ALBERT

Description: Point mutations within the BCR-ABL kinase domain represent the most common mechanism of resistance to imatinib in patients with CML. Preclinical studies have shown that dasatinib (BMS-354825) is effective at inhibiting the kinase activity of imatinib-resistant BCR-ABL mutants with the notable exception of the T315I mutation, which remains highly resistant to imatinib, dasatinib, and AMN107 (Gorre et al, Science 2001; Shah et al, Science 2004; Weisberg et al, Cancer Cell, 2005). Clinical data from Phase I and II studies of dasatinib in CML confirms the in vitro findings. Each of three imatinib-resistant patients bearing the T315I mutation (CP=1; AP=2) did not achieve objective hematologic or cytogenetic responses during treatment with dasatanib on a Phase I study. Additionally, each of two phase II patients with the T315I mutation (CP=1; LBC=1) treated at UCLA showed no evidence of objective response. We have also detected the T315I mutation in each of two cases of acquired resistance in a phase II (LBC =2) study, and in seven of nine patients with acquired resistance to dasatinib in phase I and II studies (CP=1; MBC=3; LBC=2; Ph+ ALL=1). Notably, we detected a novel BCR-ABL mutation, T315A, in one of the two patients who relapsed without a detectable T315I mutation. The patient is a 53 year-old female whose chronic phase CML had progressed to myeloid blast phase while being treated with imatinib. The imatinib-resistant mutation M244V was identified prior to dasatinib treatment. The patient achieved a major hematologic response (

Description: A critical question in the targeted therapy era relates to whether treatment outcomes will be optimized by sequential or combinatorial use of targeted agents. Selection for CML cells with BCR-ABL kinase domain mutations is the main mechanism responsible for loss of response to imatinib. Dasatinib is an ABL tyrosine kinase inhibitor that has activity against nearly all imatinib-resistant mutations and is approved for the treatment of imatinib-resistant and -intolerant BCR-ABL-associated leukemias. Acquired clinical resistance to sequential use of dasatinib following imatinib failure has been observed. We analyzed the BCR-ABL kinase domain at the time of relapse in 15 patients who lost an initial response to dasatinib, and found evolution of a total of three new mutations at the time of relapse in all cases. The highly resistant BCR-ABL/T315I mutation was detected in 11 cases. The four remaining cases were associated with the evolution of novel mutations (V299L, 3 cases; T315A, 1 case). V299L was also detected in a fourth case that had also evolved T315I. These three dasatinib-resistant mutations were part of a small number of amino acid substitutions previously isolated in a preclinical mutagenesis screen for dasatinib resistance-conferring BCR-ABL mutations. While the T315I mutation is highly resistant to imatinib, V299L and T315A retain sensitivity to imatinib in vitro and have not been previously described in imatinib-resistant cases, raising the potential utility of imatinib rechallenge in select dasaitinib-resistant cases. A significant finding of our studies is the evolution of five unique “compound” mutations (i.e. greater than one mutation on a DNA strand) in the BCR-ABL kinase domain of patients treated sequentially with imatinib and dasatinib. It is noteworthy that although the imatinib-sensitive V299L and T315A mutations evolved in five cases, they were detected in the context of a pre-existing imatinib-resistant mutation in three of these cases, and these cases are therefore unlikely to respond to rechallenge with IM. The T315A mutation was detected in the context of 2 pre-existing IM-resistant mutations (M244V/L364I). Interestingly, in bone marrow transformation assays, the clinically-identified dasatinib-resistant M244V/L364I/T315A mutation was more potently oncogenic than non-mutated BCR-ABL, in contrast to the baseline imatinib resistant M244V/L364I, which like T315A in isolation, was less potent than native BCR-ABL Our studies of CML cases resistant to sequential kinase inhibitor therapy reinforce BCR-ABL kinase domain mutation as the predominant mechanism of resistance to kinase inhibitor therapy, and provide evidence that compound mutations acquired as a result of sequential therapy can not only limit further therapeutic options, but also create more biologically aggressive isoforms of BCR-ABL. Together, these findings provide a strong rationale for early treatment of CML with combinations of kinase inhibitors that have the capacity to collectively prevent selection of resistant kinase domain mutations.

Description: The success of imatinib for the treatment of chronic myeloid leukemia (CML) has created a need for a sensitive and accurate method of monitoring disease response and burden. Quantitative PCR (Q-PCR) has been previously shown to correlate well with cytogenetic response in patients treated with imatinib, whereby a one-log reduction in BCR-ABL transcript level corresponded well with attainment of a major cytogenetic response (MCyR) and a two-log reduction correlated with a complete cytogenetic response (CCyR) (Branford et al, Leukemia17:2401, 2003). BMS-354825 is a novel orally bioavailable SRC/ABL kinase inhibitor with impressive activity against imatinib-resistant BCR-ABL mutant isoforms in vitro (Shah et al, Science305:399, 2004). The compound is presently undergoing evaluation in phase I clinical trials (see Sawyers et al, Talpaz et al, abstracts submitted for this meeting). We sought to address whether cytogenetic responses in patients treated with BMS-354825 correlated with reduction in BCR-ABL transcript levels as determined by Q-PCR. Of 13 evaluable imatinib-resistant/intolerant patients with chronic phase CML treated at UCLA, four have attained a MCyR. Achievement of MCyR corresponded with a one to two-log reduction in BCR-ABL transcript as assessed by Q-PCR. In the majority of cases, a substantial reduction in BCR-ABL transcript was detected four weeks after initiation of BMS-354825. Overall, the median reduction in BCR-ABL transcript level after four weeks of therapy was 32%. Three of these patients had developed resistance to imatinib, and two harbored the common imatinib-resistant M351T mutation. Of the nine patients who have failed to achieve a MCyR, none have achieved a one log reduction in BCR-ABL transcript level. We conclude that similar to imatinib, BMS-354825-associated MCyR in chronic phase CML is highly associated with a one to two-log reduction in BCR-ABL transcript level. Furthermore, Q-PCR offers a rapid and reliable method to assess for disease response in this setting, which promises to be of significant clinical value. Although the maximal tolerated dose of BMS-354825 has yet to be determined, the compound is clearly capable of substantially reducing disease burden in patients with imatinib-resistant CML. Updated Q-PCR data from all chronic, accelerated, and blast crisis-phase patients on study at UCLA will be presented.

Description: Dasatinib (Sprycel; formerly BMS-354825) is an oral multi-targeted SRC/ABL tyrosine kinase inhibitor that is approximately 325-fold more potent than imatinib, and has been recently approved by the US FDA for the treatment of imatinib-resistant and -intolerant Philadelphia chromosome-associated leukemias. In contrast to other tyrosine kinase inhibitors, pharmacokinetic analyses reveal that dasatinib has a short half-life (5–6 hours), with near-complete loss of BCR-ABL kinase activity inhibition eight hours after drug administration in patients with CML. Remarkably, patients treated with dasatinib as infrequently as once daily, five days per week, have achieved complete cytogenetic remission (CCyR). We tested transient BCR-ABL inhibition in vitro by assessing the sensitivity of the CML cell line K562 to short-term exposure of dasatinib. After as short as a 20-minute exposure to a clinically-relevant dasatinib concentration (100 nM), the majority of cells undergo apoptosis when analyzed after 48 hours, whereas a clinically-relevant concentration of imatinib (5 uM) failed to result in substantial cytotoxicity under these conditions. When higher concentrations (〉=12.5 uM) of imatinib were used that correct for differences in potency between imatinib and dasatinib, cytotoxicity at 48 hours after a 20-minute exposure was similar to that observed with dasatinib. These results exclude SRC family inhibition as a potential mediator of this effect, and were reproducible in a second CML cell line, KU-812. Importantly, cytotoxicity was not observed in BCR-ABL-negative leukemia cell lines under these conditions. Signiificantly, similar phenomenon was observed when an erlotinib-sensitive non-small cell lung cancer was exposed to high concentrations of erlotinib for 20 minutes. We assessed the degree of BCR-ABL kinase inhibition achieved in 20 patients with chronic phase CML who were treated with dasatinib once daily as part of a phase I clinical trial by quantifying the ratio of phospho-CRKL to CRKL (a BCR-ABL substrate) achieved in PBMCs harvested four hours after the first dose of dasatinib. We found a strong correlation between the magnitude of BCR-ABL kinase inhibition and the depth of response achieved. Notably, CCyR was achieved exclusively in four patients who experienced the deepest inhibition of BCR-ABL kinase activity activity. The current medical management of CML involves assessing the degree of cytogenetic response after 6–12 months of imatinib therapy before considering dose modifications of imatinib. While our findings will need to be validated prospectively in larger cohorts of patients, they suggest that it may be feasible to make early dose modifications based upon the degree of target inhibition following the initial dose of dasatinib, and thus maximize the likelihood of clinical benefit. Together, these findings have potentially significant implications not only for the optimal clinical management of CML patients, but also for the rational development of small molecule inhibitors of other cancer-associated tyrosine kinases, as well as our global understanding of cancer cell biology.

Description: Selection for CML cells with BCR-ABL kinase domain mutations represents the predominant molecular mechanism responsible for loss of response to imatinib. Similarly, we have found acquired resistance to dasatinib to be associated with kinase domain mutations in 100% of cases (n=15). However, unlike the multitude of imatinib resistant mutations, which to a large extent occur at non-contact residues and destabilize the inactive confirmation to which imatinib binds, only two mutations appear to be responsible for nearly all cases of dasatinib resistance, T315I and V299L (Shah et al, submitted, ASH 2006). Both of these mutations occur at critical contact residues between the ABL kinase domain and dasatinib. Successful treatment of dasatinib-resistant cases will therefore require strategies to successfully eliminate cells that harbor these mutations. Use of a combination of kinase inhibitors with the ability to collectively suppress all BCR-ABL kinase domain mutants would be predicted to lead to profoundly minimize disease resistance and relapse on targeted therapy. Although the combination of imatinib and dasatinib may prevent selection of the dasatinib-resistant V299L mutation, these agents share many targets, and their combination may therefore result in substantial toxicity. Moreover, the combination of imatinib and dasatinib is not predicted to effectively inhibit the growth of cells harboring BCR-ABL/T315I. We previously have shown that the Aurora kinase inhibitor VX-680 can bind to the ABL kinase domain and inhibit the kinase activity of the T315I mutation at low micromolar concentration. VX-680 is showing early signs of efficacy in CML cases associated with the T315I mutation. Interestingly, the co-crystal structure of VX-680 reveals that V299 is one of 14 contact residues within the ABL kinase domain. Substitution of leucine at this residue might therefore be expected to diminish the potential affinity of VX-680 for BCR-ABL/V299L. However, analysis of the amino acid sequence of Aurora-A revealed divergence from native BCR-ABL at this the corresponding amino acid position due to the presence of a leucine in Aurora-A. BCR-ABL/V299L is therefore a mimetic of Aurora-A, and as a result, predicted to retain sensitivity to VX-680. We therefore assessed VX-680 for its ability to inhibit the kinase activity of BCR-ABL/V299L in Ba/F3 cells and found effective inhibition of the kinase activity at low micromolar concentration. Consistent with predictions based upon structural considerations, the V299L mutation is somewhat more sensitive to VX-680 than BCR-ABL/T315I. We confirmed these results in an analysis of primary human PBMCs obtained from a dasatinib-resistant patient who had evolved the V299L mutation on therapy. Our findings suggest that early combination therapy with two kinase inhibitors, dasatinib and VX-680, may successfully suppress resistant disease by collectively eliminating BCR-ABL kinase domain mutation as a mechanism of resistance, and thereby achieve effective long-term disease control in the vast majority of patients.