Christopher A. Eide

AP24534, a pan-BCR-ABL inhibitor for chronic myeloid leukemia, potently inhibits the T315I mutant and overcomes mutation-based resistance.

Inhibition of BCR-ABL by imatinib induces durable responses in many patients with chronic myeloid leukemia (CML), but resistance attributable to kinase domain mutations can lead to relapse and a switch to second-line therapy with nilotinib or dasatinib. Despite three approved therapeutic options, the cross-resistant BCR-ABL(T315I) mutation and compound mutants selected on sequential inhibitor therapy remain major clinical challenges. We report design and preclinical evaluation of AP24534, a potent, orally available multitargeted kinase inhibitor active against T315I and other BCR-ABL mutants. AP24534 inhibited all tested BCR-ABL mutants in cellular and biochemical assays, suppressed BCR-ABL(T315I)-driven tumor growth in mice, and completely abrogated resistance in cell-based mutagenesis screens. Our work supports clinical evaluation of AP24534 as a pan-BCR-ABL inhibitor for treatment of CML.

Oncogenic CSF3R mutations in chronic neutrophilic leukemia and atypical CML

BACKGROUND The molecular causes of many hematologic cancers remain unclear. Among these cancers are chronic neutrophilic leukemia (CNL) and atypical (BCR-ABL1-negative) chronic myeloid leukemia (CML), both of which are diagnosed on the basis of neoplastic expansion of granulocytic cells and exclusion of genetic drivers that are known to occur in other myeloproliferative neoplasms and myeloproliferative-myelodysplastic overlap neoplasms. METHODS To identify potential genetic drivers in these disorders, we used an integrated approach of deep sequencing coupled with the screening of primary leukemia cells obtained from patients with CNL or atypical CML against panels of tyrosine kinase-specific small interfering RNAs or small-molecule kinase inhibitors. We validated candidate oncogenes using in vitro transformation assays, and drug sensitivities were validated with the use of assays of primary-cell colonies. RESULTS We identified activating mutations in the gene encoding the receptor for colony-stimulating factor 3 (CSF3R) in 16 of 27 patients (59%) with CNL or atypical CML. These mutations segregate within two distinct regions of CSF3R and lead to preferential downstream kinase signaling through SRC family-TNK2 or JAK kinases and differential sensitivity to kinase inhibitors. A patient with CNL carrying a JAK-activating CSF3R mutation had marked clinical improvement after the administration of the JAK1/2 inhibitor ruxolitinib. CONCLUSIONS Mutations in CSF3R are common in patients with CNL or atypical CML and represent a potentially useful criterion for diagnosing these neoplasms. (Funded by the Leukemia and Lymphoma Society and others.).

BCR-ABL1 compound mutations in tyrosine kinase inhibitor–resistant CML: frequency and clonal relationships

BCR-ABL1 compound mutations can confer high-level resistance to imatinib and other ABL1 tyrosine kinase inhibitors (TKIs). The third-generation ABL1 TKI ponatinib is effective against BCR-ABL1 point mutants individually, but remains vulnerable to certain BCR-ABL1 compound mutants. To determine the frequency of compound mutations among chronic myeloid leukemia patients on ABL1 TKI therapy, in the present study, we examined a collection of patient samples (N = 47) with clear evidence of 2 BCR-ABL1 kinase domain mutations by direct sequencing. Using a cloning and sequencing method, we found that 70% (33/47) of double mutations detected by direct sequencing were compound mutations. Sequential, branching, and parallel routes to compound mutations were common. In addition, our approach revealed individual and compound mutations not detectable by direct sequencing. The frequency of clones harboring compound mutations with more than 2 missense mutations was low (10%), whereas the likelihood of silent mutations increased disproportionately with the total number of mutations per clone, suggesting a limited tolerance for BCR-ABL1 kinase domain missense mutations. We conclude that compound mutations are common in patients with sequencing evidence for 2 BCR-ABL1 mutations and frequently reflect a highly complex clonal network, the evolution of which may be limited by the negative impact of missense mutations on kinase function.

Wnt/Ca2+/NFAT signaling maintains survival of Ph+ leukemia cells upon inhibition of Bcr-Abl.

Although Bcr-Abl kinase inhibitors have proven effective in the treatment of chronic myeloid leukemia (CML), they generally fail to eradicate Bcr-Abl(+) leukemia cells. To identify genes whose inhibition sensitizes Bcr-Abl(+) leukemias to killing by Bcr-Abl inhibitors, we performed an RNAi-based synthetic lethal screen with imatinib mesylate in CML cells. This screen identified numerous components of a Wnt/Ca(2+)/NFAT signaling pathway. Antagonism of this pathway led to impaired NFAT activity, decreased cytokine production, and enhanced sensitivity to Bcr-Abl inhibition. Furthermore, NFAT inhibition with cyclosporin A facilitated leukemia cell elimination by the Bcr-Abl inhibitor dasatinib and markedly improved survival in a mouse model of Bcr-Abl(+) acute lymphoblastic leukemia (ALL). Targeting this pathway in combination with Bcr-Abl inhibition could improve treatment of Bcr-Abl(+) leukemias.

Targeting the BCR-ABL Signaling Pathway in Therapy-Resistant Philadelphia Chromosome-Positive Leukemia

Beginning with imatinib a decade ago, therapy based on targeted inhibition of the BCR-ABL kinase has greatly improved the prognosis for chronic myeloid leukemia (CML) patients. The recognition that some patients experience relapse due to resistance-conferring point mutations within BCR-ABL sparked the development of the second-generation ABL kinase inhibitors nilotinib and dasatinib. Collectively, these drugs target most resistant BCR-ABL mutants, with the exception of BCR-ABLT315I. A third wave of advances is now cresting in the form of ABL kinase inhibitors whose target profile encompasses BCR-ABLT315I. The leading third-generation clinical candidate for treatment-refractory CML, including patients with the T315I mutation, is ponatinib (AP24534), a pan-BCR-ABL inhibitor that has entered pivotal phase 2 testing. A second inhibitor with activity against the BCR-ABLT315I mutant, DCC-2036, is in phase 1 clinical evaluation. We provide an up-to-date synopsis of BCR-ABL signaling pathways, highlight new findings on mechanisms underlying BCR-ABL mutation acquisition and disease progression, discuss the use of nilotinib and dasatinib in a first-line capacity, and evaluate ponatinib, DCC-2036, and other ABL kinase inhibitors with activity against BCR-ABLT315I in the development pipeline. Clin Cancer Res; 17(2); 212–21. ©2010 AACR.

Nilotinib and MEK inhibitors induce synthetic lethality through paradoxical activation of RAF in drug-resistant chronic myeloid leukemia.

We show that imatinib, nilotinib, and dasatinib possess weak off-target activity against RAF and, therefore, drive paradoxical activation of BRAF and CRAF in a RAS-dependent manner. Critically, because RAS is activated by BCR-ABL, in drug-resistant chronic myeloid leukemia (CML) cells, RAS activity persists in the presence of these drugs, driving paradoxical activation of BRAF, CRAF, MEK, and ERK, and leading to an unexpected dependency on the pathway. Consequently, nilotinib synergizes with MEK inhibitors to kill drug-resistant CML cells and block tumor growth in mice. Thus, we show that imatinib, nilotinib, and dasatinib drive paradoxical RAF/MEK/ERK pathway activation and have uncovered a synthetic lethal interaction that can be used to kill drug-resistant CML cells in vitro and in vivo.

The ABL Switch Control Inhibitor DCC-2036 Is Active against the Chronic Myeloid Leukemia Mutant BCR-ABLT315I and Exhibits a Narrow Resistance Profile

Acquired point mutations within the BCR-ABL kinase domain represent a common mechanism of resistance to ABL inhibitor therapy in patients with chronic myeloid leukemia (CML). The BCR-ABL(T315I) mutant is highly resistant to imatinib, nilotinib, and dasatinib, and is frequently detected in relapsed patients. This critical gap in resistance coverage drove development of DCC-2036, an ABL inhibitor that binds the switch control pocket involved in conformational regulation of the kinase domain. We evaluated the efficacy of DCC-2036 against BCR-ABL(T315I) and other mutants in cellular and biochemical assays and conducted cell-based mutagenesis screens. DCC-2036 inhibited autophosphorylation of ABL and ABL(T315I) enzymes, and this activity was consistent with selective efficacy against Ba/F3 cells expressing BCR-ABL (IC(50): 19 nmol/L), BCR-ABL(T315I) (IC(50): 63 nmol/L), and most kinase domain mutants. Ex vivo exposure of CML cells from patients harboring BCR-ABL or BCR-ABL(T315I) to DCC-2036 revealed marked inhibition of colony formation and reduced phosphorylation of the direct BCR-ABL target CrkL. Cell-based mutagenesis screens identified a resistance profile for DCC-2036 centered around select P-loop mutations (G250E, Q252H, Y253H, E255K/V), although a concentration of 750 nmol/L DCC-2036 suppressed the emergence of all resistant clones. A decreased concentration of DCC-2036 (160 nmol/L) in dual combination with either nilotinib or dasatinib achieved the same zero outgrowth result. Further screens for resistance due to BCR-ABL compound mutations (two mutations in the same clone) identified BCR-ABL(E255V / T315I) as the most resistant mutant. Taken together, these findings support continued evaluation of DCC-2036 as an important new agent for treatment-refractory CML.

SGX393 inhibits the CML mutant Bcr-AblT315I and preempts in vitro resistance when combined with nilotinib or dasatinib

Imatinib inhibits Bcr-Abl, the oncogenic tyrosine kinase that causes chronic myeloid leukemia. The second-line inhibitors nilotinib and dasatinib are effective in patients with imatinib resistance resulting from Bcr-Abl kinase domain mutations. Bcr-AblT315I, however, is resistant to all Abl kinase inhibitors in clinical use and is emerging as the most frequent cause of salvage therapy failure. SGX393 is a potent inhibitor of native and T315I-mutant Bcr-Abl kinase that blocks the growth of leukemia cell lines and primary hematopoietic cells expressing Bcr-AblT315I, with minimal toxicity against Bcr-Abl-negative cell lines or normal bone marrow. A screen for Bcr-Abl mutants emerging in the presence of SGX393 revealed concentration-dependent reduction in the number and range of mutations. Combining SGX393 with nilotinib or dasatinib preempted emergence of resistant subclones, including Bcr-AblT315I. These findings suggest that combination of a T315I inhibitor with the current clinically used inhibitors may be useful for reduction of Bcr-Abl mutants in Philadelphia chromosome-positive leukemia.

Foretinib is a potent inhibitor of oncogenic ROS1 fusion proteins

Significance ROS1 fusion kinases are critical oncogenes in several malignancies, suggesting that ROS1 inhibitors are likely to be effective molecularly targeted therapies in these patients. Although phase I/II clinical trials using the ALK/ROS1 inhibitor crizotinib to treat ROS1 fusion-harboring non–small-cell lung cancer patients demonstrate early success, evidence of clinical resistance to crizotinib due to the acquired ROS1G2032R mutation was recently reported. Here, we demonstrate that foretinib is a more potent ROS1 inhibitor than crizotinib in vitro and in vivo and remains effective against crizotinib-resistant ROS1 kinase domain mutations, including ROS1 G2032R. Taken together, our findings establish foretinib as a highly promising therapeutic candidate for treating patients with ROS1-driven malignancies and provide rationale for rapid clinical translation. The rapidly growing recognition of the role of oncogenic ROS1 fusion proteins in the malignant transformation of multiple cancers, including lung adenocarcinoma, cholangiocarcinoma, and glioblastoma, is driving efforts to develop effective ROS1 inhibitors for use as molecularly targeted therapy. Using a multidisciplinary approach involving small molecule screening in combination with in vitro and in vivo tumor models, we show that foretinib (GSK1363089) is a more potent ROS1 inhibitor than crizotinib (PF-02341066), an ALK/ROS inhibitor currently in clinical evaluation for lung cancer patients harboring ROS1 rearrangements. Whereas crizotinib has demonstrated promising early results in patients with ROS1-rearranged non–small-cell lung carcinoma, recently emerging clinical evidence suggests that patients may develop crizotinib resistance due to acquired point mutations in the kinase domain of ROS1, thus necessitating identification of additional potent ROS1 inhibitors for therapeutic intervention. We confirm that the ROS1G2032R mutant, recently reported in clinical resistance to crizotinib, retains foretinib sensitivity at concentrations below safe, clinically achievable levels. Furthermore, we use an accelerated mutagenesis screen to preemptively identify mutations in the ROS1 kinase domain that confer resistance to crizotinib and demonstrate that these mutants also remain foretinib sensitive. Taken together, our data strongly suggest that foretinib is a highly effective ROS1 inhibitor, and further clinical investigation to evaluate its potential therapeutic benefit for patients with ROS1-driven malignancies is warranted.

New Bcr-Abl inhibitors in chronic myeloid leukemia: keeping resistance in check

Targeted therapy with the Abl kinase inhibitor imatinib has markedly improved the outlook for patients with chronic myeloid leukemia (CML). Breakpoint cluster region (Bcr)-Abl signaling is reactivated at the time of resistance, predominantly due to mutations in the kinase domain of Bcr-Abl that interfere with drug binding. This discovery prompted the development of new Abl kinase inhibitors, among which nilotinib and dasatinib have gained regulatory approval. Despite excellent results in patients with imatinib-resistant or imatinib-intolerant CML treated with nilotinib or dasatinib, early indications suggest that: the cross-resistant Bcr-AblT315I mutant is disproportionately represented among patients who relapse on these therapies; each Abl inhibitor exhibits vulnerabilities to certain kinase domain mutations. We review new inhibitors of Bcr-Abl, including preliminary information on inhibitors of Bcr-AblT315I and discuss the potential of combined Abl kinase inhibitor therapy to pre-empt resistance. Improved Abl inhibitor therapies will be useful in achieving maximum disease control but a clinical T315I inhibitor remains a high priority.