Mutation-specific control of BCR-ABL T315I positive leukemia with a recombinant yeast-based therapeutic vaccine in a murine model.
ABSTRACT: Chromosomal translocations generating the BCR-ABL oncogene cause chronic myeloid leukemia (CML) and a subset of acute lymphoblastic leukemia. The BCR-ABL(T315I) mutation confers drug resistance to FDA-approved targeted therapeutics imatinib mesylate, dasatinib, and nilotinib. We tested the ability of a recombinant yeast-based vaccine expressing the T315I-mutated BCR-ABL antigen to stimulate an anti-BCR-ABL(T315I) immune response. The yeast-based immunotherapy significantly reduced or eliminated BCR-ABL(T315I) leukemia cells from the peripheral blood of immunized animals and extended leukemia-free survival in a murine model of BCR-ABL(+) leukemia compared to animals receiving sham injection or yeast expressing ovalbumin. With immunization, leukemic cells harboring BCR-ABL(T315I) were selectively eliminated after challenge with a mixed population of BCR-ABL and BCR-ABL(T315I) leukemias. In summary, yeast-based immunotherapy represents a novel approach against the emergence of cancer drug resistance by the pre-emptive targeted ablation of tumor escape mutants.
Project description:The BCR-ABL T315I mutation causes resistance to imatinib, nilotinib and dasatinib in chronic myeloid leukemia. Forty BCR-ABL positive patients with imatinib resistance were analyzed for T315I mutated clones after six months on nilotinib or dasatinib treatment by quantitative allele-specific ligation polymerase chain reaction with a sensitivity of 0.05%. Ligation polymerase chain reaction revealed 10 patients with more than 10(-5) BCR-ABL(T315I%)/GUS (high levels), none of whom achieved major molecular response after 12 months, and a further 8 patients with 10(-5) or below BCR-ABL(T315I%)/GUS (low levels) who all achieved major molecular response (P<0.001). A second independent group showed molecular response in one of 12 patients with high levels and 5 of 8 patients with low levels (P=0.018). Combining the groups resulted in a sensitivity and specificity of 92.9% and 87.5%, respectively. We conclude that the quantitative level of mutant T315I allele is predictive of major molecular response at 12 months on second-line nilotinib or dasatinib treatment. www.clinicaltrials.gov: CT00109707, NCT00384228, CA180013, CA180005 CA180006.
Project description: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.
Project description:The second generation of Bcr-Abl inhibitors nilotinib, dasatinib, and bosutinib developed to override imatinib resistance are not active against the T315I "gatekeeper" mutation. Here we describe a type-II T315I inhibitor 2 (GNF-7), based upon a 3,4-dihydropyrimido[4,5-d]pyrimidin-2(1H)-one scaffold which is capable of potently inhibiting wild-type and T315I Bcr-Abl as well as other clinically relevant Bcr-Abl mutants such as G250E, Q252H, Y253H, E255K, E255V, F317L, and M351T in biochemical and cellular assays. In addition, compound 2 displayed significant in vivo efficacy against T315I-Bcr-Abl without appreciable toxicity in a bioluminescent xenograft mouse model using a transformed T315I-Bcr-Abl-Ba/F3 cell line that has a stable luciferase expression. Compound 2 is among the first type-II inhibitors capable of inhibiting T315I to be described and will serve as a valuable lead to design the third generation Bcr-Abl kinase inhibitors.
Project description: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-Abl(T315I), 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-Abl(T315I), 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-Abl(T315I). 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.
Project description:Chronic myeloid leukemia (CML) is characterized by a constitutive activation of Bcr-Abl tyrosine kinase. Bcr-Abl/T315I is the predominant mutation that causes resistance to Imatinib. In the present study, we synthesized a novel Bcr-Abl inhibitor, HS-543, and investigated its effect on cell survival or apoptosis in CML cells bearing Bcr-Abl/T315I (BaF3/T315I) or wild-type Bcr-Abl (BaF3/WT). HS-543 showed anti-proliferative effects in the BaF3/WT cells as well as the BaF3/T315I cells with resistance to Imatinib and strongly inhibited the Bcr-Abl signaling pathway in a dose-dependent manner. Furthermore, it significantly increased the sub G1 phase associated with early apoptosis, with increased levels of cleaved PARP and cleaved caspase-3, as well as the TUNEL-positive apoptotic cells. In addition, we found that HS-543 induced apoptosis with the loss of mitochondrial membrane potential by decreasing the expression of Mcl-1 and survivin, together with increasing that of Bax. In BaF3/T315I xenograft models, HS-543 significantly delayed tumor growth, unlike Imatinib. Our results demonstrate that HS-543 exhibits the induction of apoptosis and anti-proliferative effect by blocking the Bcr-Abl signaling pathway in the T315I-mutated Bcr-Abl cells with resistance to Imatinib. We suggest that HS-543 may be a novel promising agent to target Bcr-Abl and overcome Imatinib resistance in CML patients.
Project description:Tyrosine kinase BCR-ABL fusion protein is the driver in patients with chronic myeloid leukemia (CML). The gate-keeper mutation T315I is the most challenging mutant due to its resistance to most tyrosine kinase inhibitors (TKIs). The third generation TKI ponatinib is the only effective TKI to treat CML patients harboring T315I-BCR-ABL mutation, but with high rate of major arterial thrombotic events. Alternative strategies to specifically target T315I-BCR-ABL are needed for the treatment of CML patients harboring such a mutation. Given that Sp1 is a fundamental transcriptional factor to positively regulate WT-BCR-ABL fusion oncogene, the purpose of this investigation was aimed at evaluating the anti-tumor activity and the underlying mechanism in terms of Sp1 regulational effect on the transcription of T315I-BCR-ABL fusion oncogene. Like in WT-BCR-ABL, we identified enrichment of Sp1 on the promoter of T315I-BCR-ABL fusion gene. Treatment of WT- and T315I-BCR-ABL-expressing CML cells by niclosamide diminished such an enrichment of Sp1, and decreased WT- and T315I-BCR-ABL transcription and its downstream signaling molecules such as STAT5 and Akt. Further, niclosamide significantly inhibited the proliferation and induced apoptosis through intrinsic pathway. The in vivo efficacy validation of p-niclosamide, a water soluble derivative of niclosamide, showed that p-niclosamide significantly inhibited the tumor burden of nude mice subcutaneously bearing T315I-BCR-ABL-expressing CML cells, and prolonged the survival of allografted leukemic mice harboring BaF3-T315I-BCR-ABL. We conclude that niclosamide is active against T315I-BCR-ABL-expressing cells, and may be a promising agent for CML patients regardless of T315I mutation status.
Project description: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.
Project description:The emergence of resistance to imatinib mediated by mutations in the BCR-ABL has become a major challenge in the treatment of chronic myeloid leukemia (CML). Alternative therapeutic strategies to override imatinib-resistant CML are urgently needed. In this study, we investigated the effect of AKI603, a novel small molecule inhibitor of Aurora kinase A (AurA) to overcome resistance mediated by BCR-ABL-T315I mutation. Our results showed that AKI603 exhibited strong anti-proliferative activity in leukemic cells. AKI603 inhibited cell proliferation and colony formation capacities in imatinib-resistant CML cells by inducing cell cycle arrest with polyploidy accumulation. Surprisingly, inhibition of AurA by AKI603 induced leukemia cell senescence in both BCR-ABL wild type and T315I mutation cells. Furthermore, the induction of senescence was associated with enhancing reactive oxygen species (ROS) level. Moreover, the anti-tumor effect of AKI603 was proved in the BALB/c nude mice KBM5-T315I xenograft model. Taken together, our data demonstrate that the small molecule AurA inhibitor AKI603 may be used to overcome drug resistance induced by BCR-ABL-T315I mutation in CML.
Project description:BACKGROUND: Chronic myelogenous leukemia (CML) is characterized by the chimeric tyrosine kinase Bcr-Abl. Bcr-Abl-T315I is the notorious point mutation that causes resistance to imatinib and the second generation tyrosine kinase inhibitors, leading to poor prognosis. CML blasts have constitutive p65 (RelA NF-kappaB) transcriptional activity, and NF-kappaB may be a potential target for molecular therapies in CML that may also be effective against CML cells with Bcr-Abl-T315I. RESULTS: In this report, we discovered that pristimerin, a quinonemethide triterpenoid isolated from Celastraceae and Hippocrateaceae, inhibited growth and induced apoptosis in CML cells, including the cells harboring Bcr-Abl-T315I mutation. Additionally, pristimerin inhibited the growth of imatinib-resistant Bcr-Abl-T315I xenografts in nude mice. Pristimerin blocked the TNFalpha-induced IkappaBalpha phosphorylation, translocation of p65, and expression of NF-kappaB-regulated genes. Pristimerin inhibited two steps in NF-kappaB signaling: TAK1TauIKK and IKKTauIkappaBalpha. Pristimerin potently inhibited two pairs of CML cell lines (KBM5 versus KBM5-T315I, 32D-Bcr-Abl versus 32D-Bcr-Abl-T315I) and primary cells from a CML patient with acquired resistance to imatinib. The mRNA and protein levels of Bcr-Abl in imatinib-sensitive (KBM5) or imatinib-resistant (KBM5-T315I) CML cells were reduced after pristimerin treatment. Further, inactivation of Bcr-Abl by imatinib pretreatment did not abrogate the TNFalpha-induced NF-kappaB activation while silencing p65 by siRNA did not affect the levels of Bcr-Abl, both results together indicating that NF-kappaB inactivation and Bcr-Abl inhibition may be parallel independent pathways. CONCLUSION: To our knowledge, this is the first report to show that pristimerin is effective in vitro and in vivo against CML cells, including those with the T315I mutation. The mechanisms may involve inhibition of NF-kappaB and Bcr-Abl. We concluded that pristimerin could be a lead compound for further drug development to overcome imatinib resistance in CML patients.
Project description:BACKGROUND: Philadelphia positive leukemias are characterized by the presence of Bcr-Abl fusion protein which exhibits an abnormal kinase activity. Selective Abl kinase inhibitors have been successfully established for the treatment of Ph (+) leukemias. Despite high rates of clinical response, Ph (+) patients can develop resistance against these kinase inhibitors mainly due to point mutations within the Abl protein. Of special interest is the 'gatekeeper' T315I mutation, which confers complete resistance to Abl kinase inhibitors. Recently, GNF-2, Abl allosteric kinase inhibitor, was demonstrated to possess cellular activity against Bcr-Abl transformed cells. Similarly to Abl kinase inhibitors (AKIs), GNF-2 failed to inhibit activity of mutated Bcr-Abl carrying the T315I mutation. METHODS: Ba/F3 cells harboring native or T315I mutated Bcr-Abl constructs were treated with GNF-2 and AKIs. We monitored the effect of GNF-2 with AKIs on the proliferation and clonigenicity of the different Ba/F3 cells. In addition, we monitored the auto-phosphorylation activity of Bcr-Abl and JAK2 in cells treated with GNF-2 and AKIs. RESULTS: In this study, we report a cooperation between AKIs and GNF-2 in inhibiting proliferation and clonigenicity of Ba/F3 cells carrying T315I mutated Bcr-Abl. Interestingly, cooperation was most evident between Dasatinib and GNF-2. Furthermore, we showed that GNF-2 was moderately active in inhibiting the activity of JAK2 kinase, and presence of AKIs augmented GNF-2 activity. CONCLUSIONS: Our data illustrated the ability of allosteric inhibitors such as GNF-2 to cooperate with AKIs to overcome T315I mutation by Bcr-Abl-independent mechanisms, providing a possibility of enhancing AKIs efficacy and overcoming resistance in Ph+ leukemia cells.