Imatinib-resistant Ph Research Articles

HnRNPK/Beclin1 signaling regulates autophagy to promote imatinib resistance in Philadelphia chromosome-positive acute lymphoblastic leukemia cells

This study sought to clarify the role of heterogeneous nuclear ribonucleoprotein K (hnRNPK) as a regulator of imatinib resistance in Philadelphia chromosome-positive acute lymphoblastic leukemia (Ph+ ALL). Expression of hnRNPK was assessed in Ph+ ALL leukemia cells in vitro and in vivo, and imatinib susceptibility was assessed via CCK-8 assay. In cells in which hnRNPK levels had or had not been modulated, LC3Ⅰ/Ⅱ and mTOR/p-ERK/Beclin1 levels were assessed via Western blotting, while electron microscopy was used to evaluate autophagic vacuole formation. Interactions between hnRNPK and Beclin1 were assessed through an RNA binding protein immunoprecipitation assay. Imatinib-resistant Ph+ ALL cell lines and patient bone marrow samples exhibited significant hnRNPK overexpression. Knockdown of hnRNPK increased the imatinib sensitivity of these tumor cells and decreased in vivo tumor burden in a xenograft model system as evidenced by a reduction in tumor volume. Levels of LC3Ⅰ/Ⅱ and Beclin1, but not p-ERK and mTOR, were consistent with the regulatory activity of hnRNPK. Electron microscopy revealed that imatinib-resistant cells harbored significantly more autophagic vacuoles relative to wild-type cells, while hnRNPK knockdown reduced the number of these vacuoles. In an RNA-binding protein immunoprecipitation assay, anti-hnRNPK was able to precipitate the Beclin1 mRNA. These results suggest that the hnRNPK/Beclin1 signaling pathway may play a role in shaping imatinib resistance in Ph+ ALL cells.

PS930 SENSITIZATION OF PH+ ALL RESISTANT TO IMATINIB BY TARGETING SPHINGOLIPID METABOLISM

Background:Acute lymphoblastic leukemia (ALL) is formed by accumulation of lymphoblasts in bone marrow and peripheral blood. The most common subtype of ALL is philadelphia positive ALL (Ph+ ALL) which is characterized by having BCR/ABL protein. For the treatment of Ph+ALL tyrosine kinase inhibitors (TKI) such as imatinib are used. However, 60–75% of the patients can still develop resistance against the TKIs and die within 5 years. Bioactive sphingolipids are a group of lipid family having ceramide as the central molecule, that play roles in many cellular mechanisms including proliferation, apoptosis, inflammation, metastasis. Ceramide is known to induce apoptosis which makes this sphingolipid a useful tool in cancer treatment. Sphingolipids can be generated through different pathways including de novo synthesis, recycling and salvage pathways. Previous studies showed that bioactive sphingolipids and genes in the pathway have been involved in response to TKI treatment in Ph+ALL.Aims:Here, we studied the roles of bioactive sphingolipids on the growth inhibitory effects of imatinib and exploit sphingolipid metabolism by majorly inhibiting glucosylceramide synthase (GCS) to further sensitize cells to imatinib and/or overcome resistance to imatinib in Ph+ ALL.Methods:Imatinib resistant cell line SD‐1R is generated by clonal selection from parental SD‐1 cell line by exposing the cells to gradually increased concentration of imatinib. Intracellular lipid levels were measured by HPLC/MS/MS. Cell viability is detected by MTT and trypan blue exclusion assay. Gene and protein expression levels were detected by real time PCR and western blotting, respectively.Results:We detected that intracellular levels of sphingolipids: ceramide, sphingosine and sphingomyelin is increased in response to imatinib in parental SD‐1 cells but not in newly generated SD‐1R cells. The data showed that these changes are not detectable in SD‐1 cells when imatinib is applied in presence of myriocin which is a palmitoyltransferase inhibitor that blocks the first step of de novo synthesis pathway in sphingolipids suggesting that imatinib induced changes in sphingolipids are due to induction of de novo synthesis pathway and a defect in this pathway is contributing the regulation of imatinib resistance in SD‐1R cells. Interestingly, another sphingolipid hexosylceramide which is a product of further metabolization of ceramide by the enzyme GCS, levels were elevated in both SD‐1 and SD‐1R cells. Knowing that, an FDA approved drug eliglustat, which is a GCS inhibitor used in Gaucher's disease is used to block the conversion of ceramide to hexosylceramide to accumulate apoptotic ceramide levels to sensitize cells to imatinib. Indeed, at the lipid levels, this combination resulted in a greater increase in ceramide and sphingosine levels. The reflection of these changes on cell growth was also detected and in parental SD‐1 cells, eliglustat application with imatinib is sensitized the cells to imatinib induced growth inhibition by 50% whereas eliglustat and imatinib cotreatment was able to overcome imatinib resistance in SD‐1R cells and inhibiting cell growth by 60–70% at 48 h.Summary/Conclusion:Taken together for the first time in the literature, we showed that pharmacological inhibition of GCS by eliglustat and imatinib cotreatment has synergistic cytostatic effects on imatinib resistant Ph+ ALL cells. Additionally, inducing the accumulation of ceramide (and/or sphingosine) by this cotreatment might be used as an approach to sensitize intrinsically / acquired resistant Ph+ ALL.

BCL2 inhibitor ABT-199 and JNK inhibitor SP600125 exhibit synergistic cytotoxicity against imatinib-resistant Ph+ ALL cells

Imatinib (IMT), a specific tyrosine kinase inhibitor (TKI), has drastically changed the treatment strategy for Ph+ ALL (Philadelphia chromosome-positive acute lymphoblastic leukemia). However, TKI resistance remains a serious problem for patient prognosis. Here, a Ph+ ALL cell line NphA2 and the IMT-resistant subline NphA2/STIR were analyzed to identify a potential novel treatment strategy. We also examined other Ph+ ALL cells, MR87 and its IMT-resistant subline, MR87/STIR. IMT induced apoptosis of NphA2 and MR87 but had no effect on resistant sublines. Increased phosphorylated ERK and BCL2, but not BCL-XL, were observed in NphA2/STIR compared with NphA2. NphA2/STIR but not NphA2 was moderately sensitive to U0126, an ERK inhibitor. Interestingly, SP600125, a JNK inhibitor, was potent in cell growth inhibition and apoptosis induction of both parental and IMT-resistant NphA2 and MR87 cells. Moreover, NphA2 and MR87 and their IMT-resistant sublines were sensitive to ABT-199, a specific BCL2 inhibitor. The combination of SP600125 and ABT-199 synergistically suppressed both parental and IMT-resistant cells, including one with T315I mutation, suggesting that Ph+ ALL exhibits high sensitivity to ABT-199 and SP600125 regardless of TKI resistance. This combination might be a possible therapeutic strategy for Ph+ ALL in the future.

Niemann-Pick disease type C1(NPC1) is involved in resistance against imatinib in the imatinib-resistant Ph+ acute lymphoblastic leukemia cell line SUP-B15/RI

Niemann-Pick disease type C1 (NPC1) is involved in cholesterol trafficking and may normally function as a transmembrane efflux pump. Previous studies showed that its dysfunction can lead to cholesterol and daunorubicin accumulation in the cytoplasmic endosomal/lysosomal system, lead to Niemann-Pick disease and resistance to anticancer drugs. In the present study, NPC1 was shown by microarray analysis to be more highly expressed in the Ph+ acute lymphoblastic leukemia cell line SUP-B15/RI, an imatinib-resistant variant of SUP-B15/S cells without bcr-abl gene mutation established in our lab. Further investigation revealed a defect in the functional capacity of the NPC1 protein demonstrated by filipin staining accompanied by a lower intracellular imatinib mesylate(IM) concentration by high-performance liquid chromatography in SUP-B15/RI compared with SUP-B15/S cells. Furthermore, U18666A, an inhibitor of NPC1 function, was used to block cholesterol trafficking to imitate the NPC1 defect in SUP-B15/S cells, leading to higher NPC1 expression, stronger filipin fluorescence, lower intracellular IM concentrations and greater resistance against IM. Samples from non-mutated relapsed Ph+ ALL patients also showed higher NPC1 expression compared with IM-sensitive patients. Our experiment may reveal a new mechanism of IM resistance in Ph+ ALL.

Targeting BCL6-Mediated Resistance to BCR-ABL Targeted Tyrosine Kinase Inhibitors (TKIs) in Philadelphia Chromosome Positive Acute Lymphoblastic Leukemia (Ph+ ALL) through the Addition of Histone Deacetylase (HDAC) Inhibitors

Introduction: Patients who harbor the Philadelphia (Ph+) chromosome t(9;22) translocation account for approximately 20-30% of adult ALL and 2-5% of pediatric ALL. Prior to approval and use of imatinib, a small molecule TKI which targets the Ph+ chromosome BCR-ABL1, these patients had poor survival & EFS - with long term survival rates in the 20% range. With the addition of imatinib and later generation TKIs to chemotherapy backbones and bone marrow transplant, EFS & survival rates have substantially improved - surpassing 50% in studies in adults and even higher in children. However, resistance to imatinib and other TKIs has become a significant problem in Ph+ ALL, especially in adults. ABL1 kinase domain mutations are the dominant form of TKI resistance, however other resistance mechanisms include upregulation of parallel pathways such as SRC family kinases, MAPK and BCL6 pathways. BCL6 is an oncogene that suppresses transcription of tumor suppressor genes such as p53 and CDNK1A. Interestingly, BCL6 has been shown to be upregulated and activated through deacetylation following imatinib treatment in Ph+ ALL, likely leading to its role in resistance. Histone deacetylase inhibitors (HDACi) have been shown to act synergistically with TKIs in imatinib sensitive and resistant Ph+ leukemia though multiple mechanisms including attenuation of BCR-ABL1 levels and other downstream proliferation promoting pathways. We have shown that HDACi treatment acetylates (and thus inactivates) BCL6 in Ph+ ALL, and that the combination of HDACis and TKIs leads to synergistic effects in vitro and in vivo (using xenograft models).Methods: In vitro WST-1 cell viability assays were carried out on TOM1 cells (non-ABL1 mutant, imatinib sensitive Ph+ ALL) and NALM1 cells (non-ABL1 mutant, imatinib resistant CML lymphoid blast crisis) with imatinib and entinostat (a HDACi). Synergy was assessed using Calcusyn software. Western blots were performed assessing BCL6 expression and acetylation, and expression of downstream effectors of apoptosis. Two separate in vivo xenograft mouse experiments were performed transplanting TOM1 and NALM1 cells into Nod SCID Gamma (NSG) mice. Cohorts of TOM1 mice were treated with imatinib 50mg/kg BID, entinostat 15mg/kg QD, imatinib plus entinostat combination, or vehicle control. In the NALM1 mice we added a higher dose imatinib cohort (100 mg/kg BID) due to known imatinib resistance.Results: In vitro, there was substantially more synergy of the imatinib/entinostat combination in imatinib-resistant NALM1 cells vs. the imatinib-sensitive TOM1 cells. Average Combination Index (CI) values in TOM1 cells across multiple entinostat and imatinib doses was 1.2 (CI: =1 suggest additive effect, <1 = synergy, >1 = antagonism), while the CI in NALM1 cells at the same dose combinations was 0.53. We noted BCL6 upregulation and decreased BCL6 acetylation - signs correlating with resistance - in Western blots of NALM1 and TOM1 cells treated with imatinib, while exposure to entinostat caused increased acetylation of BCL6 and increased expression of downstream tumor suppressors.In the imatinib-sensitive TOM1 xenograft trial, the combination displayed a significant reduction in bone marrow leukemic blast involvement versus control following 6 weeks of dosing as measured by flow cytometry (36.9% mean decrease, p=0.001). There was a trend toward decreased bone marrow involvement between the combination treatment and other active treatment arms. There was no difference in peripheral blood blast percentage between arms. In the imatinib-resistant NALM1 xenograft trial, the combination showed a significant decrease in peripheral blood blast percentage in the combination arms versus all other arms after only two weeks of therapy (p=0.0008).Conclusions: Upregulation of activated BCL6 is a known mechanism of resistance in Ph+ ALL that may be abrogated by acetylation of BCL6 with HDACi, as our in-vitro data suggests. Further, we have shown in xenograft models of Ph+ acute lymphoblastic leukemia that combination therapy with HDACi + imatinib, even in imatinib-resistant leukemia, has significant activity. Interestingly, the combination appears more active in resistant disease than in imatinib-sensitive disease. This combination could prove a viable strategy to attenuate imatinib- (and perhaps other TKI-) resistance in Ph+ ALL relapse, particularly in cases not driven by ABL1 kinase domain mutations. DisclosuresNo relevant conflicts of interest to declare.

The study of resistant mechanisms and reversal in an imatinib resistant Ph+ acute lymphoblastic leukemia cell line

In this study, we established an imatinib resistant Ph+ acute lymphoblastic leukemia (ALL) cell line SUP-B15/RI in vitro and studied the mechanism of imatinib resistance. Our results showed that the BCR-ABL1 fusion gene and the mdr1 gene were 6.1 times and 1.7 times, respectively, as high as that of parental SUP-B15 cell line. We found no mutation in the Abl kinase domain of SUP-B15/RI. Furthermore, the detection of cell signaling pathway of PI3K/AKT/mTOR, RAS/RAF, NF-κB, JNK and STAT showed the up-regulation of phosphorylation of AKT, mTOR, P70S6K, and RAF, ERK, and MEK, down-regulation of PTEN and 4EBP-1, and no change in other cell signaling pathways in SUP-B15/RI. However, dasatinib and nilotinib showed partial resistance. Interestingly, bortezomib had no resistance. Imatinib combination with rapamycin had synergistic effect on overcoming the resistance. Altogether, over-expression of BCR-ABL1 and mdr1 gene were involved in the resistance mechanisms, and up-regulation of the cell signaling pathways of PI3K/AKT/mTOR, RAS/RAF in SUP-B15/RI cell line may be correlated with them. The SUP-B15/RI cell line was also resistant to the second generation tyrosine kinase, dasatinib, and nilotinib, not bortezomib. The combination of imatinib with rapamycin can partially overcome the resistance and blockade of the ubiquitin–proteasome can be also a promising pathway to overcome imatinib resistance.

PD0332991, a CDK4/6 Inhibitor, Has An Anti-Leukemic Activity Against Ph+ Leukemia with T315I Mutation in Bcr-Abl; In Vivo Analysis Using Xenograft Mice Model

Abstract 2577Imatinib and second generation of TKIs such as dasatinib and nilotinib are effective for controlling CML and Ph+ ALL. However, Ph+ leukemia having T315I mutation of BCR-ABL shows resistance not only to imatinib but also to second generation of TKIs. Thus, it is urgent to clarify new strategies controlling Ph+ leukemia having T315I mutation. For this purpose, we assumed that effecter molecules for cell cycle progression regulated by BCR-ABL could be attractive targets for controlling Ph+ leukemia even with T315I mutant. Thus, we performed screening of cell cycle machineries in Ph+ leukemia cell lines whose expression is downregulated by imatinib, and found that CDK4 expression was downregulated by imatinib in most of Ph+ ALL cell lines. Consistently, PD0332991, a potent CDK4/6 inhibitor that is under phase II clinical study for solid tumor patients, showed significantly higher anti-leukemic activity against Ph+ ALL cell lines with intact Rb expression (n=8) (IC50; < 20 nM) in comparison with Ph− ALL cell lines with intact Rb expression (n=24) (IC50; 100nM). We next tested anti-leukemic activity of PD0332991 against Ph+ ALL cell line that has T315I mutant in BCR-ABL. SU/SR is an imatinib-resistant Ph+ ALL cell with T315I mutant (IC50 for imatinib >5,000 nM), which was established from SU-Ph2, an imatinib-sensitive Ph+ ALL cell line (IC50 for imatinib <100 nM), after long-term culture in the presence of gradually increasing concentration of imatinib. As expected, PD0332991 potently showed anti-leukemic activity to SU/SR (IC50; 30 nM) as well as SU-Ph2 (IC50; <25 nM), suggesting that PD0332991 is an attractive agent for controlling Ph+ ALL even with T315I mutation (Nemoto A and Inukai T et al). Based on these in vitro findings, we next analyzed in vivo activity of PD0332991 against SU-Ph2 and SU/SR in xenograft model using NOD/SCID/γc null (NOG) mice. NOG mice were transplanted with SU-Ph2 or SU/SR cells (1×106cells) through the tail vein. After the flow cytometric confirmation of bone marrow (BM) engraftment on day 14, PD0332991 (150 mg/kg) or vehicle (distilled water) was given for 2 weeks (5 days on, 2 days off) from day 21. The chimerism analyses revealed that PD0332991 prevented leukemia growth compared with vehicle in both SU-Ph2 and SU/SR on Day 35 in BM (0.4% vs. 49%, 37% vs. 89% in SU-Ph2 and SU/SR) and in peripheral blood (PB) (0.05% vs. 19%, 0.7% vs. 44% in SU-Ph2 and SU/SR), respectively. As a result, xenograft NOG mice treated with PD0332991 demonstrated higher overall survival compared with xenograft NOG mice treated with vehicle. Next, we tested whether PD0332991 could induce the dephosphorylation of pRb in vivo. PD0332991 (150 mg/kg) or vehicle were given to xenograft NOG mice transplanted with SU/SR or SU-Ph2 cells two times on day 29 and 4 hours before BM aspiration on day 30. Isolated BM cells were stained with anti-human CD19 plus anti-phospho-pRb antibodies. Flow cytometric analysis showed a significant reduction in pRb phosphorylation of SU/SR cells by PD0332991 in comparison with vehicle (9.7% vs. 57.1%, PD0332991 vs. vehicle) and SU-Ph2 cells (17.1% vs. 74.2%, PD0332991 vs. control), respectively. In summary, CDK4/6 inhibitor PD0332991 prevented the growth of Ph+ leukemia with T315I mutation in BCR-ABL in vivo and improved overall survival dramatically. We also demonstrated that PD0332991 showed a significant reduction in pRb phosphorylation in vivo. Our findings provide a rationale for efficacy of CDK4/6 inhibitor in the context of anti-leukemic therapy for Ph+ leukemia patients with T315I mutation in BCR-ABL. Disclosures:No relevant conflicts of interest to declare.

PD0332991, a CDK4/6 Inhibitor, Has An Anti-Leukemic Activity Against Lymphoid Crisis of CML and Ph+ALL Even with T315I Mutation in BCR-Abl; in Vitro Analysis

Abstract 1511Since BCR-ABL plays a central role in cell cycle progression of Philadelphia-chromosome positive (Ph+) leukemia cells and CDK4/6 critically involves in G1-progression of cell cycle, we analyzed sensitivity of Ph+ leukemia cell lines to compounds that act as specific CDK4/6 inhibitors. H3-thymidine uptake assay showed that both PD183812 and CBC219476 significantly inhibited cell growth of Ph+ lymphoid leukemia cell lines (n=9) in comparison with Ph+ myeloid leukemia cell lines (n=7) and Ph- ALL cell lines (n=26). Thus, we next tested the anti-leukemic activity of PD0332991, a potent CDK4/6 inhibitor that is under phase II clinical study for solid tumor patients, and found that 8 of 9 Ph+ lymphoid leukemia cell lines showed extremely higher sensitivity to PD0332991; median IC50 was <25 nM. IC50 of Ph+ lymphoid leukemia cell lines was significantly lower than that of Ph+ myeloid cell lines (200 nM, n=7) and Ph-ALL cell lines (100nM, n=25). PD0332991 effectively dephosphorylated Rb protein (pRb), and subsequently induced G1 arrest on all of Ph+ lymphoid leukemia cell lines. Moreover, PD0332991 gradually induced cell death in 4 Ph+ lymphoid leukemia cell lines. Since CDK4/6 inhibitor acts depending on intact pRb, we analyzed protein and gene expression status of Rb. Of note, all Ph+ lymphoid leukemia cell lines expressed intact pRb except for one cell line that showed relative resistance to PD0332991. In contrast, pRb was almost undetectable in Ph+ myeloid cell lines in spite of comparable level of Rb gene expression, which might be mechanism for resistance to PD0332991. However, most of Ph- ALL cell lines had intact pRb expression in spite of their relative resistance to PD0332991, indicating that Rb status alone did not explain higher PD0332991-sensitivity of Ph+ lymphoid leukemia cell lines. Thus, we assumed that Ph+ lymphoid leukemia cells showed higher PD0332991-sensitivity probably because BCR-ABL regulates CDK4/6 expression for cell cycle progression. To clarify this assumption, we treated Ph+ lymphoid leukemia cell lines with imatinib and performed immunoblot analysis of cell cycle machineries such as CDKs, cyclines, and CDK inhibitors. Of note, CDK4 expression level was frequently downregulated by imatinib in Ph+ lymphoid leukemia cell lines. Moreover, imatinib-induced downregulation of CDK4 in Ph+ lymphoid leukemia cell line was abrogated by the addition of IL-7 and FLT3 ligand, which stimulated cell cycle progression of imatinib-treated Ph+ ALL cell line. LY294002, a PI3K inhibitor, but not U0126, a MAPK inhibitor, and AG490, an inhibitor for JAK/STAT pathway, efficiently downregulated CDK4 expression in Ph+ lymphoid leukemia cell lines. Gene expression level of CDK4 in Ph+ lymphoid leukemia cell lines was downregulated by imatinib, and lactastatin, an inhibitor of protein degradation, partially inhibited imatinib-induced downregulation of CDK4 protein in Ph+ lymphoid leukemia cell lines, indicating that BCR-ABL regulates CDK4 expression both in gene expression level and in protein degradation level. These findings indicated that Ph+ lymphoid leukemia cell lines showed higher sensitivity to PD0332991 since BCR-ABL induces cell cycle progression of Ph+ lymphoid leukemia cells by regulating CDK4 as one of downstream pathways. Accordingly, we tested if PD0332991 shows anti-leukemic activity in Ph+ lymphoid leukemia cells that have a T315I mutation of BCR-ABL. SU/SR is an imatinib-resistant Ph+ ALL cell line with T315I mutation (IC50 for imatinib >10 mM), which was established from SU-Ph2, an imatinib-sensitive Ph+ ALL cell line (IC50 for imatinib <0.1 mM), after long-term culture in the presence of gradually increasing concentration of imatinib. Of note, PD0332991 effectively dephosphorylated pRb and inhibited cell growth of both SU/SR and SU-Ph2. Our findings provide a rationale for efficacy of PD0332991 in the context of anti-leukemic therapy for lymphoid crisis of CML and Ph+ ALL patients even with T315I mutation in BCR-ABL. Disclosures:No relevant conflicts of interest to declare.

To Study Resistant Mechanisms and Reversal In An Imatinib Resistant Ph+ Positive Acute Lymphoblastic Leukemia Cell Line

AbstractAbstract 2135 ObjectiveTo establish an imatinib resistant Bcr-Abl positive acute lymphoblastic leukemia (ALL) cell line in vitro and to study imatibin resistance in Ph+ ALL. The reversal of the imatinib resistance by rapamycin, the second generation tyrosine kinase inhibitor and proteasome inhibitor was studied. MethodsPh(+) ALL SUP-B15 cell line was cultured in gradually increasing concentrations of imatinib to generate the imatinib resistant cell line at 6 μM imatinib. The cytotoxic effect of imatinib and other drugs was analyzed by MTT assay. RT-PCR, flow cytometry, Western blot analyses of proteins, DNA sequence analysis of ABL kinase domain were used to clarify the possible mechanisms of the imatinib resistance in the SUP-B15/RI cell line. ResultsWe established the imatinib resistant Ph+ ALL cell line. The fusion bcr-abl gene was 6.1 times as high as that of the parental sensitive cell, and the mdr1 gene also increased 1.7 times in SUP-B15/RI cell line by the RT-PCR detection. However, the expression of hoct1 Abcl–2 and topoIIα gene were no difference between two cell lines by the RT-PCR detection. A K362S point mutation in the Abl kinase domain of SUP-B15/RI was found. The detection of cell signaling pathway of PI3K/AKT/mTOR, RAS/RAF, NF-κBA JNK and STAT showed the expression of PTEN and 4EBP-1 was down-regulated, AKT, mTOR and P70S6K was up-regulated and the expression of other cell signaling pathways in SUP-B15/RI was similar to its parental sensitive cell line. Dasatinib, nilotinib, and bortezomib could inhibit proliferation of SUP-B15/RI cells at nM concentration. SUP-B15/RI cell line also showed partial resistance to dasatinib and nilotinib, but not bortezomib. The combination of imatinib with rapamycin had synergistic effect to the resistance cell line. ConclusionIn vitro, we establish imatinib resistant Ph + ALL cell line. Overexpression of bcr-abl and mdr1 gene, K362S point mutation in ABL kinase domain and up-regulation of the cell signaling pathways of PI3K/AKT/mTOR, RAS/RAF in SUP-B15/RI cell line were involved in the resistance mechanisms. The SUP-B15/RI cell line was also resistant to the second generation tyrosine kinaeses dasatinib and nilotinib,not bortezomib in vitro. However, the combination of imatinib with rapamycin can partially overcome the resistance. Blockade of the ubiquitin-proteasome could be a promising pathway to overcome resistance to imatinib. Disclosures:No relevant conflicts of interest to declare.

Treatment with mTOR Inhibitor, Everolimus (RAD001) Overcomes Resistance to Imatinib in Ph-Leukemia Quiescent or T315I-Mutated Cells.

Abstract 3277Poster Board III-1Recent studies suggest that leukemia stem cells (LSCs) are responsible for relapse of leukemia following conventional or targeted agents and that eradication of LSCs might be necessary to cure the disease. Aberrant activation of mTOR signaling has also been reported to be involved in LSCs. In order to examine mechanisms of drug resistance in Ph-positive (Ph+) LSCs and to seek strategies to overcome the resistance, we've previously established in vivo-murine and ex vivo-culture models using murine hematopoietic pluripotent progenitors transduced with BCR-ABL (Minami, et al., Proc Natl Acad Sci USA, 2008). Furthermore, Ph+ leukemia (including T315I-, F311I-mutated CML-BC, or Y253H-mutated Ph-ALL) patient cells were serially xenotransplanted into immunodeficient NOD/SCID/IL2rγnull (NOG) mice. Engrafted bone marrow and spleen cells were almost identical to the original leukemia cells as to phenotypes including karyotypes and distribution of primitive populations. Spleen cells derived from leukemic NOG mice were co-cultured with S17 stromal cells and treated with imatinib and the mTOR inhibitor, everolimus (RAD001, Novartis Pharmaceuticals). While quiescent (Hoechst-33342low/Pyronin-Ylow) CD34+ cells were insensitive to imatinib in spite of BCR-ABL- and CrkL-dephosphorylation, substantial cell death including CD34+ population was induced with nM level of everolimus. In imatinib-resistant Ph+ leukemia cell lines harboring T315I-mutation (Baf3p210/T315I and TCC-Y/T315I), everolimus induced cell death with low IC50 values in PI-exclusion assays. We are also investigating detailed biomarkers in the cell death (such as phosphorylation of 4E-BP1 or p70 S6K) and effects of theses drugs in the leukemic NOG mice systems. These results imply that treatment with everolimus can overcome the resistance to imatinib in Ph+ LSCs or T315I-mutated cells. Disclosures:Kiyoi:Kyowa Hakko Kirin: Consultancy. Naoe:Kyowa Hakko Kirin, Wyeth and Chugai: Research Funding.

Recent advances in the treatment of Philadelphia chromosome-positive acute lymphoblastic leukemia

The advent of imatinib, a selective inhibitor of the ABL tyrosine kinase, has revolutionized the treatment of Philadelphia chromosome-positive acute lymphoblastic leukemia (Ph+ ALL). Combined with chemotherapy, imatinib exerts remarkable efficacy in patients with newly diagnosed disease with a complete remission (CR) rate of 95% and a survival rate of 55% at 3 years. Profound eradication of leukemia cells not only provides patients with a better chance for receiving allogeneic hematopoietic stem cell transplantation during first CR but also contributes to durable CR even without transplantation. Despite such improvement, however, relapse does occur, mainly owing to acquisition of resistance. Growing comprehension of the molecular mechanisms of resistance to imatinib has led to the development of novel BCR-ABL inhibitors that yield higher affinity for BCR-ABL and/or potent inhibitory activity against other target molecules such as SRC family kinases. The second-generation ABL kinase inhibitors, namely dasatinib and nilotinib, are already showing clinical activity in patients with imatinib-resistant Ph+ ALL, and other novel agents are undergoing preclinical and early clinical evaluation. Further improvement in treatment results will be achieved by identifying each patient's disease profile based on information obtained before and during treatment and by optimizing subsequent treatment accordingly.

KW-2449, a Novel Multi-Kinase Inhibitor, Suppresses the Growth of Imatinib-Resistant Ph+ Leukemia Including BCR-ABL/T315I Both in Vitro and in Vivo.

Background: KW-2449 is a multi-kinase inhibitor against FLT3, ABL and ABL/T315I and Aurora kinases with IC50 values of 0.007, 0.014, 0.004 and 0.048 micro mol/L, respectively. We reported a possible mode of action of KW-2449 with respect to its anti-leukemic effects on FLT3-mutated and FLT3-wild type leukemia cells via FLT3 and Aurora inhibition, respectively (1). Currently KW-2449 is being investigated in a Phase 1/2 study in patients with acute myeloid leukemia. In this report, we investigated the activity of KW-2449 or imatinib in imatinib-resistant leukemia with the T315I mutation.Methods and results: We evaluated the effects of KW-2449 in vitro and in vivo on imatinib-resistant Ph+ leukemia. While imatinib suppressed the growth of K562 (Ph+CML with wild-type BCR-ABL) and TCC-Y (Ph+ALL with wild-type BCR-ABL) with GI50 values of 0.20 and 0.18 micro mol/L, it had little inhibitory effects on TCC-Y/sr (Ph+ALL with BCR-ABL/T315I) with a GI50 value of 24 micro mol/L. On the other hand, KW-2449 showed equivalent growth inhibitory activities against K562, TCC-Y and TCC-Y/sr giving the GI50 values of 0.2–0.6 micro mol/L. In addition, KW-2449 showed potent growth inhibitory activity against IL-3 dependent cells transfected with BCR-ABL and BCR-ABL/T315I with GI50 values below 0.50 micro mol/L, whereas imatinib had no growth inhibition in BCR-ABL/T315I cells. When we examined the ABL-signaling pathway, imatinib had no effects on the expression of phosphorylated BCR-ABL (P-BCR-ABL) and STAT5 (P-STAT5), a key downstream signal molecules of BCR-ABL in TCC-Y/sr cells. Furthermore, no obvious apoptosis or cell cycle effects were observed in BCR-ABL/T315I cells after imatinib treatment. In addition, the exposure to KW-2449 induced reduction of P-BCR-ABL and P-STAT5 at 0.25 micro mol/L and induced G2/M arrest and apoptosis over the GI50 value (0.50–1.0 micro mol/L). These data provide the evidence that BCR-ABL inhibition at a lower concentration of KW-2449 modulates its signaling pathway and that Aurora inhibition at a higher concentration may play a critical role in the anti-proliferative effects in imatinib-resistant CML and Ph+ALL. To assess the anti-leukemia activity of KW-2449 in vivo, the SCID mice intravenously inoculated with TCC-Y/sr leukemia were orally treated with KW-2449 or imatinib. While KW-2449 prolonged the survival, imatinib treatment had no effects in this model. Furthermore, anti-proliferative activity of KW-2449 was examined in primary samples from blast crisis CML patients who had BCR-ABL/T315I mutation. After inoculation of blast cells into NOG mice, KW-2449 or imatinib treatment started. In this model, oral treatments with KW-2449 decreased peripheral copy number of BCR-ABL mRNA and CD45+ blast cells in the bone marrow, though imatinib treatment showed limited activity.Conclusion: KW-2449 demonstrated anti-leukemia activity against imatinib resistant leukemia both in vitro and in vivo. These results suggest that KW-2449 would be effective against imatinib-resistant CML or Ph+ALL because of its potent and unique kinase inhibition profile.

Preclinical Evaluation of Adjuvant Therapy with AMD3100 for Drug Resistant Philadelphia Chromosome Positive and Negative ALL

Despite advances in chemotherapeutic treatment of acute lymphoblastic leukemia (ALL), 20% of children relapse with high death rates, and adults show a long-term survival of only 60%, so new treatment modalities are needed. AMD3100 is being tested in clinical trails for use as a mobilizing agent for hematopoieitic stem cells and blocks the interaction of CXCL12 (SDF-1a) and its receptor, CXCR4, which are involved in retaining hematopoietic cells in primary lymphoid organs. CXCR4 expressed on bone marrow fibroblasts is involved in retaining pre-B cells in the bone marrow. Blocking the CXCL12 – CXCR4 interaction makes murine acute lymphoblastic leukemia cells more sensitive to drug treatment in vitro. Therefore, we hypothesized, that AMD3100 may sensitize drug resistant leukemia cells to chemotherapy. To test this hypothesis, we established a pre-clinical xenograft model of B-cell precursor-ALL, allowing us to monitor progression of leukemia in vivo with non-invasive bioimaging. Frozen samples of drug resistant adult patients, who did not respond to chemotherapy (drug-resistant Philadelphia chromosome (Ph) positive (Ph+) or negative (Ph−) pre-B ALL leukemia cells), were engrafted via tail-vein injections into female NOD/SCID or NOD/SCID IL2Rγ−/− mice. Primary passages were serially expanded up to three passages. Analysis of the phenotype by flow cytometry and morphology by histochemistry of the xenografts confirmed that the initial characteristics of the patient were retained or not altered throughout the passages. In an effort to sensitize those drug resistant leukemia cells to chemotherapy, we have next proceeded to test AMD3100 in our established preclinical models. Primary ALL cells of an Imatinib-resistant Ph+ patient were transduced with luciferase and injected into sublethally irradiated NOD/SCIDIL2Rγ −/− mice (0.7×106 cells/recipient). Mice were treated per os with saline (n=3), Imatinib (n=4;75 mg/kg/d), AMD3100 via an osmotic pump (n=4;10 mg/kg/d) or Imatinib plus AMD3100 (n=8) for 28 days, after detection of engraftment by bioluminescent imaging. Survival of the group treated with Imatinib + AMD3100 (Median survival time, MST=49 days) was significantly prolonged compared to the group treated with Imatinib only (MST=38 days) (p<0.05). Next, primary ALL cells of a Ph− patient were transduced with luciferase and injected into NOD/SCID mice (0.4×106 cells/animal). Upon detection of engraftment by bioimaging, xenografted mice were treated with saline (n=2), vincristine-dexamethasone-L-asparaginase (VDL; n=3), AMD3100 (n=3) or AMD3100 plus VDL (n=6). Again, significantly prolonged survival of the group treated with the combination of VDL + AMD3100 (MST=61.5 days) compared to the VDL only treated group (MST=54 days) (p<0.05) was observed. In summary, there was a clear beneficial effect of the combination of AMD3100 with two different chemotherapeutic regimens resulting in a significant improvement of survival. Further preclinical evaluation of AMD3100 as a novel adjuvant has the potential to lead to translation into clinical trials.

Safety and Efficacy of Nilotinib Dose Escalation 600mg BID in Patients (pts) with Imatinib-Resistant or -Intolerant Chronic Myeloid Leukemia (CML) in Chronic Phase (CP).

Background: Nilotinib, a 2nd-generation BCR-ABL inhibitor, is 20–30 times more potent than imatinib in vitro. A phase II, open-label study of safety/efficacy of nilotinib in pts with imatinib-resistant/intolerant Ph+ CML-CP or -AP was reported. Planned starting dose was 400mg BID, with escalation to 600mg BID if response was inadequate. The objective of the post-hoc analysis was to evaluate safety/efficacy of nilotinib 600mg BID in CML-CP pts who had dose escalation.Methods: Starting dose was 400mg BID. If response was inadequate (failure to achieve complete hematologic response [CHR] by 3 mos, minimal cytogenetic response [CyR] by 6 mos, or major cytogenetic response [MCyR] by 12 mos; loss of HR, CyR, or progression), nilotinib to 600mg BID dosing was permitted. Imatinib resistance was defined as treatment with imatinib ≥600 mg/d with disease progression (≥50% increase in WBC, blasts, basophils, or platelets) or no HR in peripheral blood and/or bone marrow after 4 wks.Results: Of 320 enrolled pts in CP, 269 remained on nilotinib 400mg BID; 51 pts were dose-escalated to 600mg BID for inadequate responses (mean age 57.7 yrs; 68.6% male). All pts met the study criteria for imatinib resistance. Prior imatinib dose was ≥600mg QD in 98% pts and ≥800 mg QD in 53%. 43/51 (84%) pts had no baseline CHR; 38% had no prior history of complete cytogenetic response (CCyR). 35% had prior MCyR. Median time to dose escalation was 169 (114–211) d. 14 (27.5%) pts remain on therapy; median time to discontinuation from study was 307 (235–414) d. 5 pts with disease progression and no deaths were observed among pts who received 600mg BID. CHR was achieved in 58% of evaluable pts without CHR at baseline (n=43). MCyR occurred in 29% of evaluable pts (15/51) on 600mg BID; 10% (5/51) CCyR. Disease progression was low, occurring in 10% (5/51) of pts on 600mg BID. Median time to progression for CP pts on 600mg BID was 12 mos, substantially less than for pts who stayed on 400mg BID (median not observed yet, at least 20 mo). Nonhematologic AEs in 600mg BID pts were similar to those reported for the entire CP cohort from the phase II study. Grade 3/4 hematologic lab abnormalities were: anemia 13.8%, neutropenia 7.9%, thrombocytopenia 13.7%. Hyperglycemia and elevated bilirubin occurred in 6% and 10% of pts, respectively, on 600mg BID.Conclusions: Some HR and CyR after dose escalation to 600mg BID were observed. Safety/tolerability for 600mg BID were similar to the entire CP cohort. Based on these preliminary data, nilotinib dose escalation to 600mg BID in imatinib-resistant Ph+ CML-CP pts may be effective in pts with inadequate responses to 400mg BID. As might be expected in a more resistant population, response duration was shorter than reported for the phase II CP cohort. Additional follow-up is necessary. Use of 600mg BID is not currently recommended for imatinib-resistant CML-CP pts outside clinical studies.

Nilotinib in Patients (pts) with Relapsed/Refractory Philadelphia Chromosome-Positive Acute Lymphoblastic Leukemia (Ph+ ALL) Who Are Resistant or Intolerant to Imatinib.

Background: Ph+ ALL occurs in about 15% of adults with ALL and is characterized by a poor prognosis. Imatinib in combination with systemic chemotherapy has become the standard therapy for adults with Ph+ ALL. Nilotinib is a highly specific BCR-ABL inhibitor, which is 30-fold more potent than imatinib and active in vitro against 32 of a panel of 33 BCR-ABL mutations. Nilotinib was demonstrated in a phase I study to be active against a small subset of imatinib-resistant Ph+ ALL adult pts. Based on this preliminary data, a phase II, open-label study was designed to further evaluate the safety and efficacy of nilotinib in pts with relapsed/refractory Ph+ ALL.Methods: The initial dose of nilotinib was 400 mg twice daily (BID) with escalation to 600 mg BID, when there was an inadequate response and no safety concerns.Results: The safety and efficacy data were reported for 41 Ph+ ALL pts in which 38 had relapsed and 3 were refractory [(37 had active disease; 4 had minimal residual disease (MRD)]. The median age at study entry was 46 (18–75) years and 54% were men. At study entry 31% of pts had >35% Ph+ metaphases; chromosomal abnormalities other than Ph+ were noted in 32% (13/41) pts, and extramedullary involvement was present in 3 pts. The median time since initial diagnosis of Ph+ ALL was 15 months. Treatment with nilotinib is ongoing for 2 pts (5%). Most discontinuations were due to disease progression (68%). The median treatment duration was 53 (1–563) days and the median dose intensity was 800 mg/day. Thirty-one percent of pts had >35% of Ph+ metaphases, chromosomal abnormalities other than Ph+ were noted in 32% of the pts, and extramedullary involvement was present in 7% of pts. Complete response (CR) was reported in 10 (24%) pts including 1 patient with MRD. Hematological improvement was observed in 1 (3%) pt, stable disease was observed in 9 pts, 14 pts had progressive disease. The most common Grade 3/4 laboratory abnormalities were thrombocytopenia (60%), neutropenia (43%), anemia (21%), and hypophosphatemia (22%).Conclusions: Nilotinib as a monotherapy appears to have promising activity and a favorable safety profile in relapsed/refractory Ph+ ALL pts. The use of nilotinib in combination with chemotherapy for Ph+ ALL merits further investigation.

Linking the hematopoietic microenvironment to imatinib-resistant Ph+ B-ALL: Figure 1.

Linking the hematopoietic microenvironment to imatinib-resistant Ph⁺ B-ALL: Figure 1.

Phase I study of nilotinib in patients (pts) from Japan with imatinib-resistant Ph+ chronic myelogenous leukemia (CML) or acute lymphocytic leukemia (ALL)

17511 Background: Nilotinib is a highly selective Bcr-Abl tyrosine kinase inhibitor designed to be more potent than imatinib. Results from a phase I study in Japanese pts with imatinib-resistant/intolerant Ph+CML/ALL are reported. Methods: Japanese pts with imatinib-resistant/intolerant Ph+CML (5 CP; 2 AP; 2 BC) or ALL (2) were enrolled in an open-label, dose-escalation study evaluating safety, efficacy and pharmacokinetics (PK) of oral nilotinib. A standard three-pt-per-cohort design was used; initial cohorts included 200mg QD, 400mg QD, and 400mg BID. Serum samples were collected on days 1 and 15 of cycle 1 in all 11 pts. Results: Peak serum concentration was achieved at 4 hrs in most pts. Systemic exposure increased with dose. Serum level reached steady-state within 1 week and was stable over time. The accumulation ratio (AUC day15/day1) was 2 to 3. Nilotinib 400mg BID maintained serum levels &gt;/= 30 times higher than the IC50 required to inhibit Bcr-Abl phosphorylation. No pt experienced dose-limiting toxicity. 7/11 pts had Grade 3/4 AEs, only 1 pt had an AE (thrombocytopenia) suspected as being related to nilotinib treatment. No pt had EKG abnormalities. 57% of all pts and 75% of CML CP pts achieved MCyR. 3 pts in the 200mg QD cohort discontinued due to disease progression, all other pts completed the study. PK results are summarized in Table 1 . Conclusions: Nilotinib PK profile in Japanese pts is comparable to that reported for pts outside Japan. Based on these data, the recommended starting dose of nilotinib in Japanese pts with imatinib-resistant/intolerant Ph+ CML/ALL is 400mg BID. [Table: see text] No significant financial relationships to disclose.

Cell Death Mechanisms Induced by INNO-406, a Novel Tyrosine Kinase Inhibitor for Imatinib-Resistant Ph+ Leukemia.

The blockade of Bcr-Abl signaling suppresses cellular growth and induces cell death in Bcr-Abl-positive (Bcr-Abl+) cells. We herein assessed the cell death mechanisms induced by INNO-406 (formerly NS-187; Kimura et al, Blood 2005), in four CML-derived Bcr-Abl+ cell lines (K562, KT-1, BV173 and MYL), and Ba/F3 harboring wild type bcr-abl (Ba/F3/wt bcr-abl). When cells are treated by INNO-406, the accumulation of subG1 fraction was seen in all five cell lines. This cell death was accompanied by loss of mitochondrial membrane potential and was inhibited by over-expression of Bcl-2, indicating that INNO-406-induced cell death is mainly mediated by mitochondria-dependent apoptosis. Caspase-3 activation in INNO-406-treated cell was also common among all cell lines. However, the inhibition of caspase activity by ZVAD-fmk (ZVAD), a pan-caspase inhibitor, was variable in the cell lines tested. In K562, KT-1 and BV173 cells treated with INNO-406, ZVAD almost completely prevented apoptosis (i.e. showing atypical feature for apoptosis, no DNA fragmentation and no accumulation of subG1 fraction), with cell death resulting from morphologically non-apoptotic cell death. The percentages of non-apoptotic cells under ZVAD co-treated with INNO-406 varied among the three cell lines, suggesting that the dependence on non-apoptotic cell death is variable. While, in MYL and Ba/F3/wt bcr-abl cells, despite the sufficient inhibition of caspases' activity, the inhibition of the cell death by ZVAD was only partial and these cell lines still underwent apoptosis (i.e. showing DNA fragmentation and the accumulation of subG1 population), suggesting the presence of caspase-independent apoptotic machineries. In addition, assay data for apoptosome activities (complex of Apaf-1, cytochrome c and caspase-9 that initiates and drives cysteine protease activities of caspase in mitochondrial-mediated pathway) suggested that cell types could be largely subdivided into two groups, namely those cells with high apoptosome activity (K562, KT-1 and BV173) that undergo non-apoptotic, and, those cells with low apoptosome activity (MYL and Ba/F3/wt bcr-abl.) that undergo caspase-independent apoptosis when caspase activity was blocked by ZVAD. These data indicate that there is a common initial pathway for cell death due to INNO-406, while the pathway for cell death commitment (i.e. dependence on apoptosome/caspases-mediated apoptosis pathway that has been commonly believed to be central for apoptosis execution) vary among cellular context in Bcr-Abl+ leukemic cells. Moreover, in a mouse model of primary human CML in blast crisis, INNO-406 caused cell death with fragmented nuclei typical to apoptosis and “necklace-like” nuclei not typical of apoptosis, further implicating the significance of involvement of caspase-independent, non-apoptotic cell death in vivo. Further studies of the role of caspase-independent cell death in patient-derived Bcr-Abl+ cells and the molecular mechanisms that lead to mitochondrial-depolarization and caspase-independent apoptotic and/or non-apoptotic cell death may help the development of novel therapeutic strategies against Bcr-Abl+ leukemias.

Advances and potential treatment for Philadelphia chromosome-positive adult acute lymphoid leukaemia

Philadelphia chromosome-positive (Ph+) acute lymphoblastic leukaemia (ALL) is the most common subtype of ALL in adults. Conventional chemotherapy-based approaches that are effective in other precursor B cell ALL cases have a poor chances of cure in patients with a Ph+ diagnosis. Therefore, allogeneic stem cell transplantation performed during the first remission is the recommended therapy. Recently, the availability of imatinib mesylate and other tyrosine kinase inhibitors and small molecules that affect the BCR/ABL signalling pathways has introduced a new therapeutic opportunity, and could change the treatment paradigm and prognosis for these patients. In this article, the results from clinical trials using imatinib in relapsed/refractory patients and as front-line therapy are described. In addition, preliminary experiences with novel tyrosine kinase inhibitors in imatinib-resistant Ph+ ALL are discussed.

Bcr-Abl mutations in imatinib-resistant CML and Ph+ALL patients (pts) enrolled in a phase I study of AMN107

6527 Background: AMN107 is a potent, highly selective, aminopyrimidine inhibitor which in vitro is 30-fold more potent than imatinib and active against 32/33 imatinib resistant Bcr-Abl mutations. Methods: The spectrum of mutations in Bcr-Abl was evaluated by direct sequencing of the kinase domain and surrounding regions on samples from pts with imatinib-resistant Ph+ CML or ALL in a phase I study of AMN107 (total daily dose range 50 to 1200 mg administered qd or bid). The template was created by semi-nested PCR using primers in the BCR and ABL regions of the gene. Mutations were correlated with clinical response. Results: Among 119 pts, 86 had both pre and post tx analyses: 39 (45%) had mutations at baseline, of whom 27 (69%) responded. 47 (55%) had no mutation at baseline, of whom 34 (72%) responded. The most common baseline mutations were G250E, E255K/V, E355G, F317L, H396R and M351T. New mutations were found during median follow-up of 112 (6 - 350) days in 37 pts (26 without baseline mutations). New mutations most commonly included F349V, E255K/V, E355G, G250E, M244V and T315I. Of the 37 pts, 30 had evaluations after mutations emerged, and 15 continued to respond for median of 160 (41–351) days. Fourteen mutations not previously reported occurred, 6 at codons with known imatinib resistance mutations resulting in novel amino acid substitutions. Novel mutations in multiple pts and/or multiple timepoints included E344G, F311I, E453K, E459Q and L248L. The T315I mutation was present at baseline in 1 pt who failed to respond and emerged in 4 pts, of whom follow-up was available for 3. Two continued to respond ≥ 80 days after developing the mutation and one progressed when the mutation emerged. Conclusions: AMN107 has clinical activity in pts with Ph+CML/ALL with nonmutated and mutated Bcr-Abl. At AMN107 doses used in this phase I study, new mutations often emerged, but were not a reliable predictor of clinical relapse. [Table: see text]