase mutations in the CD34+ cells from 12 of 12 patients regardless of their subsequent IM responses (20–80%). Interestingly, a higher incidence of BCR-ABL kinase domain mutations was found in 5 IM-nonresponders (33–80% of transcripts showed ≥1 BCR-ABL kinase domain mutation) as compared to 5 IM-responders (values of 20-30%, P<0.02). A higher frequency of BCR-ABL kinase domain mutations was also detected in extracts of colonies generated from assays of cells harvested from 3-week suspension cultures initiated with the same starting CD34+ CML cells (21–68% vs 10–43%). A high incidence of BCR-ABL kinase domain mutations was also documented in freshly isolated or cultured CD34+ cells from 2 patients who developed sudden blast crisis (50–63% and 17–83%). Overall, 38 different mutations were identified from freshly isolated CD34+ CML cells and >50 additional mutations were identified in the progeny of CD34+ CML cells cultured ± IM. These included 15 point mutations frequently associated with clinical IM resistance (including G250, Q252, E255, T315, M351, F359 and H396) and >40 mutations not previously described. Furthermore, freshly isolated CD34+ cells from IM-nonresponders (including the 2 patients who developed blast crisis, n=10) showed a greater resistance to IM in vitro (∼2 fold, P< 0.001 with 5 μM and P<0.02 with 10 μM IM) as compared to CD34+ cells from IM-responders (n=8) in the presence of 5 and 10 μM IM, as determined by colony-forming cell (CFC) assays. Although more IM-resistant CFCs were obtained in the presence of IM from 3-week cultures initiated with CD34+ cells from the same IM-nonresponders than from IM responders, these latter differences were not significantly different (P= 0.28). These results suggest that the CD34+ leukemic cells from individual chronic phase CML patients harbor differences in their biologic properties that are predictive of how they will respond to IM therapy and that assessment of these differences may form the basis of rapid, practical and quantitative tests to assist in optimized patient management.

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Optimal Treatment for Patients after Dasatinib or Nilotinib: Comparison of Preclinical Activities of Approved and Promising Investigational Kinase Inhibitors Against BCR-ABL Kinase Domain Mutations That Confer Clinical Resistance to Dasatinib or Nilotinib.

Blood ◽

10.1182/blood.v110.11.4589.4589 ◽

2007 ◽

Vol 110 (11) ◽

pp. 4589-4589

Author(s):

Corynn Kasap ◽

Christopher Weier ◽

Neil P. Shah

Keyword(s):

Second Generation ◽

Kinase Inhibitors ◽

Kinase Inhibitor ◽

Kinase Domain ◽

Inhibitor Therapy ◽

Drug Resistant ◽

Imatinib Resistance ◽

Abl Kinase ◽

Clinical Resistance ◽

Kinase Domain Mutations

Abstract The optimal management of patients with chronic myeloid leukemia (CML) is increasingly reliant upon molecular studies. Loss of response to imatinib in CML is most commonly associated with selection for a limited number of BCR-ABL kinase domain mutations that impair the ability of imatinib to effectively bind to BCR-ABL Molecular understanding of imatinib resistance mechanisms has led to the development of effective “second generation” BCR-ABL kinase inhibitors, such as dasatinib and nilotinib, which have clinical activity against most, but not all, drug-resistant mutations. Analysis of the BCR-ABL kinase domain in patients who develop resistance to second-generation inhibitors has implicated further selection of drug-resistant BCR-ABL kinase domain mutants in nearly all cases reported to date. Encouragingly, the number of resistant mutations capable of conferring clinical resistance to the most clinically-advanced second-generation agents, dasatinib (approved by the US FDA and EMEA) and nilotinib (approved in Mexico and Switzerland), appears to be restricted to a relatively small number of amino acid substitutions. As clinical experience with dasatinib and nilotinib grows, an understanding of the relative sensitivities of dasatinib- and nilotinib-resistant BCR-ABL mutants to other kinase inhibitors, both approved and investigational, is critical to optimize clinical outcomes in patients with resistance to dasatinib or nilotinib. At the present time, kinase inhibitor therapy options for patients with resistance to one of these agents include the investigational options bosutinib and MK-0457 (VX-680), as well as dasatinib and nilotinib (for patients not yet exposed to one of these agents) and re-exposure imatinib. It is likely that the success of therapeutic intervention in these cases can be predicted based upon the preclinical sensitivity of the mutation(s) involved with the agent chosen. We have therefore conducted a thorough biochemical and biological cross-analysis of the activities of each of these clinically-useful kinase inhibitors against mutations that confer clinical resistance to dasatinib or nilotinib. These studies provide clinicians with a useful reference for choosing an appropriate kinase inhibitor based upon the identity of the resistant BCR-ABL kinase domain mutation(s) detected at the time of relapse when faced with a patient who has lost response to dasatinib or nilotinib. It is hoped that the application of such “personalized medicine” strategies to the clinical management of CML cases will further improve outcomes in patients treated with kinase inhibitor therapy.

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Screening of Mutations in BCR-ABL Kinase Domain in Chronic Myeloid Leukemia (CML) Patients Treated with Kinase Inhibitors by Denaturing High-Performance Liquid Chromatography (D-HPLC).

Blood ◽

10.1182/blood.v110.11.4580.4580 ◽

2007 ◽

Vol 110 (11) ◽

pp. 4580-4580

Author(s):

Cintia C. Mascarenhas ◽

Anderson F. Cunha ◽

Katia B.B. Pagnano ◽

Rosana A. Silveira ◽

Fernando F. Costa ◽

...

Keyword(s):

Bone Marrow ◽

Bone Marrow Transplantation ◽

Marrow Transplantation ◽

Kinase Inhibitors ◽

Blast Crisis ◽

Point Mutations ◽

Kinase Domain ◽

Molecular Response ◽

Abl Kinase ◽

Clinical Resistance

Abstract Point mutations within the ABL kinase domain are the most frequent mechanism for reactivation of kinase activity of the BCR-ABL gene and have been associated with clinical resistance to tyrosine kinases (TK) inhibitors in CML patients conferring in some of them a poor prognosis. The T315I (Treonine → Isoleucine) is a mutation described in exon 6 of BCR-ABL gene that makes the protein resistant to all kinase inhibitors most currently used for treating CML (imatinib, nilotinib and dasatinib). D-HPLC allows for high throughput mutation screening. This technique is based on heteroduplex formation by PCR products amplified from wild type and mutant alleles. Under optimized denaturing conditions, these heteroduplexes can be distinguished from homoduplex. In this study we screened mutations in exon 6 of BCR-ABL gene in patients treated with kinase inhibitors, in different phases of the disease. We evaluated 85 patients: 9 at diagnosis, 81 in chronic phase, 3 in accelerated phase, one in blast crisis. Thirty four were resistant to imatinib, 10 of them to dasatinib and three had suboptimal response to imatinib. In 9 of 85 (10,5%) samples, D-HPLC showed an abnormal elution profile suggesting the presence of nucleotide changes. Automated sequencing confirmed the presence of two point mutations: T315I (two patients) and F359V (two patients). Five patients requires sequencing confirmation. Patients with T315I mutation failed to imatinib and dasatinib. One of them relapsed after bone marrow transplantation in blast crisis. Patients with F359V mutation were resistant to imatinib. One of them has partial hematological response with dasatinib and the other is in complete molecular response after bone marrow transplantation. D-HPLC seems to be a ship and practical method for routine clinical monitoring for emergence of kinase domain mutations and may be useful for optimizing therapy in CML. Early detection of emerging mutant clones may help in decision-making of alternative treatment.

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Comparison of Imatinib, AMN107 and Dasatinib in an Accelerated Cell-Based Mutagenesis Screen.

Blood ◽

10.1182/blood.v106.11.691.691 ◽

2005 ◽

Vol 106 (11) ◽

pp. 691-691 ◽

Cited By ~ 1

Author(s):

Michael W.N. Deininger ◽

Heather Bradeen ◽

Taiping Jia ◽

Thomas O’Hare ◽

Stephanie G. Willis ◽

...

Keyword(s):

Structural Changes ◽

Kinase Inhibitors ◽

Kinase Domain ◽

Abl Kinase ◽

Drug Concentrations ◽

A Cell ◽

Vitro Model ◽

Proliferation Assays ◽

Abl Kinase Inhibitors

Abstract Background. Mutations in the Bcr-Abl kinase domain (KD) are the leading cause of acquired imatinib (IM) resistance. Dasatinib (BMS354825) and AMN107 are potent alternate Abl inhibitors with activity at nanomolar levels against wild type Bcr-Abl and most KD mutants, with the exception of T315I. In a cell-line based mutagenesis assay we compared incidence and type of Bcr-Abl mutants emerging in the presence of IM, AMN107 and dasatinib. Methods. BaF3-p210Bcr-Abl cells were mutagenized by 24 hours exposure to 0.42 μM N-ethyl-N-nitrosourea (ENU), a dose with minimal cytotoxicity. After ENU washout cells were seeded at 5 x 105/well in 96-well plates and observed for growth for up to 4 weeks. Cells from wells with growth were expanded and subjected to BCR-ABL KD sequencing. Results: The frequency of wells with growth decreased with higher doses of all 3 inhibitors (table 1) and growth tended to occur later. Only isolated wells had growth without ENU exposure. At ≥2 μM IM (2-fold the IC90 in cell proliferation assays) 18 different mutations were seen, with highly resistant mutants prevailing at higher concentrations (table 2). At 50 nM AMN107 (2-fold the IC90) Y253H, G250E, F359C, E255K, L384M, L387F, E292V and T315I were detected, at 500 nM Y253H, E255V and T315I were recovered and only T315I at 2000 nM. At 5 nM dasatinib (2-fold the IC90), E255K, L284V, F317V were detected in addition to T315I, at 10 nM T315I, F317V/I and V299L were found and at 25 nM only T315I. All resistant clones growing out at ≥4 μM IM, 500 nM AMN107 or 10 nM dasatinib were KD mutant, suggesting that KD mutations were the sole cause of the observed resistance. Conclusions: (i) At drug concentrations corresponding to 2-fold IC9018 different mutations were recovered with IM, 9 with AMN107 and 6 with dasatinib, suggesting that the conformational requirements for dasatinib binding to Abl may be least stringent. If free plasma trough levels ≥25 nM dasatinib or ≥2000 nM AMN107 are achievable, the only mutant predicted to emerge clinically is T315I. (ii) No additional mutations were observed with AMN107 compared to IM, suggesting the structural changes in AMN107 compared to IM did not generate novel vulnerable sites. (iii) At least in this in vitro model, resistance to Abl kinase inhibitors is entirely dependent on Bcr-Abl, despite the fact that ENU treatment is expected to induce multiple additional mutations. Thus a T315I inhibitor combined with AMN107 or dasatinib may be effective at preventing the emergence of resistance to Abl kinase inhibitors. Table 1 Recovery of resistant clones (representative experiment) Imatinib (microM) Wells with mutations/wells sequenced/wells with growth Dasatinib (nM) Wells with mutations/wells sequenced/wells with growth AMN107 (nM) Wells with mutations/wells sequenced/wells with growth 2 62/62/82 5 8/24/96 10 0/24/96 4 74/74/74 10 38/38/56 50 20/24/96 8 27/27/42 25 22/22/24 500 45/45/46 16 12/12/12 100 20/20/21 2000 23/23/24 Table 2 Percentage of resistant clones with T315I mutations Imatinib (μM) number of different mutations/% T315I Dasatinib (nM) number of different mutations/% T315I AMN107 (nM) number of different mutations/% T315I 2 16/27.8 5 4/16.7 10 0/0.0 4 7/43.2 10 3/63.2 50 8/20.8 8 4/37.4 25 1/100 500 3/62.0 16 4/50.0 100 1/100.0 2000 1/100.0

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Generation of Specific Resistance Profiles in FLT3-ITD and FIP1L1-PDGFRalpha towards Specifically Acting Tyrosine Kinase Inhibitors.

Blood ◽

10.1182/blood.v108.11.1376.1376 ◽

2006 ◽

Vol 108 (11) ◽

pp. 1376-1376

Author(s):

Nikolas von Bubnoff ◽

Silvia Thoene ◽

Sivahari P. Gorantla ◽

Jana Saenger ◽

Christian Peschel ◽

...

Keyword(s):

Tyrosine Kinase ◽

Tyrosine Kinase Inhibitors ◽

Kinase Inhibitors ◽

Kinase Inhibitor ◽

Point Mutations ◽

Kinase Domain ◽

Myelogenous Leukemia ◽

Resistance Mutations ◽

Abl Kinase ◽

Mechanism Of Resistance

Abstract BCR-ABL kinase domain mutations constitute the major mechanism of resistance in patients with chronic myelogenous leukemia treated with the ABL kinase inhibitor imatinib. Mutations causing resistance to therapeutic kinase inhibition were also identified in other target kinases in various malignant diseases, such as FLT3-ITD in acute myelogenous leukemia, cKit in gastrointestinal stromal tumors, EGFR in patients with lung cancer, and FIP1L1-PDGFRalpha in hypereosinophilic syndrome. Thus, mutations in kinase domains seem to be a general mechanism of resistance to therapeutically applicated tyrosine kinase inhibitors. We recently developed a cell-based screening strategy that allows one to predict the pattern and relative abundance of BCR-ABL resistance mutations emerging in the presence of imatinib, and the novel ABL kinase inhibitor AMN107 (nilotinib). We therefore intended to determine, if this method would also allow the generation of resistant cell clones with other oncogeneic tyrosine kinases as targets in the presence of specifically acting kinase inhibitors. When FLT3-ITD and su5614 were used as drug/target combination in our cell-based method, the frequency of resistant clones in the presence of su5614 at 10 times the IC50 was 0.17 per million cells. In 40 per cent of resistant clones, point mutations were detected leading to amino acid exchanges within the FLT3-ITD split kinase domain. The yield of resistant clones was increased by the factor of 14 to 2.37 per million cells by adding ethyl-nitrosourea (ENU), a potent inducer of point mutations. Also, the proportion of mutant clones increased from 40 to 74 per cent. In 83 mutant clones that were examined so far, we detected eight exchanges affecting kinase domain two (TK2) of the split kinase domain within or shortly behind the FLT3-ITD activation loop (A-loop). We did not detect exchanges affecting TK1. We next examined whether resistant clones would also come up with FIP1L1-PDGFRalpha-transformed cells in the presence of imatinib. Again, the yield of resistant clones increased when cells were pretreated with ENU, and a proportion of resistant clones contained mutations in the FIP1L1-PDGFRalpha kinase domain, affecting the nucleotide-binding loop (P-loop) and A-loop. We conclude that cell-based resistance screening is a simple and powerful tool that allows prediction of resistance mutations towards kinase inhibitors in various relevant oncogeneic kinases.

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Rac GTPases Are Potential Therapeutic Targets in p210-BCR-ABL-Induced Myeloproliferative Disease (MPD).

Blood ◽

10.1182/blood.v110.11.465.465 ◽

2007 ◽

Vol 110 (11) ◽

pp. 465-465

Author(s):

Emily K. Thomas ◽

Jose A. Cancelas ◽

Heedon Chae ◽

Adrienne D. Cox ◽

Patricia J. Keller ◽

...

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Median Survival ◽

Peripheral Blood ◽

Kinase Inhibitors ◽

Abl Kinase ◽

Rac Gtpases ◽

Imatinib Resistant ◽

Abl Kinase Inhibitors

Abstract The p210-BCR-ABL fusion protein is a constitutively active tyrosine kinase that is necessary and sufficient for the development of chronic myelogenous leukemia (CML). ABL-kinase inhibitors such as imatinib mesylate (Gleevec, STI571) potently block BCR-ABL activation, but the continued presence of leukemic stem cells and the emergence of imatinib-resistant BCR-ABL mutants suggest that ABL kinase inhibitors alone cannot completely eradicate disease. Rac GTPases have been implicated in BCR-ABL-mediated proliferation in cell lines and regulate many of the same signaling pathways as BCR-ABL, suggesting that these proteins could be additional therapeutic targets in CML. We have found that Rac1, Rac2, and, to a lesser extent, Rac3 were hyperactivated in CD34+ cells purified from the peripheral blood of two CML patients. To better study the role of Rac in BCR-ABL disease development, murine hematopoietic stem cells (HSC) genetically deficient in Rac1 and/or Rac2 were transduced with a retroviral vector expressing p210-BCR-ABL. Wild type (WT) and Rac1−/− mice experienced similar disease progression [median survival 23 ± 6 days (n=30) and 22 ± 4 days (n=8), respectively], Rac2−/− mice exhibited significantly attenuated development of BCR-ABL-mediated MPD [median survival 43 ± 27 days (n=18); p<0.001], and Rac1−/−;Rac2−/− animals showed markedly prolonged survival [median survival 92 ± 34 days (n=19); p<0.001]. p210-BCR-ABL WT, Rac1−/−, and Rac2−/− mice had elevated circulating myeloblasts 30 days post-transplant, while Rac1−/−;Rac2−/− mice had normal peripheral blood morphology. Attenuation of disease in Rac2- and Rac1/Rac2-deficient animals correlated with severely diminished activation of BCR-ABL-induced signaling pathways, including p44/42 and p38 ERK, JNK, CrkL, and Akt. The leukemogenesis impairment induced by Rac deficiency did not appear to be due to loss of p210-BCR-ABL vector integration, as clonal analysis of leukemic bone marrow from mice in each genotype by LAM-PCR showed similar, oligoclonal reconstitution of p210-BCR-ABL expressing cells. Interestingly, bone marrow cells obtained from Rac1/Rac2-deficient animals that developed late leukemia showed marked hyperactivation of Rac3 and initiated disease in recipients with a latency of three weeks, suggesting that leukemia-initiating cells were able to engraft, in spite of Rac1/Rac2 deficiency. Treatment of BCR-ABL-expressing murine HSC with NSC23766, a rationally-designed Rac-specific small molecule antagonist, potently inhibited cell proliferation in vitro and increased the survival of leukemic animals treated in vivo, compared to PBS control-treated animals (p<0.05). NSC23766 also inhibited the growth of an imatinib-resistant p210-BCR-ABL-T315I-expressing Ba/F3 leukemic cell line by 90%, compared to <5% by imatinib alone, blocked the growth of primary human chronic phase Rac-hyperactivated CML blast colonies by 80% in vitro, and inhibited survival of these cells in NOD-SCID mice. These results suggest that individual Rac proteins play both unique and combinatorial roles in stem cell transformation and may represent unique targets for therapy of BCR-ABL-persistent and imatinib-resistant CML.

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