Chronic Myeloproliferative Neoplasias

The gatekeeper mutation T315I confers resistance against small molecules by increasing or restoring the ABL-kinase activity accompanied by aberrant transphosphorylation of endogenous BCR, even in loss-of-function mutants of BCR/ABL

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In Philadelphia chromosome-positive (Ph+) leukemia BCR/ABL induces the leukemic phenotype. Targeted inhibition of BCR/ABL by kinase inhibitors leads to complete remission. However, patients with advanced Ph+ leukemia relapse and acquire resistance, mainly due to point mutations in BCR/ABL. The ‘gatekeeper mutation’ T315I is responsible for a general resistance to small molecules. It seems not only to decrease the affinity for kinase inhibitors, but to also confer additional features to the leukemogenic potential of BCR/ABL. To determine the role of T315I in resistance to the inhibition of oligomerization and in the leukemogenic potential of BCR/ABL, we investigated its influence on loss-of-function mutants with regard to the capacity to mediate factor independence. Here, we show that T315I (i) requires autophosphorylation at tyrosine 177 in the BCR-portion to mediate resistance against the inhibition of oligomerization; (ii) restores the capacity to mediate factor-independent growth of loss-of-function mutants due to an increase in or activation of ABL-kinase; (iii) leads to phosphorylation of endogenous BCR, suggesting aberrant substrate activation by BCR/ABL harboring the T315I mutation. These data show that T315I confers additional leukemogenic activity to BCR/ABL, which might explain the clinical behavior of patients with BCR/ABL–T315I-positive blasts.


The BCR/ABL fusion protein is the hallmark of Philadelphia chromosome-positive (Ph+) leukemia. BCR/ABL is characterized by deregulated and constitutively activated ABL tyrosine kinase activity that determines its transformation potential.1 Cellular transformation and leukemogenesis are strictly dependent on the tyrosine kinase activity of BCR/ABL.2, 3, 4, 5 Molecular-targeted therapy with selective ABL-kinase inhibitors (AKI) such as imatinib, dasatinib, or nilotinib induces complete hematological and cytogenetic remission in the majority of Ph+ leukemia patients.6 In advanced disease stages, relapse frequently occurs and is accompanied by resistance to further treatment with the used compound. In the majority of the cases, clones harboring point mutations in the ABL portion of the fusion protein that interfere with the binding affinity for the inhibitors are selected upon exposure to AKI.7, 8 The most frequent mutations are the ‘P-loop’ mutations Y253F and E255K, the ‘activation loop’ mutation H396P, the ‘catalytic domain’ mutation M351 T and the ‘gatekeeper’ mutation T315I.9 The activity spectrum of actually available AKI covers 14/15 of these mutations. T315I is the only mutation that confers resistance against virtually all ATP competitors.10

There is increasing evidence that these mutations not only interfere with binding affinity to the kinase inhibitors, but also modify the biological functions of BCR/ABL. The observation that additional factors influence the transformation potential of the AKI-resistant BCR/ABL T315I mutant was supported by our recent findings on the inhibition of tetramerization of BCR/ABL and its AKI-resistant mutants. Tetramerization of ABL through the N-terminal coiled-coil region (CC) of BCR is essential for aberrant ABL-kinase activation. Targeting the CC-domain forces BCR/ABL into a monomeric conformation, abolishes its transformation potential by interfering with its kinase activity and increases sensitivity to Imatinib.11, 12 We showed that factor-dependent hematopoietic progenitors expressing BCR/ABL harboring the Y253F and E255K mutations but not the T315I mutation responded to the inhibition of tetramerization. Together with previous findings, these results led to the hypothesis that BCR/ABL harboring the T315I mutation exhibits additional functions contributing to its leukemogenic potential that are not present in the unmutated BCR/ABL. In fact, it has been shown that the BCR/ABL mutants Y253F and E255K exhibited increased transformation potential and the M351T and H396P mutations showed reduced transformation potential, whereas the potential of T315I was assay-dependent.14 Interestingly, the transformation potentials of these BCR/ABL mutants were not consistently correlated with intrinsic ABL-kinase activity. These findings strongly suggest the presence of additional factors influencing the transformation potential of BCR/ABL harboring E255K or T315I.

Therefore, in this study we investigated why inhibition of oligomerization is ineffective in the case of cells expressing the BCR/ABL–T315I mutant and whether T315I confers additional features to the leukemogenic activities of BCR/ABL that may account for its activity as a monomer and also for its resistance to AKI. This is of particular importance considering the high frequency of resistance to AKI occurring, especially in patients with Ph+ ALL. Therefore, we focused our study on the Ph+ ALL associated p185BCR/ABL.

Materials and methods


All cDNAs encoding p210BCR/ABL as well as p185BCR/ABL and its mutants have been described earlier.11, 12, 13 All retroviral expression vectors used in this study were based on the bi-cistronic vectors PINCO or PAULO converted into Gateway-destination vectors by the introduction of a Gateway cassette according to the manufacturer's instructions (Invitrogen, Karlsruhe, Germany). All related inserts were available in the Gateway entry-vector (pENTR1A) for recombination into the destination vectors using the ‘LR-clonase’ enzyme kit (Invitrogen). ΔCCp185–Y253F, ΔCCp185–E255K, ΔCCp185–T315I, ΔCCp185–T315I, ΔCCp210 and ΔCCp210–T315I were generated by substituting the sequence encoding the first 63 aa of p185BCR/ABL by a Kozak consensus by using the following primers: 5′-IndexTermatctacctgcagacgacgatggccaag-3′ and 5′-IndexTermatggcccttgcggatccgctcg-3′. The resulting fragments were cloned into BamHI-digested p185–Y253F, p185–E255K, p185–T315I and p210BCR/ABL sequences. BCC/ABL-T315I was cloned by ‘megaprimer’ PCR. First, PCR was performed with the primers Pr1 5′-IndexTermccaccatggtggacccggtgggcttc-3′ and Pr2 5′-IndexTermcgctgaagggcttcttccagcaacgtctgcaggt-3′ (ABL sequences, BCR-sequences). The resulting ‘megaprimer’ and the primer Pr3 -5′-IndexTermaggcccatggtaccaggagtg-3′ were used in a second PCR round, and the resulting fragment was cloned into a KpnI-digested pEp185–T315I. For constructing the ABL part of BCR/ABL (#ABL and #ABL–T315I), the start codon and Kpn1 sites were introduced at the ABL-part by PCR using the primers Pr4 5′-IndexTermaaggtaccaccatggaagcccttcagcggccagtagcatctgactttgagc-3′ and Pr3 5′-IndexTermaggcccatggtaccaggagtg-3′. The resulting PCR products were controlled by sequencing and cloned into the KpnI-digested p185 and p185–T315I sequences, respectively. ΔSH3–ABL and ΔSH3–ABL–T315I were constructed by digesting the constructs pEΔCCp185 and pEΔCCp185 –T315I with HincII followed by recircularization. Linker 5′-IndexTermgaccaccatggagaaacactcctggtac was ligated to the recircularized plasmids with Kpn1 and HincII to introduce a Kozak consensus sequence.

To construct BCR(1–196)/ABL and BCR(1–196)/ABL–T315I, two PCR rounds were performed, first with primers pr5 5′-IndexTermttctaaagcttcacctctttgtcgttgacc-3′ and pr6 5′-IndexTermaaatggatccggtaccatggtggacccggtgg-3′ and then with pr7 5′-IndexTermatcatgaagcttgaagaagcccttcagcgg3′ and Pr3 5′-IndexTermaggcccatggtaccaggagtg-3′. The resulting products were controlled by sequencing and ligated and subcloned into KpnI-digested pEp185 and pEp185–T315I. To construct p185Y177F and p185Y177F–T315I, three PCR rounds were performed: first with the primers pr8 5′-IndexTermcctgttagttagttacttaagctcg-3′ and pr9 5′-IndexTermtgaaactcgacgttcacgaagaagggcttctcg-3′, second with the primers pr10 5′-IndexTermagaagcccttcttcgtgaacgtcgagtttcacc-3′ and Pr3-5′-IndexTermaggcccatggtaccaggagtg-3′ and finally, PCR products 1 and 2 were used as primers without a template. The full-length p185Y177F and p185Y177F–T315I products were completed by ligating the third PCR products using the KpnI-site of p185BCR/ABL p185–T315I cDNA. To construct double mutant ΔCCp185–Y177F and ΔCCp185 Y177F–T315I, pEΔCC p185 was cloned into BamHI-digested p185Y177F and p185Y177F–T315I. All other constructs have been described earlier.11, 12

Cell lines

Ba/F3 and 32D cells were obtained from the German Collection of Microorganisms and Cell Cultures (DSMZ, Braunschweig, Germany) and maintained in RPMI-1640 medium supplemented with 10% fetal calf serum (FCS) (Invitrogen) containing 10 ng/ml IL-3 (Cell Concepts, Umkirch, Germany). Ecotropic Phoenix cells and Rat-1 cells were cultured in DMEM supplemented with 10% FCS. Cell growth was assessed by dye exclusion using Trypan-blue.

Western blotting

Western blot analyses were performed according to widely established protocols. The following antibodies were used: anti-ABL (α-ABL) Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-phosphorylated ABL specific for the phosphorylated tyrosine-residues 245 (α-p-ABL–Y245) (Upstate-Biotechnology, Lake Placid, NY, USA), anti-BCR (α-BCR) (Santa Cruz Biotechnology) and anti-phosphorylated BCR specific for the phosphorylated tyrosine residue 177 (α-p-BCR–Y177) (Cell Signaling, Boston, USA). Blocking and antibody incubation were performed in 5% low-fat dry milk followed by washing in Tris-buffered saline (TBS) (10 mM Tris-HCl pH 8, 150 mM NaCl) containing 0.1% Tween-20 (TBS-T).

Retroviral infection and transfection

Ecotropic retroviral supernatants were obtained as described earlier.15 For infection of target cells, retronectin (Takara Bio Inc., Otsu, Japan) was used to enhance infection efficiency according to the manufacturer's instructions. Then, 2 × 105 target cells per well were seeded. Infection efficiency was measured after 48 h by determining the percentage of GFP- or ΔNGFR-positive cells by FACS analysis.

Proliferation–competition assay

The proliferation–competition assay was performed as described earlier.12 Briefly, Ba/F3 cells were infected with PINCO vectors harboring unmutated or mutant p185BCR/ABL IL-3 was removed from the media of infected Ba/F3 cells by washing the cells twice with phosphate-buffered saline. The cells were continuously cultivated in the absence of IL-3 and super-infected with GFP and Helix-2–GFP. Day 4 GFP-expression levels were used to normalize the expression levels of different experiments. Proliferation–competition among single- and double-infected cell fractions was monitored by FACS analysis of the GFP expression.

Transformation assays

Soft-agar and focus formation assays were performed using Rat-1 fibroblasts retrovirally transduced with PAULO vectors harboring unmutated or mutant p185BCR/ABL. Six-well plates were filled with DMEM supplemented with 10% FCS and 0.5% bacto-agar (DIFCO Laboratories, Detroit, MI, USA) (2 ml per well). Then, 5 × 103 transduced Rat-1 cells were suspended in ‘top-agar’ (DMEM supplemented with 10% FCS and 0.25% bacto-agar) (1 ml per well) and then stacked in the wells. Colonies were counted after 15 days incubation at 37 °C and 5% CO2. For focus-formation assays in a 24-well plate format, 4 × 104 transduced Rat-1 cells were plated per well. Foci were stained after 15 days using 1% crystal violet (Sigma-Aldrich, Steinheim, Germany).


AKI-resistance mutations restore both transformation potential and the capacity to mediate factor independence of oligomerization-deficient p185BCR/ABL

Targeting the oligomerization interface of p185BCR/ABL using competitive peptides reduced the factor independence of hematopoietic progenitors expressing either unmutated p185BCR/ABL or p185BCR/ABL harboring the Y253F or E255K but not the T315I mutations. Furthermore, it restored Imatinib sensitivity to p185BCR/ABL harboring the Y253F and E255K mutations, but not to p185BCR/ABL-T315I.12 To determine whether T315I and the other two most clinically relevant mutations Y253F and E255K influence the leukemogenic potential of p185BCR/ABL, we created oligomerization-deficient p185BCR/ABL lacking the N-terminal CC-domain (ΔCCp185)11 and harboring the Y253F, the E255K or the T315I mutations. The aberrant kinase activity of unmutated BCR–ABL induces signaling pathways able to replace factor-induced survival signaling of hematopoietic precursors such as the IL-3 signaling in the Ba/F3 cell line. We first investigated the capacity of the mutated ΔCCp185 to mediate factor independence of Ba/F3 cells. Therefore, we retrovirally expressed the indicated constructs (Figure 1a) in Ba/F3 cells and controlled the transgene expression by western blotting. As reported in Figure 1b, ‘unmutated’ ΔCCp185 did not grow upon factor withdrawal, in contrast with ΔCCp185 harboring Y253F and T315I, which exhibited growth curves identical to unmutated p185BCR/ABL. The ΔCCp185–E255Y cells showed a 2-day delay in growth (Figure 1b). Similar results were obtained in 32D cells (data not shown). Nearly identical results were obtained with the CML-associated p210BCR/ABL where T315I also restored the factor-independent growth of the oligomerization-deficient ΔCCp210 (see Supplementary Figure 1).

Figure 1

The influence of the resistance mutations on the transformation potential of oligomerization-deficient BCR/ABL. (a) Modular organization of the p185BCR/ABL mutants lacking the CC oligomerization interface and expression of the transgenes in Ba/F3 cells. Tubulin was used as a loading control. (b) Factor-independent growth of Ba/F3 cells expressing the indicated transgenes; the graph shows the means +/− s.d. of three independent experiments. (c) Focus formation assay −4 × 104 infected Rat-1 cells per well were plated in 24-well plates, grown for 72 h to confluence and incubated for additional 12 days. The plates were then washed, dried and stained with crystal violet. One representative of each of the three experiments performed in triplicate is given ( × 34 magnification). (d) Colony formation—Rat-1 cells were retrovirally transduced with the indicated constructs and seeded at 5 × 103 cells per well in soft-agar in 6-well plates. After 15 days, the colonies were counted and the means +/− s.d. of triplicates of two representative experiments are given.

To investigate the influence of the mutations on the transformation potential of p185BCR/ABL we performed classical transformation assays in untransformed fibrobasts: focus formation assays for the determination of contact inhibition and colony assays in semi-solid medium for the determination of anchorage-dependent growth. We retrovirally transduced Rat-1 fibroblasts with the constructs indicated in Figure 1a. As shown in Figures 1c and d, the inhibition of the oligomerization by the deletion of the CC-domain completely abolished both focus and colony formation of p185BCR/ABL whereas the presence of either Y253F, E255K or T315I restored the transformation potential of ΔCCp185 as shown by the formation of foci and colonies, respectively (Figures 1c and d).

Taken together, these data indicate that the ‘p-loop mutations’ Y253F and E255K and the ‘gatekeeper’ mutation T315I not only are responsible for AKI-resistance but also confer additional properties to the oligomerization-deficient ΔCCp185, which allow the transformation potential and the capacity to mediate factor independence to be restored. Notably, the T315I mutation exhibited the most prominent effects.

Resistance of p185–T315I against inhibition of the oligomerization depends on the phosphorylation at Y177

To determine the mechanisms (i) of the resistance of T315I mutants against the inhibition of oligomerization and (ii) of how the T315I mutation influences the biology of BCR–ABL itself, we investigated the effects of T315I on BCR/ABL mutants that have no capacity or a reduced capacity to mediate factor-independent growth of hematopoietic progenitors. First, we focused on mutations interfering with functional domains in the BCR portion of the fusion protein, particularly with the autophosphorylation at the Tyr-residue in position 177 (Y177). The autophosphorylation at Y177 is indispensable for the transformation potential of BCR/ABL. Thus, we retrovirally transduced Ba/F3 cells with the indicated constructs and investigated cell growth upon factor withdrawal (Figures 2a–c). The expression of the transgenes was controlled by western blotting (Figures 2a–c). To study the response of these mutants to the inhibition of the oligomerization, we performed proliferation–competition assays upon exposure to either the helix-2 peptide fused to GFP or to GFP alone as a control as described earlier,12 (Figures 2a–c).

Figure 2

Role of Y177 in the resistance of T315I mutants against the oligomerization inhibition by helix-2. For the determination of factor-independent growth and for the proliferation–competition assays, Ba/F3 cells were retrovirally transduced with the indicated constructs. Transgene expression was confirmed by western blotting with the indicated antibodies. Molecular mass references (kDa) are given, and α-Tubulin was used as a loading control. For factor-independent growth, the number of viable cells was daily determined by Trypan blue dye-exclusion. For the CPAs, the cells were adapted to factor independence, infected with GFP or helix-2–GFP and seeded. The proportion of the GFP-positive cell fraction was followed by FACS for 11 days and normalized to the GFP expression at day 4 (rel. GFP expression). The graphs show the means +/− s.d. of three independent experiments. (a) BCC/ABL—the N-terminal CC-domain of BCR/ABL directly fused to the ABL-portion of the fusion protein +/−T315I. (b) BCR(1–196)/ABL—the N terminus of BCR comprising the CC-domain and the Y177 phosphorylation site fused to the ABL-portion of the fusion protein +/−T315I. (c) p185BCR/ABL with a point mutation at Y177 (Y177F) +/−T315I.

We first investigated the effects of T315I on BCC/ABL, in which the CC-domain is directly fused to the ABL-portion of the fusion protein.11 As shown in Figure 2a, T315I strongly improved the capacity of BCC/ABL to mediate factor-independent growth of Ba/F3. In fact, BCC/ABL–T315I grew to the same extent as unmutated p185BCR/ABL, whereas BCC/ABL showed the expected delay in growth (Figure 2a). The capacity of both BCC/ABL and BCC/ABL–T315I to mediate factor-independent growth was completely abolished by the inhibition of the BCC-mediated oligomerization through the competitive helix-2 peptide (Figure 2a).

In BCR(1–196)/ABL, in which the Y177 phosphorylation is present in addition to the CC-domain, the T315I mutation improved the capacity to mediate factor-independent growth to the level of unmutated p185BCR/ABL (Figure 2b). Notably, the presence of T315I rendered the BCR(1–196)/ABL resistant against the inhibition of oligomerization by helix-2 (Figure 2b).

To confirm the hypothesis that the phosphorylation of Y177 is indispensable for the resistance of p185BCR/ABL harboring the T315I mutation against the helix-2, we studied a p185–T315I mutant in which phosphorylation at Y177 was abolished by a point mutation to phenylalanine (p185–Y177F–T315I). As shown in Figure 2c, the absence of phosphorylation at Y177 strongly delayed growth upon factor withdrawal, which was completely restored by the presence of T315I. Furthermore, the p185–Y177F–T315I turned sensitive to the oligomerization inhibition by helix-2 (Figure 2c). These data were also confirmed in 32D cells (data not shown).

In summary, our results show that (i) the phosphorylation of Y177 is critical for the resistance of p185–T315I against the inhibition of the oligomerization and (ii) the effects of the T315I mutation are not limited to oligomerization-deficient p185BCR/ABL mutants.

Autophosphorylation at Y177 is not affected by the oligomerization inhibition, but phosphorylation at Y177 of endogenous BCR parallels the effects of T315I

The differences between unmutated p185BCR/ABL and p185–T315I regarding the apparent importance of the phosphorylation at Y177 for the resistance against the inhibition of oligomerization prompted us to investigate the tyrosine phosphorylation at Y177 in the presence/absence of T315I. We used a specific antibody directed against BCR phosphorylated at Y177 on lysates of Ba/F3 cells expressing the different p185BCR/ABL mutants with or without the T315I mutation. To avoid the interference of stress-signaling induced by the selection factor withdrawal, we performed these experiments on newly transduced cells in the presence of IL-3. We showed that unmutated p185BCR/ABL and all mutants in which Y177 was present (ΔCCp185, ΔCCp185–T315I, BCR(1–196)/ABL, BCR(1–196)/ABL–T315I) were fully phosphorylated at the Y177 (Figures 3a–c) independently of the inhibition of oligomerization through either the helix-2 or the deletion of the CC-domain (Figures 3a and b). The antibody also revealed the Y177-phosphorylation of endogenous BCR. The inhibition of oligomerization either by the deletion of the CC-domain or by the competitive helix-2 peptides reduced or abolished Y177 phosphorylation of endogenous BCR in cells expressing unmutated p185BCR/ABL BCR(1–196)/ABL or BCC/ABL (Figures 3a–c). Notably, in all mutants, the T315I mutation restored the phosphorylation of BCR at Y177 independently of targeting the oligomerization (Figures 3a–c and d).

Figure 3

Y177-phosphorylation in the autophosphorylation of p185BCR/ABL and its mutants and the transphosphorylation of endogenous BCR in relationship to the presence of T315I upon the inhibition of oligomerization. Western blotting using antibodies directed against BCR (which did not detect BCR/ABL) (α-BCR) and BCR phosphorylated at Y177 (α-p-BCR–Y177). p185BCR/ABL and its mutants were detected by an α-ABL-antibody. Molecular mass references (kDa) are given. Tubulin or endogenous ABL was used as a loading control. (a) Ba/F3 cells expressing p185BCR/ABL or p185–T315I in the presence of either helix-2 (H2) or control GFP. (b) Ba/F3 cells expressing p185BCR/ABL lacking the N-terminal CC-oligomerization interface (ΔCCBCR/ABL) in the presence of either helix-2 (H2) or control GFP. (c) Ba/F3 cells expressing BCC/ABL in the presence of either helix-2 (H2) or control GFP. (d) Ba/F3 cells expressing BCR(1–196)/ABL.

Taken together, these data indicate that the biological effects of the T315I mutation may be mediated through an aberrant trans-phosphorylation of endogenous BCR as an expression of a restored or increased substrate phosphorylation by the ABL-kinase in the presence of the T315I mutation.

The effects of T315I are associated with an intact ABL-kinase activity

Our data suggest a direct relationship between positive effects of T315I on p185BCR/ABL-mutants and the phosphorylation of endogenous BCR. To confirm this hypothesis and to disclose whether the effects of T315I depend on an intact ABL-kinase activity, we first explored the effects of T315I on the isolated ABL portion of the BCR/ABL fusion proteins (#ABL) regarding factor-independent growth. As shown in Figure 4a, T315I was unable to confer to #ABL the capacity to induce factor independence. In these cells, endogenous BCR was not phosphorylated (Figure 4a).

Figure 4

Role of the ABL-kinase in the activity of T315I in p185BCR/ABL and its mutants. For the determination of factor-independent growth, Ba/F3 cells were retrovirally transduced with the indicated constructs and grown upon factor withdrawal. Transgene expression was confirmed by western blotting with the indicated antibodies against ABL and BCR and BCR phosphorylated at Y177. α-Tubulin was used as a loading control. (a) #ABL—the ABL-portion of the BCR/ABL fusion protein +/−T315I. (b) ΔSH3-ABL—ABL lacking the N-terminal SH3 domain +/−T315I. (c) p185BCR/ABL lacking the N-terminal CC-domain together with a point mutation at the Y177 (Y177F) +/− T315I. As control Ba/F3 expressing p185BCR/ABL or p185–T315I in the presence of either helix-2 (H2) or GFP were used.

In the past, it has been described that the deletion or inactivation of the SH3 domain of ABL by point mutations confers transformation potential to c-ABL.16, 17 The SH3 domain seems to be responsible for the functional inactivity of the ABL-portion of BCR/ABL. Hence, we created a mutant that lacks a functional SH3 domain (ΔSH3–ABL) alone and in combination with the T315I. As shown in Figure 4b, upon factor-withdrawal, ΔSH3–ABL allowed the survival of the cells but the cells did not proliferate. In contrast, cells expressing ΔSH3–ABL–T315I proliferated to the same extent as p185BCR/ABL In both ΔSH3–ABL- and ΔSH3–ABL–T315I-expressing cells, endogenous BCR was phosphorylated (Figure 4b).

To definitively prove the effects of T315I on functionally completely inactive BCR/ABL mutants, we created an oligomerization-deficient mutant lacking the phosphorylation site at Y177 (ΔCCp185–Y177F). Interestingly, even in this mutant, which lacks two functions considered indispensable for the activity of BCR/ABL, in the presence of T315I the capacity to mediate factor-independent growth was almost completely restored (Figure 4c). Also, the presence of T315I was accompanied by the Y177 phosphorylation of endogenous BCR.

These data show that T315I exerts its effects in the presence of an intact ABL-kinase activity and confirms the relationship between the phosphorylation of endogenous BCR and the capacity of BCR/ABL and ABL mutants to mediate factor-independent growth induced by the presence of T315I. Furthermore, it seems that activation of the ABL-kinase activity is enough to ensure survival of cells upon factor withdrawal but not to make them proliferate.

The presence of T315I is associated with an increased ABL-kinase activity also in mutants unable to induce Y177 phosphorylation of endogenous BCR

To determine whether the Y177 phosphorylation in the presence of T315I is due to increased substrate phosphorylation of the ABL-kinase, we investigated whether the T315I was able to interfere with the ABL-kinase activity. Therefore, we studied the autophosphorylation at Y245 in the different mutants used in this study. As shown in Figure 5, T315I increased the autophosphorylation in all mutants as revealed by the specific antibody. Interestingly, T315I induced a slight increase in kinase activity in the #ABL mutant, which was unable to rescue the capacity of this mutant to mediate factor-independent growth or the Y177 phosphorylation of endogenous BCR (Figure 4a).

Figure 5

Influence of T315I on the kinase activity p185BCR/ABL and its mutants—autophosphorylation. Western blotting on lysates of Ba/F3 cells expressing the indicated transgenes using antibodies directed against c-ABL and ABL–Y245 (α-p-ABL–Y245). Molecular mass references (kDa) are given and Tubulin was used as a loading control.

These data show that T315I induces increased ABL-kinase activity, an effect that is probably masked in the unmutated forms of BCR/ABL. Interestingly, there is no direct relationship between the ABL-kinase activity and the capacity to mediate factor independence as revealed by the #ABL–T315I mutant, which was unable to induce Y177 phosphorylation of BCR.


The aim of the study was to determine (i) why p185BCR/ABL can be inhibited by the exposure to competitive peptides in the presence of the Y253F or the E255K mutations, but not in the presence of the T315I,12 and (ii) whether the AKI-resistance mutations confer additional features to the biology of p185BCR/ABL that modify its leukemogenic potential.

Through our experiments on the oligomerization-deficient p185BCR/ABL we found that Y253F and E255K are not inert regarding the response to the inhibition of oligomerization. In contrast to T315I, which induces complete resistance, Y253F and E255K confer a partial resistance that is stronger in the case of Y253F as compared with E255K. This effect against oligomerization inhibition is hardly revealed in the available models of AKI-resistant BCR/ABL, and only by deleting the CC-domain were the effects of Y253F and E255K unmasked. The fact that these mutations were able to restore not only factor independence but also full transformation potential indicates that they probably induce conformational changes within the molecule that allow the activation of BCR/ABL as a monomer.

T315I requires the autophosphorylation at Y177 of BCR/ABL for the resistance against the inhibition of oligomerization. In fact, all mutants in which autophosphorylation at the Y177 was present exhibited resistance against the competitive helix-2 peptide. The phosphorylated Y177 represents the GRB-2-binding site, which is at the top of the RAS-signaling pathway initiated by BCR/ABL,1 suggesting an important role for RAS-signaling in the inhibition of the oligomerization.

Interestingly the T315I was able to restore the activity of an oligomerization-deficient BCR/ABL lacking also the Y177 phosphorylation site, the ΔCCp185–Y177F. Seemingly at odds with this finding is our observation that autophosphorylation at Y177 is a prerequisite for T315I-mediated resistance against the treatment with helix-2 suggesting that a mutant in which oligomerization is inhibited by the deletion of CC would behave like the p185–Y177F–T315I in the presence of helix-2. A possible explanation of this discrepancy is the ability of activated BCR to substitute the autophoshorylation of BCR/ABL. In addition, the activation of BCR by the T315I does not appear to require the interaction between BCR/ABL and BCR, suggesting that in the case of the p185–Y177F–T315I there are two relevant targets of the helix-2, being one the BCR/ABL mutants itself and the other endogenous BCR. Thus the helix-2 might inhibit not only the oligomerization of BCR/ABL, but also that of BCR as well as the hetero-oligomerization between BCR/ABL and BCR.

Until now, the grade of transformation activity of BCR/ABL was thought to be directly correlated to the degree of its kinase activity.2, 18 Recently, the first evidence was raised that BCR/ABL harboring the ‘gatekeeper’ mutation T315I, responsible for the resistance against kinase inhibitors, such as imatinib, nilotinib or dasatinib exhibits ABL-kinase activity that may be higher or lower than that of unmutated BCR/ABL depending on the model.14 These findings suggested that the kinase activity might not be the decisive parameter causing T315I-positive cells to overgrow in the presence of cells whose unmutated BCR/ABL-kinase activity is efficiently suppressed by inhibitors. Our findings on the different mutants clearly showed that the capacity of T315I to improve the functionality of the respective mutants is closely related to functional ABL-kinase activity. Thus, we can exclude the possibility that T315I confers activities that are independent of the ABL-kinase activity of BCR/ABL. In fact, in all cases, the presence of T315I led to an increase in or an activation of the kinase activity of the respective mutant. These data confirm recent findings showing that mutations of the highly conserved ‘gatekeeper’ threonine, which lies within the hinge region of the enzymatic cleft of many kinases, represent a crucial structural feature. The mutation of threonine promotes kinase activation sufficient to confer the capacity to mediate factor-independent growth for several native non-receptor and receptor tyrosine kinases, such as c-SRC, PDGFRA and EGFR.19 In the case of the isolated ABL-portion of BCR/ABL, either this activation was unable to reach levels high enough for biological activity or, although the intensity of this activation reached the level of other biologically active mutants, it is not sufficient by itself to exert full biological activity. In fact, in cells expressing #ABL–T315I, endogenous BCR was not phosphorylated. Our results indicate a relationship between the phosphorylation of endogenous BCR and the biological activity of T315I. The BCR-phosphorylation is most likely the expression of an increased substrate phosphorylation activity of the ABL-kinase in the presence of the T315I an effect exerted by all mutants with biological activity. The phosphorylation seems to be independent of the overall kinase activity of the respective mutants, which was variable. One can speculate on the existence of a still unknown mechanism involved in the phosphorylation of BCR by the ABL-kinase, the inhibition of which is released in the case of the deletion of the SH3 domain of ABL.

In summary, our data show that T315I not only interferes with the affinity of ATP-analogs for the ABL-kinase, but also increases the kinase activity of different ABL and BCR/ABL-mutants, conferring strong biological activity that seems to be related to the phosphorylation of endogenous BCR. These findings introduce possible new approaches for the molecular therapy of patients harboring AKI-resistant BCR/ABL.


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This project was supported by a grant from Deutsche Forschungsgemeinschaft (DFG-RU 728/3-1) to MR. MR is further funded by grants from Alfred und Angelika Gutermuth Foundation, Deutsche Krebshilfe e.V. (DKH-107063 and DKH-107741) and Deutsche José Carreras Leukämie-Stiftung e.V. (DJCLS - R 07/27f).

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Correspondence to T Beissert or M Ruthardt.

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Supplementary Information accompanies the paper on the Leukemia website (

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Mian, A., Schüll, M., Zhao, Z. et al. The gatekeeper mutation T315I confers resistance against small molecules by increasing or restoring the ABL-kinase activity accompanied by aberrant transphosphorylation of endogenous BCR, even in loss-of-function mutants of BCR/ABL. Leukemia 23, 1614–1621 (2009) doi:10.1038/leu.2009.69

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  • Philadelphia chromosome-positive leukemia
  • imatinib-resistance
  • ‘gatekeeper’ mutation T315I
  • inhibition of oligomerization

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