T315I Gatekeeper Mutation Research Articles

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

BackgroundDevelopment 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. ResultsWe 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. ConclusionsIn 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.

EPIC: A phase III randomized, open-label study of ponatinib versus imatinib in adult patients with newly diagnosed chronic myeloid leukemia in chronic phase.

TPS7129 Background: The hallmark genetic abnormality of chronic myeloid leukemia (CML), known as the Philadelphia chromosome, generates the BCR-ABL fusion gene; expression of BCR-ABL in hematopoietic stem cells gives rise to CML. Ponatinib is a potent oral pan–BCR-ABL tyrosine kinase inhibitor (TKI) that is active against native and mutated forms of BCR-ABL, including the T315I gatekeeper mutant. Results from the phase 1 and phase 2 studies of ponatinib demonstrated that ponatinib is generally well tolerated and has substantial anti-leukemic activity in patients with CML who are resistant or intolerant to prior TKI therapy, regardless of baseline mutation status. In addition, multivariate analyses suggest that ponatinib has greater activity in younger patients who are less heavily pretreated and have a shorter time since diagnosis. The phase 3 EPIC (Evaluation of Ponatinib vs Imatinib in CML) study is testing the hypothesis that ponatinib is an effective treatment for newly diagnosed chronic phase (CP) CML patients when compared with standard imatinib therapy. Methods: EPIC is a multicenter, international, phase 3, two-arm, open-label trial of ponatinib (45 mg once daily) versus imatinib (400 mg once daily) in patients with newly diagnosed CP-CML. Patients ≥18 years of age with CP-CML (diagnosed within 6 months prior to study entry) and adequate renal, hepatic, and pancreatic function are eligible for enrollment. Enrolled patients are assigned to receive ponatinib or imatinib in a 1:1 fashion, stratified by Sokal Risk score (low vs intermediate vs high). The primary efficacy endpoint for this trial is major molecular response (MMR) rate at 12 months. Secondary endpoints include MMR rate at 5 years, BCR-ABLIS&lt;10% rate at 3 months, CCyR rate at 12 months, progression-free survival, overall survival, and safety. A sample size consisting of 480 patients will provide 90% power to detect a 15% absolute increase in MMR rate at 12 months using an unstratified Fisher exact 2-sided test at an alpha level of 0.05. Assuming a 10% dropout rate, approximately 528 patients will be enrolled. The first patient was enrolled in August 2012. Clinical trial information: NCT01650805.

Abstract SY19-04: Design and development of ponatinib, a pan-BCR-ABL inhibitor for CML.

Abstract SY19-04: Design and development of ponatinib, a pan-BCR-ABL inhibitor for CML.

Enhanced SH3/Linker Interaction Overcomes Abl Kinase Activation by Gatekeeper and Myristic Acid Binding Pocket Mutations and Increases Sensitivity to Small Molecule Inhibitors*

Multidomain kinases such as c-Src and c-Abl are regulated by complex allosteric interactions involving their noncatalytic SH3 and SH2 domains. Here we show that enhancing natural allosteric control of kinase activity by SH3/linker engagement has long-range suppressive effects on the kinase activity of the c-Abl core. Surprisingly, enhanced SH3/linker interaction also dramatically sensitized the Bcr-Abl tyrosine kinase associated with chronic myelogenous leukemia to small molecule inhibitors that target either the active site or the myristic acid binding pocket in the kinase domain C-lobe. Dynamics analyses using hydrogen exchange mass spectrometry revealed a remarkable allosteric network linking the SH3 domain, the myristic acid binding pocket, and the active site of the c-Abl core, providing a structural basis for the biological observations. These results suggest a rational strategy for enhanced drug targeting of Bcr-Abl and other multidomain kinase systems that use multiple small molecules to exploit natural mechanisms of kinase control.

Ponatinib Induced Thrombocytopenia Might Inhibit Proplatelet Formation of Megakaryocytes Via the Pathways Including Rho/Rock and Rac.

Abstract 2186 Background:Ponatinib (AP24534) is identified as a pan-BCR-ABL inhibitor that potently inhibits the T315I gatekeeper mutant, and has advanced into clinical development for the treatment of refractory or resistant CML (Chronic Myeloid Leukemia). Ponatinib potently inhibited in vitro proliferation of Ba/F3 cells expressing BCR-ABL T315I mutation (IC 50; 11 nM). In PACE (Ponatinib Ph+ ALL and CML Evaluation) clinical trial indicated that 57% had CCyR (complete cytogenetical response), and 47% had MMR (major molecular response) in CML-chronic phase with T315I mutation. Ponatinib has substantial activity in heavily pretreated patients and those with refractory T315I mutation. However, approximately one third of ponatinib-treated patients had moderate to severe thrombocytopenia (J.E. Cortes et al. 2011 ASH Annual Meeting). The mechanism of ponatinib-induced thrombocytopenia remains unknown. In this study, we evaluated the effects of ponatinib on megakaryocytic progenitor cells, megakaryocytopoiesis, megakaryocyte and platelet production in mice. Method:All animal procedures were approved by the Institutional Animal Care and Use Committee of Iwate Medical University. Male ddY mice at 8 weeks of age were used in all experiments. We studied in vitro culture of megakaryocytic colonies (CFU-Meg), megakaryocyte ploidy analyses in vitro culture and proplatelet formation (PPF) assay in vitro. Murine bone marrow cells were cultured in methylcellulose with mIL-3, mIL-6 and TPO at 37°C in 5% CO2 and 20% O2 for 7 days in the presence of ponatinib (0.01, 0.1, 1, 10, 100 nM). PPF: Murine megakarocytes were partially purified from bone marrow cells using BSA gradient. They were plated in 96 micro-well culture plates (300 megakaryocyte/well) and cultured in IMDM, supplemented with 1% ITS-G (serum-free medium) in the presence of ponatinib (0.01, 0.1, 1, 10, 100 nM), at 37°C in 5% CO2and 20% O2. After 24 hr incubation, the megakaryocytes with proplatelets in each well were counted. Activated Rho and activated Rac in murine platelets were measured by the Western blot using Rhotekin-binding domain (RBD) beads and PAK-PBD Affinity beads respectively. The phosphorylation of Lyn (Src family kinase) in murine platelets was also evaluated by the Western blots. Results:CFU-Megs did not decrease significantly at 0.01, 0.1, 1, 10 and 100 nM (31.8 +/− 1.4 to 42.3 +/− 2.4 cells) and decreased significantly (17.0 +/− 1.6 cells p<0.01) at 1000 nM of ponatinib. PPF were decreased significantly at 0.1, 1, 10, 100 nM ponatinib (0 nM: 26.4 ± 0.8 %, 0.1 nM: 19.2 ± 1.7% p<0.05, 1 nM: 19.4 ± 2.1 % p<0.05, 10 nM: 17.9 ± 1.1% p<0.01, 100 nM: 12.5 ± 1.1 % p<0.001, 1000 nM: 11.6 ± 0.9 % p<0.001). The decreases in PPF were cancelled significantly by the addition of Y27632, Rho-associate kinase ROCK inhibitor (ponatinib 10 nM; 17.9 ± 1.1%, ponatinib 10 nM + Y27632 10μM; 29.8 ± 1.7% p<0.001, ponatinib 100 nM; 12.5 ± 1.1%, ponatinib 100 nM + Y27632 10μM; 19.3 ± 1.4% p<0.001). There was no difference in DNA ploidy of cultured megakaryocytes in the presence of ponatinib (0.01 to 100 nM). Next we have tried to understand the precise role of Rho/Rock and Rac pathways in platelets. Our data showed that Rho was upregulated and that activated Rac was downregulated at 50 nM of ponatinib. Ponatinib reduced the levels of phosphorylated Lyn (p-Lyn). Discussion and Conclusion:The Rho/ROCK pathway was reported to be negative regulators (Blood 2007 109; 4229) and positive regulators (Blood 2007 110; 3637) in PPF. Ponatinib induced thrombocytopenia might not be due to the inhibition of megakaryocyte colony formations but the inhibition of PPF of megakaryocytes via pathways including Rho/Rock and Rac. Disclosures:No relevant conflicts of interest to declare.

Abstract 853: Ponatinib, a potent pan-BCR-ABL inhibitor, retains activity against gatekeeper mutants of FLT3, RET, KIT, PDGFRα/β and FGFR1

Abstract Introduction: Ponatinib was designed as a highly potent inhibitor of native BCR-ABL as well as the T315I gatekeeper mutant that is resistant to all approved therapies, and has demonstrated substantial activity in patients with native or mutant BCR-ABL in a phase 1 trial. Activity against T315I is due to a key design feature, the triple bond linker, which enforces ligand linearity in this sterically demanding region of the binding site. Ponatinib also inhibits the in vitro kinase activity of a number of other clinically relevant kinases (FLT3, RET, KIT, PDGFRα, PDGFRα, and FGFR1; IC50s of 0.2 to 13 nM), many of which have developed resistance to existing therapies via mutation of the gatekeeper residue. Here we assessed whether ponatinib might represent a more general solution to gatekeeper mutant inhibition of these and other clinically important kinases by virtue of its design features. Results: The cellular activity of ponatinib was assessed using Ba/F3 cells transformed to IL-3 independence by expression of an activated kinase (via fusion to a TEL dimerization domain). Ponatinib potently inhibited viability of cells expressing activated FLT3, RET, KIT, PDGFRα, PDGFRα and FGFR1 with IC50s of 15, 8, 7, 1, 2 and 21 nM, respectively. In contrast, ponatinib did not affect viability of parental Ba/F3 cells grown in the presence of IL-3 (IC50 &amp;gt;1000 nM). Kinase variants in which the gatekeeper residue was mutated to isoleucine, leucine or methionine (all by a single nucleotide change from the native sequence) were then transformed into Ba/F3 cells. All of these variants have been shown previously, or in our studies, to confer substantial resistance (i.e. &amp;gt;50-fold) to other targeted agents. Ponatinib maintained substantial activity against all variants tested. The smallest potency reduction (2- to 9-fold) was seen in variants mutated to isoleucine, with FLT3F691I, KITT670I, PDGFRαT674I, and PDGFRαT681I being inhibited with IC50s of 62, 26, 2 and 17 nM, respectively. A slightly greater potency reduction (10- to 22-fold) was seen in sterically more demanding leucine or methionine mutations, with RETV804M, RETV804L and FGFR1V561M being inhibited with IC50s of 84, 178 and 202 nM, respectively. In all cell lines, cell viability effects were accompanied by inhibition of receptor phosphorylation. Conclusion: Ponatinib is a potent inhibitor of activated variants of FLT3, RET, KIT, PDGFRα, PDGFRα, and FGFR1, in addition to BCR-ABL. Ponatinib also maintains substantial activity against gatekeeper variants shown to mediate resistance to other targeted agents, inhibiting their activity with IC50s that are within range of known clinically achievable plasma concentrations. These results suggest that ponatinib may offer a more general solution to gatekeeper mutant inhibition and support clinical testing in a variety of cancers driven by activated kinases. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 853. doi:1538-7445.AM2012-853

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A drug that keeps the ABL1 kinase in its inactive conformation has efficacy against the ABL1 T315I gatekeeper mutation.

Recent Developments in the Third Generation Inhibitors of Bcr-Abl for Overriding T315I Mutation

In the treatment of chronic myeloid leukemia (CML) with Bcr-Abl kinase inhibitors, the T315I gatekeeper mutant has emerged as resistant to all currently approved agents, such as imatinib, nilotinib and dasatinib, by discrupting important contact interactions between the inhibitors and the enzyme. To overcome this particular resistance, several different strategies have been explored and many molecules have been investigated as capable of potently inhibiting Bcr-Abl T315I. Herein, this review reports on some predominant examples of third generation inhibitors of Bcr-Abl active against the T315I mutation, and special attentions are paid to the "hybrid-design" strategy for creating type-II class ATP-competitive inhibitors.

Discovery of 5-(arenethynyl) hetero-monocyclic derivatives as potent inhibitors of BCR–ABL including the T315I gatekeeper mutant

Ponatinib (AP24534) was previously identified as a pan-BCR-ABL inhibitor that potently inhibits the T315I gatekeeper mutant, and has advanced into clinical development for the treatment of refractory or resistant CML. In this study, we explored a novel series of five and six membered monocycles as alternate hinge-binding templates to replace the 6,5-fused imidazopyridazine core of ponatinib. Like ponatinib, these monocycles are tethered to pendant toluanilides via an ethynyl linker. Several compounds in this series displayed excellent in vitro potency against both native BCR-ABL and the T315I mutant. Notably, a subset of inhibitors exhibited desirable PK and were orally active in a mouse model of T315I-driven CML.

Discovery of 3-[2-(Imidazo[1,2-b]pyridazin-3-yl)ethynyl]-4-methyl-N-{4-[(4-methylpiperazin-1-yl)methyl]-3-(trifluoromethyl)phenyl}benzamide (AP24534), a Potent, Orally Active Pan-Inhibitor of Breakpoint Cluster Region-Abelson (BCR-ABL) Kinase Including the T315I Gatekeeper Mutant

In the treatment of chronic myeloid leukemia (CML) with BCR-ABL kinase inhibitors, the T315I gatekeeper mutant has emerged as resistant to all currently approved agents. This report describes the structure-guided design of a novel series of potent pan-inhibitors of BCR-ABL, including the T315I mutation. A key structural feature is the carbon-carbon triple bond linker which skirts the increased bulk of Ile315 side chain. Extensive SAR studies led to the discovery of development candidate 20g (AP24534), which inhibited the kinase activity of both native BCR-ABL and the T315I mutant with low nM IC(50)s, and potently inhibited proliferation of corresponding Ba/F3-derived cell lines. Daily oral administration of 20g significantly prolonged survival of mice injected intravenously with BCR-ABL(T315I) expressing Ba/F3 cells. These data, coupled with a favorable ADME profile, support the potential of 20g to be an effective treatment for CML, including patients refractory to all currently approved therapies.

Abstract 2671: Structural analysis of the inhibitory mechanism of AP24534, a pan-BCR-ABL inhibitor overriding the T315I gatekeeper mutation

Abstract Although durable response in many chronic myeloid leukemia (CML) patients can be achieved by imatinib treatment, point mutations in the kinase domain of the BCR-ABL fusion protein can lead to drug resistance and relapse. Nilotinib and dasatinib, the two second generation Abl kinase inhibitors, are active against most imatinib resistant mutants with a few exceptions, particularly the T315I gatekeeper residue mutation. Using a structure-based approach we have identified AP24534, a molecule that potently inhibits the T315I mutant and all other mutants tested, thus acting as a pan-BCR-ABL inhibitor. To elucidate the basis for this unique profile of AP24534, we have used a combination of X-ray crystallographic and structure-activity relationship analysis on AP24534 and its analogs. The crystal structure of the T315I mutant Abl in complex with the inhibitor clearly demonstrates that AP24534 binds to Abl in a DFG-out binding mode with the ethynyl chemical moiety skirting the mutated gate-keeper residue avoiding steric clashes. The X-ray co-structures of both the wild type and the T315I mutant Abl, in conjunction with the SAR analysis, further reveal that AP24534 retains potency against the T315I and other imatinib-resistant ABL mutants through optimized multiple points of contact, balancing and distributing the overall binding affinity so that mutation-based disruption of one facet of the binding network results in only a slight reduction in binding affinity. Our structural analysis enables us to predict and rationalize the different degree of sensitivity of ABL point mutants to AP24534, and to understand how AP24534 can completely abrogate resistance in cell-based mutagenesis screens at relatively low concentrations. AP24534 is currently being investigated in a Phase I clinical trial in patients with refractory and resistant CML and other hematological malignancies. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2671.

Inhibitors of the Abl kinase directed at either the ATP- or myristate-binding site

The ATP-competitive inhibitors dasatinib and nilotinib, which bind to catalytically different conformations of the Abl kinase domain, have recently been approved for the treatment of imatinib-resistant CML. These two new drugs, albeit very efficient against most of the imatinib-resistant mutants of Bcr–Abl, fail to effectively suppress the Bcr–Abl activity of the T315I (or gatekeeper) mutation. Generating new ATP site-binding drugs that target the T315I in Abl has been hampered, amongst others, by target selectivity, which is frequently an issue when developing ATP-competitive inhibitors. Recently, using an unbiased cellular screening approach, GNF-2, a non-ATP-competitive inhibitor, has been identified that demonstrates cellular activity against Bcr–Abl transformed cells. The exquisite selectivity of GNF-2 is due to the finding that it targets the myristate binding site located near the C-terminus of the Abl kinase domain, as demonstrated by genetic approaches, solution NMR and X-ray crystallography. GNF-2, like myristate, is able to induce and/or stabilize the clamped inactive conformation of Abl analogous to the SH2-Y527 interaction of Src. The molecular mechanism for allosteric inhibition by the GNF-2 inhibitor class, and the combined effects with ATP-competitive inhibitors such as nilotinib and imatinib on wild-type Abl and imatinib-resistant mutants, in particular the T315I gatekeeper mutant, are reviewed.

Structural Analysis of DFG‐in and DFG‐out Dual Src‐Abl Inhibitors Sharing a Common Vinyl Purine Template

Bcr-Abl is the oncogenic protein tyrosine kinase responsible for chronic myeloid leukemia (CML). Treatment of the disease with imatinib (Gleevec) often results in drug resistance via kinase mutations at the advanced phases of the disease, which has necessitated the development of new mutation-resistant inhibitors, notably against the T315I gatekeeper mutation. As part of our efforts to discover such mutation resistant Abl inhibitors, we have focused on optimizing purine template kinase inhibitors, leading to the discovery of potent DFG-in and DFG-out series of Abl inhibitors that are also potent Src inhibitors. Here we present crystal structures of Abl bound by two such inhibitors, based on a common N9-arenyl purine, and that represent both DFG-in and -out binding modes. In each structure the purine template is bound deeply in the adenine pocket and the novel vinyl linker forms a non-classical hydrogen bond to the gatekeeper residue, Thr315. Specific template substitutions promote either a DFG-in or -out binding mode, with the kinase binding site adjusting to optimize molecular recognition. Bcr-Abl T315I mutant kinase is resistant to all currently marketed Abl inhibitors, and is the focus of intense drug discovery efforts. Notably, our DFG-out inhibitor, AP24163, exhibits modest activity against this mutant, illustrating that this kinase mutant can be inhibited by DFG-out class inhibitors. Furthermore our DFG-out inhibitor exhibits dual Src-Abl activity, absent from the prototypical DFG-out inhibitor, imatinib as well as its analog, nilotinib. The data presented here provides structural guidance for the further design of novel potent DFG-out class inhibitors against Src, Abl and Abl T315I mutant kinases.

Serial Analysis of BCR-ABL Kinase Domain Mutations Prior to and During Imatinib Treatment for Newly Diagnosed and Recurrent BCR-ABL Positive Acute Lymphoblastic Leukemia.

Abstract 2041Poster Board II-18 Background:Patients with newly diagnosed and recurrent Philadelphia chromosome positive acute lymphoblastic leukemia (Ph+ALL) differ profoundly in their responsiveness to imatinib: whereas nearly all imatinib-naïve patients with de novo Ph+ALL initially achieve a complete remission (CR), eventually followed by secondary resistance, imatinib rarely induces a CR in patients with disease recurrence after chemotherapy. Mutations in the BCR-ABL tyrosine kinase domain (KD) are commonly associated with acquired imatinib resistance, but their role in primary resistance is not known. Recently, we determined that the same mutation found at relapse could already be detected prior to first imatinib exposure in 30% to 40% of patients with de novo Ph+ALL but surprisingly was not associated with an inferior response to imatinib monotherapy, which resulted in a CR rate exceeding 90%. Possible reasons for the inferior response of patients with recurrent Ph+ALL include a higher frequency of pre-existing mutations, a preponderance of mutations with greater transforming activity, a larger mutant clone or the contribution of non-mutational resistance mechanisms. To elucidate the potential role of these resistance mechanisms, we compared patients with de novo Ph+ALL and patients who had failed prior chemotherapy with respect to the frequency and pattern of pre-existing mutations, the level of mutant clones, the outgrowth kinetics of mutations during the first 4 weeks of imatinib monotherapy and the concordance between mutations detected prior to imatinib and at the time of relapse. Patients and methods:By denaturing high-performance liquid chromatography (D-HPLC; sensitivity 0.1-1%), ligation PCR (sensitivity 0.1-0.005%) and cDNA sequencing, we examined bone marrow and/or peripheral blood samples collected pre-treatment, during therapy and at relapse from 91 patients with Ph+ lymphoid leukemias who were enrolled in the initial phase II studies of imatinib for treatment of BCR-ABL positive leukemias after chemotherapy failure (n=65), or in a prospective, randomized clinical trial assessing front-line imatinib therapy in elderly patients with newly diagnosed Ph+ALL (n=26). Results:Prior to imatinib treatment, the frequency of TKD mutations did not differ significantly between patients with de novo and recurrent Ph+ ALL, respectively (30 % vs. 46%; p=n.s.) as detected by highly sensitive DHPLC and ligation PCR. P-loop mutations predominated in both patient groups (92% and 85% of detected mutations). The T315I gatekeeper mutation was identified in 4 patients, mutations in the activation loop were uncommon. Following only four weeks imatinib, the prevalence of detectable mutations increased substantially in patients with recurrent (to 75%) but not with de novo Ph+ALL (38%). Moreover, the median mutated bcr-abl transcript levels after 4 weeks of imatinib treatment were significantly higher in patients with recurrent than with de novo Ph+ALL (median 10%, [range 0-100] versus median 0%;range [0-7]). Conspicuously, we observed good concordance between the type of bcr-abl mutation detected prior to imatinib and at relapse. Conclusions:The incidence, pattern and levels of TKD mutations detected prior to imatinib and at relapse are similar in advanced and newly diagnosed Ph+ALL. In contrast, the outgrowth kinetics of cells with TKD mutations were far more rapid in patients with recurrent disease. These data provide clinical-translational evidence for a cooperative effect between mutational and non-mutational resistance mechanisms in Ph+ALL, and highlight the importance of early mutation monitoring using highly sensitive methodology. Disclosures:Hochhaus:Novartis : Research Funding. Ottmann:Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees; BMS: Honoraria.

Preliminary Clinical Activity in a Phase I Trial of the BCR-ABL/IGF- 1R/Aurora Kinase Inhibitor XL228 in Patients with Ph++ Leukemias with Either Failure to Multiple TKI Therapies or with T315I Mutation

XL228 is a protein kinase inhibitor with potent activity against wild-type and T315I isoforms of BCR-ABL (wild-type ABL kinase, IC50 = 5 nM; ABL T315I, 1.4 nM), Aurora A (3.1 nM), IGF-1R (1.6 nM), SRC (6.1 nM), and LYN (2 nM). A Phase 1 dose escalation clinical trial in patients (pts) with CML or Ph++-ALL who are resistant or intolerant to at least two prior standard therapies (including imatinib, dasatinib, and nilotinib) or have a known BCR-ABL T315I mutation is ongoing.XL228 is administered as a 1-hour IV infusion either once weekly or twice weekly. Twenty-seven pts have been enrolled into six cohorts with the once-weekly dosing schedule (dose range from 0.45 mg/kg to 10.8 mg/kg). All pts have failed prior imatinib therapy, and received nilotinib, dasatinib, and other therapies The majority of pts harbor mutations in BCR-ABL, with the most common mutations being T315I (n=10), F317L (n=7), and V299L (n=3). The maximum administered dose (MAD) of once-weekly IV dosing of XL228 is 10.8 mg/kg. Dose escalation of pts in the twice-weekly dosing schedule at an initial XL228 dose of 3.6 mg/kg on Days 1 and 4 of each week is ongoing.XL228 has been generally well-tolerated. Dose limiting toxicities observed with once-weekly dosing included Grade 3 syncope and hyperglycemia in two pts dosed at 10.8 mg/kg. Grade 2 adverse events reported to be possibly related to XL228 in the once-weekly dosing schedule were usually transient and manageable, and included hyperglycemia, fatigue, nausea, vomiting, and bradycardia.Pharmacokinetic analysis across five cohorts treated with once-weekly dosing of XL228 demonstrated an approximately dose-proportional exposure, with a mean terminal half life of 15 to 38 hours. In the 7.2 mg/kg cohort, the Cmax of approximately 13 μM exceeds the IC50 for modulation of phospho-CrkL levels determined in mouse K562 xenograft pharmacodynamic studies. Peak exposures of XL228 in the 7.2- and 10.8-mg/kg cohorts were associated with inhibition of peripheral blood leukocyte CrkL phosphorylation in several patients, including three harboring the T315I mutation. Transient increases in mean plasma glucose levels (up to three fold) and mean insulin levels (up to 40 fold) postinfusion are coupled, dose-related, and imply inhibition of the IGF1R and IR pathways by XL228.Preliminary evidence of clinical activity has been observed in pts treated at doses of 3.6 mg/kg and higher, including stable or decreasing white blood cell count and/or platelet count within 2 months (14 pts, 5 with T315I), and/or >1 log reduction in BCR-ABL levels by QPCR within 3 months (3 pts, 2 with T315I). Pts in the 7.2 mg/kg and higher cohorts have been followed a minimum of 1 month to a maximum of 4 months at the time of abstract submission. XL228 shows potential for treating drug-resistant CML and Ph+-ALL, including pts harboring the T315I gatekeeper mutation.

171 POSTER Inhibition of the drug-resistant T315l mutant of BCR-Abl

The ATP-competitive inhibitors dasatinib and nilotinib, which bind to catalytically different conformations of the Abl kinase domain, have recently been approved for the treatment of imatinib-resistant CML. These two new drugs, albeit very efficient against most of the imatinib-resistant mutants of Bcr–Abl, fail to effectively suppress the Bcr–Abl activity of the T315I (or gatekeeper) mutation. Generating new ATP site-binding drugs that target the T315I in Abl has been hampered, amongst others, by target selectivity, which is frequently an issue when developing ATP-competitive inhibitors. Recently, using an unbiased cellular screening approach, GNF-2, a non-ATP-competitive inhibitor, has been identified that demonstrates cellular activity against Bcr–Abl transformed cells. The exquisite selectivity of GNF-2 is due to the finding that it targets the myristate binding site located near the C-terminus of the Abl kinase domain, as demonstrated by genetic approaches, solution NMR and X-ray crystallography. GNF-2, like myristate, is able to induce and/or stabilize the clamped inactive conformation of Abl analogous to the SH2-Y527 interaction of Src. The molecular mechanism for allosteric inhibition by the GNF-2 inhibitor class, and the combined effects with ATP-competitive inhibitors such as nilotinib and imatinib on wild-type Abl and imatinib-resistant mutants, in particular the T315I gatekeeper mutant, are reviewed.