Targeting BCR-ABL Research Articles

Identification and Characterization of New Microrna Biomarkers and Target Genes in Drug-Insensitive CD34+ CML Stem/Progenitor Cells Using Global Comparative RNA-Seq Analyses

Therapeutic targeting of BCR-ABL with selective ABL tyrosine kinase inhibitors (TKIs) has led to a significant survival benefit for early phase CML. However, TKI monotherapies are rarely curative, with persistence of leukemic stem cells, emergence of resistance and relapses remaining as challenges. To identify differentially expressed and new miRNAs in CD34+ CML stem/progenitor cells that might serve as potential biomarkers and/or therapeutic targets, we have performed Illumina Deep Sequencing to obtain absolute miRNA expression profiles of highly purified CD34+ cells obtained at newly diagnosed stage from six CML patients. Three of the patients were classified retrospectively, after imatinib (IM) therapy, as IM-responders and three as IM-nonresponders. CD34+ cells isolated from five normal bone marrow (NBM) samples were similarly analyzed as controls. Bioconductor DESeq2 analysis revealed 63 differentially expressed miRNAs between CML and NBM samples (adjusted P<0.05). Most differentially expressed miRNAs identified were down-regulated in CML compared to NBM, while 17 were up-regulated. Interestingly, 12 miRNAs were found to be differentially expressed between the IM-responders and IM-nonresponders. In addition, 34 novel miRNAs were identified in the CD34+ CML stem/progenitor cells. We next validated the sequencing data in a larger cohort of samples. CD34+ cells from IM-responders (n=12), IM-nonresponders (n=10) and normal individuals (n=11) were analyzed using a high-throughput qPCR microfluidics device. These studies confirmed the differential expression in CD34+ CML cells of 32 of the 63 miRNAs (adjusted P<0.05), including an increased level of oncomirs miR-155 and miR-17-92, and a decreased level of tumor suppressors miR-145, miR-151, and miR-452. Importantly, significant changes in some of these miRNAs were detected in CD34+ cells from CML patients (n=60) after three months of nilotinib (NL) treatment compared to the same patient samples before the treatment: expression of 18 miRNAs were normalized after NL therapy, whereas 10 showed little change. To further identify potential miRNA target genes, RNA-seq analysis was performed on the same RNA samples to correlate miRNA profiles with corresponding mRNA expression changes. Bioconductor RmiR analysis was performed to match miRNA target genes whose expression was inversely correlated with the expression of deregulated miRNAs based on three of six prediction algorithms (mirBase, TargetScan, miRanda, tarBase, mirTarget2, and PicTar). We have identified 1,210 differentially expressed mRNAs that are predicted targets of the deregulated miRNAs in the comparison of CML and NBM data. Interestingly, only seven differentially expressed genes were predicted targets of the deregulated miRNAs identified in a comparison of IM-responders and IM-nonresponders. Most of the predicted target genes are involved in cell cycle regulation, MAPK signaling and TGF-beta signaling pathways according to DAVID Bioinformatics Resources analysis, which clusters predicted target genes to known KEGG pathways. To elucidate the biological significance of the differentially expressed miRNAs in TKI-insensitive CML stem/progenitor cells, a number of functional assays were performed. An initial screen of eight miRNAs, selected for their novelty and CML-related potential target genes, was performed by transiently transfecting CML cells with miRNA mimics or inhibitors, and chemically synthesized RNAs which mimic or inhibit mature endogenous miRNAs. Four of the eight miRNA mimics/inhibitors transfected cells displayed significant growth disadvantages and enhanced sensitivity to TKI treatments based on trypan-blue exclusion, thymidine incorporation, apoptosis, and colony-forming cell assays. Q-RT-PCR analysis further showed reduced expression of their predicted target genes in cells transfected with miRNA mimics. Taken together, we have identified aberrant, differentially expressed miRNAs and their target genes in TKI-insensitive CML stem/progenitor cells that may serve as useful biomarkers to predict clinical response of CML patients to TKI therapy and ultimately lead to identification of new therapeutic targets for improved treatment options in CML. DisclosuresNo relevant conflicts of interest to declare.

Beclin-1 Phosphorylation By BCR-ABL Is Crucial for CML Leukemogenesis By Suppression of Autophagy

The constitutively activated chimeric Tyrosine kinase BCR-ABL is critical for initiation, progression and maintenance of chronic myelogenous leukemia (CML). Imatinib and second generation BCR-ABL tyrosine kinase inhibitors (TKIs) serve now as standard therapies for Ph+-patients. However, disease persistence occurs frequently and insensitivity of CML stem cells to TKI treatment is discussed as one major reason for this.Recent evidence accumulates, that autophagy, a genetically-regulated process of adaptation to metabolic stress, is involved in TKI-induced cell death. It is hypothesized, that TKI-induced autophagy could allow CML stem cells to become metabolically dormant enabling their survival under conditions that may mimic growth factor deprivation and thereby "antagonize" TKI-induced cell death. However, the molecular mechanism of BCR-ABL and TKI induced autophagy as well as its role as tumor suppressor or promoter is poorly understood.In our study, we aim to identify the precise role of autophagy and its´ effector molecules in a murine CML model. To test whether BCR-ABL regulates autophagy, we measured LC3 as a marker for autophagy in BCR-ABL+-K562 cell. Interestingly, inhibition of BCR-ABL activity by nilotinib led to increased LC3-II expression and punctual LC3 accumulation, indicating, that BCR-ABL activity can suppress autophagy. Consistent with this, Ba/F3 cells expressing BCR-ABL WT induce autophagy, whereas Ba/F3 cell expressing BCR-ABL-T315I fail to induce autophagy by nilotinib treatment, pointing to a BCR-ABL specific autophagy induction than an unspecific effect of TKI treatment. Next, we investigated the proteins involved in BCR-ABL mediated autophagosome formation. Recruitment of VPS34 and ATG14 to Beclin1 was increased in case of nilotinib treatment and could thereby positively regulate autophagosome formation, whereas Rubicon, a negative regulator was less recruited to the Beclin1-complex.To further identify the impact of Beclin1 as a key regulator of autophagy in BCR-ABL-driven leukemia, we used a targeted genetic approach in a CML mouse model. Interestingly, mice transplanted with Belin1 knockdown, BCR-ABL expressing bone marrow showed a less aggressive disease with significantly lower WBC-count, leukemic burden and prolonged overall survival of the mice. In contrast, deletion of ATG5, another central regulator of autophagy, was not able to change disease onset or progression in the CML model.To further clarify the function of Beclin1, we performed biochemical binding analyses and were able to show, that Beclin1 binds to BCR-ABL independent of BCR-ABL kinase activity and Beclin1 is phosphorylated by BCR-ABL. Interestingly, Beclin1 is an exclusive target of BCR-ABL and can not be phosphorylated by other aberrantly activated tyrosine kinases like Flt3-ITD, NPM-ALK and PDGFRA-D842V. In vitro kinase assay with active ABL-kinase confirm Beclin1 as a specific substrate of BCR-ABL. GST pulldown experiments mapped the N-terminal region of Beclin1 to interact with BCR-ABL. Cloning of different phospho-deficient mutants identified tyrosine residues Y233 and Y352 of Beclin1 as the crucial sites for specific BCR-ABL phosphorylation. To test the impact of BCR-ABL mediated Beclin1-phosphorylation on autophagy induction, we generated Beclin1 phospho-mimic (Y233E/Y352E) and phospho-deficient (Y233F/Y352F) mutations. Interestingly, nilotinib treatment fails to induce autophagy in cells expressing the Beclin1 phospho-mimic mutations, thereby highlighting the necessity of Beclin1 in BCR-ABL-mediated autophagy. Expression of Beclin1 mutations in Beclin1 knockout MEFs and K562 cells show decreased binding of UVRAG, ATG14 and VPS34 to Beclin1 Y233E/Y352E, suggesting an important role of Beclin1 phosphorylation for complex stabilization and autophagy suppression.Taken together our findings identify Beclin1 as a specific substrate of BCR-ABL. Downregulation of Beclin1 is associated with a prolonged overall survival of BCR-ABL transplanted animals; direct phosphorylation of Beclin1 on Tyrosine residues Y233 and Y352 lead to LC3 inhibition and suppression of autophagy. Our results thereby highlight the importance of Beclin1 in BCR-ABL-mediated leukemogenesis and show, that autophagy induction in CML cells may be rather a specific Beclin1-BCR-ABL interaction effect than a general microenvironmental stress phenomenon. DisclosuresNo relevant conflicts of interest to declare.

A New AHI-1-DNM2-BCR-ABL Complex Regulates Leukemic Properties of Primitive CML Cells through Mediation of Cellular Endocytosis and ROS-Induced Autophagy

Tyrosine kinase inhibitor (TKI) therapies have been introduced into clinical practice with remarkable effects on chronic phase CML. However, early relapses, acquired drug resistance, and persistence of leukemic stem cells (LSCs) remain problematic. Improved treatment approaches to specifically target key molecular elements active in CML LSCs are needed. One candidate is the adaptor protein AHI-1 (Abelson helper integration site-1), an oncogene that is highly deregulated in LSCs. An AHI-1-mediated protein complex containing BCR-ABL and JAK2 has been shown to modulate transforming activity and TKI-response/resistance of CML LSCs. We have recently identified the large GTPase Dynamin-2 (DNM2) as another AHI-1 interacting protein using the AHI-1 SH3 domain as protein "bait" in immunoprecipitation (IP)/mass spectrometry. DNM2 plays key roles in the regulation of trafficking processes such as endocytosis, and is activated through tyrosine phosphorylation. However, its role in CML pathogenesis is unknown. We have now demonstrated that transcript levels of DNM2 are significantly increased in treatment-naive CD34+ cells from CML patients who were classified retrospectively, after Imatinib (IM) therapy, as IM-responders (n=11) and IM-nonresponders (n=15) in comparison to CD34+ normal bone marrow cells (n=7, p=0.013 and 0.037). In particular, DNM2 is more highly expressed in CML stem-enriched cells (lin-CD34+CD38-) and progenitor cells (lin-CD34+CD38+) than more mature cells (lin+CD34-, 2-fold). Interestingly, BCR-ABL+ human cells with stable knockdown of DNM2 exhibited significantly reduced cell growth and increased apoptosis as compared to control cells. They also showed enhanced sensitivity to single TKIs or MitMAB (DNM2 inhibitor) and these effects were greatly enhanced by combination treatments (2-3 fold). Mechanistically, co-IP and confocal co-localization analysis with mutant forms of AHI-1 and DNM2 (HA-AHI-1, HA-AHI-1 SH3Δ, Myc-DNM2 and Myc-DNM2 PRDΔ) in 293T cells indicated that the PRD domain of DNM2 is mainly responsible for the interaction between the SH3 domain of AHI-1 and DMN2. More importantly, we identified a new protein interaction between DNM2 and BCR-ABL in both BCR-ABL and BCR-ABL/AHI-1 co-transduced hematopoietic cells using co-IP/Western analysis; this interaction is enhanced in BCR-ABL/AHI-1 co-transduced cells. Moreover, DNM2 phosphorylation was decreased upon IM treatment in BCR-ABL-transduced cells, but remained unchanged in control and T315I mutant cells, suggesting that DNM2 is a direct target of BCR-ABL. Interestingly, we further observed that AHI-1 co-localizes with EEA-1 (early endosome marker) and LAMP-1 (late endosome marker) in cells co-transfected with full-length AHI-1 and DNM2, but not in AHI-1 and DNM2 mutant cells. Using transferrin uptake assays, increased transferrin signals were observed in BCR-ABL/AHI-1 co-transduced cells compared to BCR-ABL- and BCR-ABL/AHI-1 SH3Δ-transduced cells, while transferrin signals were much lower in DNM2-knockdown cells than control cells. These results suggest that the AHI-1-DNM2-BCR-ABL complex indeed improves the kinetics and increases efficiency of endocytosis, which may contribute to response/resistance of primitive CML cells to TKIs. This is further supported by observation of increased surface expression levels of CXCR4 in DNM2 knockdown cells, a key endocytotic target and known mediator of TKI response in CML. Moreover, reactive oxygen species (ROS) signals were found to be higher in BCR-ABL/AHI-1 co-transduced cells than BCR-ABL- and BCR-ABL/AHI-1 SH3Δ-transduced cells, while ROS accumulation was significantly decreased in DNM2-knockdown cells compared to control cells. More interestingly, ROS-induced autophagy was further observed in BCR-ABL/AHI-1 co-transduced cells, but reduced in DNM-2-knockdown cells, with protein expression changes in key autophagy proteins, including ULK-1, Beclin-1, LC3 and p62. To the best of our knowledge, this is the first study to implicate a new AHI-1-DNM2-BCR-ABL complex in the deregulation of endocytosis signaling and ROS production/autophagy in CML. This may play a unique and important role in the regulation of cellular properties of primitive CML cells, including their response/resistance to TKI. DisclosuresNo relevant conflicts of interest to declare.

Therapeutic Immune Monitoring of Chronic Myeloid Leukemia Patients Treated with Tyrosine Kinase Inhibitors: Focus on CD4+ CD25+ t Cells

Background and Objectives: In clinical, conventional Tyrosine Kinase Inhibitors (TKIs) including imatinib, dasatinib, and nilotinib are remarkably effective forms of therapy for certain types of solid cancers as well as Ph+ leukemias. In addition to the BCR-ABL target oncoprotein, they also inhibit certain off-target kinases (Eph, c-KIT, TEC, SRC). Some TKIs affect immune reconstitution as well as the proliferation, function, and activation of T cells. Certain TKIs have been known to have an especially strong effect on CD4+CD25+ T cells, also known as regulatory T Cells (Tregs). There is currently a gap in the clinical data available about on this area of study.Patients and methods: In this study, we collected 108 Peripheral Blood (PB) samples from patients in the Chronic Phase (CP) of Chronic Myeloid Leukemia (CML) at the time of diagnosis (n=31) and also the TKIs treatment. Groups consisted of individuals treated with TKIs like imatinib (n=12), dasatinib (n=11) and nilotinib (n=8), as well as healthy controls (n=15). We evaluated the quantity and function of Tregs from patients in the CML-CP at the time of diagnosis and during treatment with TKIs.Results: It was found that at diagnosis, patients with CML had a similar proportion and absolute number of lymphocytes compared to healthy donors. After TKIs treatment, proportions and absolute numbers of total T cellsACD4+ T cells and Tregs decreased at different degree. Moreover, thedecrease would be more and more significant as time goes on.Our results indicated that although these three TKIs show similar inhibitory effects in the proportion and number of Tregs in vivo, they have differential effects on the functions of Tregs in vitro. The proliferation, suppression, and expression of suppressive cytokines (IL-4,IL-10 and TGF-β) as well as suppression-associated molecules (FoxP3, GITR, and CTLA-4) of Tregs decreased in groups treated with imatinib and dasatinib. The decrease was not significant in the nilotinib-treated group.Conclusions: The results showed that imatinib and dasatinib have stronger inhibitory roles than nilotinib when it comes to regulating the number and functions of Tregs. These findings can be used to argue in favor of calls for personalized treatment and follow-up of CML patients during TKIs treatment, particularly for those patients who received combination therapy with allo-transplantation and post-transplant TKIs. DisclosuresNo relevant conflicts of interest to declare.

Synergistic effects of p53 activation via MDM2 inhibition in combination with inhibition of Bcl-2 or Bcr-Abl in CD34+ proliferating and quiescent chronic myeloid leukemia blast crisis cells.

The Bcr-Abl tyrosine kinase regulates several Bcl-2 family proteins that confer resistance to apoptosis in chronic myeloid leukemia (CML) cells. Given p53's ability to modulate the expression and activity of Bcl-2 family members, we hypothesized that targeting Bcr-Abl, Bcl-2, and p53 concomitantly could have therapeutic benefits in blast crisis (BC) CML and in quiescent CML CD34+ cells that are insensitive to tyrosine kinase inhibitors (TKI). We examined the effects of the MDM2 inhibitor nutlin3a and its combination with the dual Bcl-2 and Bcl-xL inhibitor ABT-737, and the Bcr-Abl inhibitor nilotinib on BC CML patient samples. We found that in quiescent CD34+ progenitors, p53 expression is significantly lower, and MDM2 is higher, compared to their proliferating counterparts. Treatment with nutlin3a induced apoptosis in bulk and CD34+CD38- cells, and in both proliferating and quiescent CD34+ progenitor CML cells. Nutlin3a synergized with ABT-737 and nilotinib, in part by inducing pro-apoptotic, and suppressing anti-apoptotic, Bcl-2 proteins. Nilotinib inhibited the expression of Bcl-xL and Mcl-1 in BC CML cells. These results demonstrate that p53 activation by MDM2 blockade can sensitize BC CML cells, including quiescent CD34+ cells, to Bcl-2 inhibitor- and TKI-induced apoptosis. This novel strategy could be useful in the therapy of BC CML.

Abstract 5389: Novel kinase inhibitor for imatinib-resistant chronic myeloid leukemia with T315I mutation

Abstract 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 pathways in a dose dependent manner. Furthermore, it significantly increased the sub G1 phase associated with early apoptosis, with increased levels of cleaved PARP and 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 the expression 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 pathways 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. Citation Format: Soon-Sun Hong, Soo Jung Kim, Kyung Hee Jung, Hong Hua Yan, Zhenghuan Fang, Joo Han Lim, Jeong Seon Ryu. Novel kinase inhibitor for imatinib-resistant chronic myeloid leukemia with T315I mutation. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5389. doi:10.1158/1538-7445.AM2015-5389

CD-200 induces apoptosis and inhibits Bcr-Abl signaling in imatinib-resistant chronic myeloid leukemia with T315I mutation.

Chronic myeloid leukemia (CML) is characterized by a constitutively active Bcr-Abl tyrosine kinase. Although Imatinib has been proven to be an effective drug against CML, its resistance has been observed with disease relapse due to T315I predominant point mutation. Liriodendron tulipifera L., one of the fastest growing hardwood tree species, exerts antioxidant activity and anti-inflammatory effects. However, its anticancer effect has been minimally reported. In this study, we extracted CD-200 from Liriodendron tulipifera L. and investigated its effect on cell survival or apoptosis in CML cells with Bcr-Abl/T315I (BaF3/T315I) as well as wild-type Bcr-Abl (BaF3/WT). CD-200 inhibited cell proliferation in the BaF3/WT cells, and also in the BaF3/T315I cells with Imatinib resistance. Moreover, it strongly inhibited Bcr-Abl signaling pathways in a dose-dependent manner. Also, it significantly increased the sub-G1 phase and the expression of cleaved PARP and caspase-3, as well as the TUNEL-positive apoptotic cells. In addition, we observed that CD-200 induced apoptosis with a loss of mitochondrial membrane potential by decreasing the expression of Mcl-1 and survivin. Furthermore, CD-200 showed a significant inhibition in tumor growth, compared to Imatinib in BaF3/T315I mouse xenograft models. Taken together, our study demonstrates that CD-200 exhibits apoptosis induction and anti-proliferative effect by blocking the Bcr-Abl signaling pathways in the Bcr-Abl/T315I with resistance to Imatinib. We suggest that CD-200 may be a natural product to target Bcr-Abl and overcome Imatinib resistance in CML patients.

Restoration of miR-424 suppresses BCR–ABL activity and sensitizes CML cells to imatinib treatment

MicroRNAs (miRNAs) are small noncoding RNAs that participate in many biological processes by posttranscriptionally regulating gene expression. Dysregulation of miRNA expression has been shown to be typical of many neoplasms. Chronic myeloid leukemia (CML) is a disorder of hematopoietic stem cells carrying the Philadelphia (Ph) chromosome and an oncogenic BCR–ABL tyrosine kinase fusion gene. While the development of tyrosine kinase inhibitors (TKIs) like imatinib has revolutionized treatment of CML, it has become increasingly clear in recent years that TKI treatment alone will not be curative in many cases. Thus, further dissection of the regulatory networks that drive BCR–ABL-induced malignant transformation may help to identify other novel therapeutic approaches that complement TKI treatment. In this study we demonstrate that the expression of miR-424 is markedly low in CML cell lines and patient samples at time of diagnosis. With the aid of bioinformatics analysis we revealed a conserved target site for miR-424 in the 3′-untranslated region (UTR) of the ABL gene. Via luciferase assays, we showed that miR-424 directly targets BCR–ABL. Overexpression of miR-424 was shown to suppress proliferation and induce apoptosis of K562 cells as well as sensitize these cells to imatinib treatment. These findings strongly suggest that miR-424 acts as a tumor suppressor by downregulating BCR–ABL expression. Up-regulation of miR-424 in CML cells may therefore have a therapeutic effect against this disease.

Targeting BCL-2 and BCR-Abl Activity in Ph+ALL

s / Biol Blood Marrow Transplant 21 (2015) S185eS205 S190 time to relapse from day 30 post-transplant was 69 days (range 41-230 days). The cumulative incidence of acute GVHD grade II-IV was 44% with 10% grade III-IV. The cumulative incidence of chronic GVHD at 2 years was 48%, with 22% extensive chronic GVHD. Conclusion: In this cohort of patients with AML/ MDS, MCP-1 levels at day 30 post allogeneic HCT in patients who had achieved complete remission and full chimerism were predictive of relapse two months on average prior to overt hematological relapse. Larger studies may find potential role of MCP-1 in predicting relapse post-transplant and development of early strategies for prevention.

Genetic Depletion of Fc Gamma Receptor 2b Affects CML Stem Cell Biology

Chronic myeloid leukemia (CML) is caused by the acquisition of the Philadelphia chromosome t(9;22) at the hematopoietic stem cell (HSC) level. The resulting Bcr-Abl protein loses the Abl autoinhibitory function and gains the Bcr oligomerization domain resulting in the constitutive activation of the Abl tyrosine kinase. The implementation of Bcr-Abl tyrosine kinase inhibitors (TKI) has greatly improved therapy outcome of CML patients. However, various mechanisms such as the acquisition of tyrosine kinase domain mutations or kinase-independent alterations have been described to result in therapy resistance. Using an inducible transgenic SCLtTAxBcr-Abl CML mouse model, we previously demonstrated that Bcr-Abl inhibition leads to eradication of mature leukemic cells but spares disease-driving leukemic stem cells (LSC) due to a lack of oncogene-addiction (Schemionek et al., BLOOD 2010; Hamilton, Helgason, Schemionek et al., BLOOD 2012). Aiming to identify new Bcr-Abl targets that could overcome therapy resistance of stem cells, we previously performed a microarray analysis using murine LSC. Expression of FcγRIIb (Fc gamma receptor 2b; CD32) was significantly increased 2.79-fold in Bcr-Abl positive vs. control Lin-Sca1+c-kit+(LSK) cells. The FcγRIIb contains an immuno receptor tyrosine-based inhibitory motif (ITIM) and inhibits intracellular signaling upon activation. Phosphorylation of the receptor is mainly mediated via Src kinase Lyn and enables the receptor to recruit Src homology 2 domain-containing phosphatases.Using qRT-PCR, we confirmed 2.83-fold upregulation of FcγRIIb within the LSK population of CML mice. Retroviral infection of 32D cells and primary BM cells with Bcr-Abl significantly increased FcγRIIb expression level (90.3-fold and 13.2-fold, resp., p<0.005 each vs. emtpy vector). TKI treatment of Bcr-Abl positive 32D cells did not affect FcγRIIb mRNA levels or surface expression. Bcr-Abl infection of lineage-depleted BM cells decreased growth factor independent colony forming capability (5.5-fold, p<0.05) and the proliferation potential of FcγRIIb-/- lineage negative bone marrow (cells) compared to wild type (wt) in the absence of cytokines. Transplantation of Bcr-Abl infected FcγRIIb-/- BM cells significantly reduced white blood cell count (WBC) of the recipients as compared to wt Bcr-Abl positive cells by 2.4-fold (p<0.05). Interestingly, WBC of Bcr-Abl positive FcγRIIb-/- mice was similar to FcγRIIb-/- empty vector control animals (p=0.875) as well as wt control mice (p=0.241). CML development was analyzed upon autopsy by FACS of BM and spleen cells and showed a 2.0-fold reduction (p<0.05) of leukemic c-kit positive cells as well as a reduction in spleen weight in recipients of Bcr-Abl transduced FcγRIIb-/- cells (293mg ± 30) vs. Bcr-Abl transduced wt controls (381mg ± 13; p<0.05). Moreover, total numbers of leukemic LSK cells were significantly decreased by 3.5-fold in BM and 3-fold in spleen of Bcr-Abl transduced FcγRIIb-/- BM recipients compared to recipients of Bcr-Abl transduced wt cells. Transplantation of empty vector transduced FcγRIIb-/- vs. empty vector transduced wt control cells did not result in a decrease of knock-out cells showing that the observed effects were specific for impairment of Bcr-Abl transforming activity by loss of FcγRIIb but not due to FcγRIIb depletion alone.In conclusion, we show that FcγRIIb is upregulated in the stem cell compartment of CML mice and that this is due to Bcr-Abl expression. The absence of FcγRIIb impairs leukemic cell growth in vitro and in vivo. Moreover, genetic depletion of the receptor decreases LSC numbers and affects CML development in vivo suggesting that FcγRIIb plays an important role in Bcr-Abl leukemogenesis, making it an attractive novel stem cell specific target for CML therapy. (*Authors contributed equally) DisclosuresBrümmendorf:Novartis: Consultancy, Honoraria, Membership on an entity’s Board of Directors or advisory committees, Patents & Royalties, Research Funding. Koschmieder*:Novartis: Consultancy, Honoraria, Other, Research Funding.

HS-543 induces apoptosis of Imatinib-resistant chronic myelogenous leukemia with T315I mutation.

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.

Nucleo-cytoplasmic transport as a therapeutic target of cancer.

Shuttling of specific proteins out of the nucleus is essential for the regulation of the cell cycle and proliferation of both normal and malignant tissues. Dysregulation of this fundamental process may affect many other important cellular processes such as tumor growth, inflammatory response, cell cycle, and apoptosis. It is known that XPO1 (Exportin-1/Chromosome Region Maintenance 1/CRM1) is the main mediator of nuclear export in many cell types. Nuclear proteins exported to the cytoplasm by XPO1 include the drug targets topoisomerase IIα (topo IIα) and BCR-ABL and tumor suppressor proteins such as Rb, APC, p53, p21, and p27. XPO1 can mediate cell proliferation through several pathways: (i) the sub-cellular localization of NES-containing oncogenes and tumor suppressor proteins, (ii) the control of the mitotic apparatus and chromosome segregation, and (iii) the maintenance of nuclear and chromosomal structures. The XPO1 protein is elevated in ovarian carcinoma, glioma, osteosarcoma, pancreatic and cervical cancer. There is a growing body of research indicating that XPO1 may have an important role as a prognostic marker in solid tumors. Because of this, nuclear export inhibition through XPO1 is a potential target for therapeutic intervention in many cancers. The best understood XPO1 inhibitors are the small molecule nuclear export inhibitors (NEIs; Leptomycin B and derivatives, ratjadones, PKF050-638, valtrate, ACA, CBS9106, selinexor/KPT-330, and verdinexor/KPT-335). Selinexor and verdinexor are orally bioavailable, highly potent, small molecules that are classified as Selective Inhibitors of Nuclear Export (SINE). KPT-330 is the only NEI currently in Phase I/II human clinical trials in hematological and solid cancers. Of all the potential targets in nuclear cytoplasmic transport, the nuclear export receptor XPO1 remains the best understood and most advanced therapeutic target for the treatment of cancer.Electronic supplementary materialThe online version of this article (doi:10.1186/s13045-014-0085-1) contains supplementary material, which is available to authorized users.

Tyrosine kinase inhibitor Thiotanib targets Bcr-Abl and induces apoptosis and autophagy in human chronic myeloid leukemia cells.

Chronic myeloid leukemia (CML) is characterized by abnormal Bcr and Abl genes and enhanced tyrosine kinase activity. Anti-CML therapy has been much improved along with the applications of tyrosine kinase inhibitors (TKIs) which selectively target Bcr-Abl and have a cytotoxic effect on CML. Recently, four-membered heterocycles as "compact modules" have attracted much interest in drug discovery. Grafting these small four-membered heterocycles onto a molecular scaffold could probably provide compounds that retain notable activity and populate chemical space otherwise not previously accessed. Accordingly, a novel TKI, Thiotanib, has been designed and synthesized. It selectively targets Bcr-Abl, inducing growth inhibition, cell cycle arrest, and apoptosis of CML cells. Meanwhile, the compound Thiotanib could also induce autophagy in CML cells. Interestingly, inhibition of autophagy promotes Thiotanib-induced apoptosis with no further activation of caspase 3, while inhibition of caspases did not affect the cell survival of CML cells. Moreover, the compound Thiotanib could inhibit phosphorylation of Akt and mTOR, increase beclin-1 and Vps34, and block the formation of the Bcl-2 and Beclin-1 complex. This indicates the probable pathway of autophagy initiation. Our results highlight a new approach for TKI reforming and further provide an indication of the efficacy enhancement of TKIs in combination with autophagy inhibitors.

Selective JAK2/ABL dual inhibition therapy effectively eliminates TKI-insensitive CML stem/progenitor cells

Imatinib Mesylate (IM) and other tyrosine kinase inhibitor (TKI) therapies have had a major impact on the treatment of chronic myeloid leukemia (CML). However, TKI monotherapy is not curative, with relapse and persistence of leukemic stem cells (LSCs) remaining a challenge. We have recently identified an AHI-1-BCR-ABL-JAK2 protein complex that contributes to the transforming activity of BCR-ABL and IM-resistance in CML stem/progenitor cells. JAK2 thus emerges as an attractive target for improved therapies, but off-target effects of newly developed JAK2 inhibitors on normal hematopoietic cells remain a concern. We have examined the biological effects of a highly selective, orally bioavailable JAK2 inhibitor, BMS-911543, in combination with TKIs on CD34+ treatment-naïve IM-nonresponder cells. Combination therapy reduces JAK2/STAT5 and CRKL activities, induces apoptosis, inhibits proliferation and colony growth, and eliminates CML LSCs in vitro. Importantly, BMS-911543 selectively targets CML stem/progenitor cells while sparing healthy stem/progenitor cells. Oral BMS-911543 combined with the potent TKI dasatinib more effectively eliminates infiltrated leukemic cells in hematopoietic tissues than TKI monotherapy and enhances survival of leukemic mice. Dual targeting BCR-ABL and JAK2 activities in CML stem/progenitor cells may consequently lead to more effective disease eradication, especially in patients at high risk of TKI resistance and disease progression.

Regulation of the Interferon regulatory factor-8 (IRF-8) Tumor Suppressor Gene by the Signal Transducer and Activator of Transcription 5 (STAT5) Transcription Factor in Chronic Myeloid Leukemia

Tyrosine kinase inhibitors such as imatinib can effectively target the BCR-ABL oncoprotein in a majority of patients with chronic myeloid leukemia (CML). Unfortunately, some patients are resistant primarily to imatinib and others develop drug resistance, prompting interest in the discovery of new drug targets. Although much of this resistance can be explained by the presence of mutations within the tyrosine kinase domain of BCR-ABL, such mutations are not universally identified. Interferon regulatory factor-8 (IRF-8) is a transcription factor that is essential for myelopoiesis. Depressed IRF-8 levels are observed in a majority of CML patients and Irf-8(-/-) mice exhibit a CML-like disease. The underlying mechanisms of IRF-8 loss in CML are unknown. We hypothesized that BCR-ABL suppresses transcription of IRF-8 through STAT5, a proximal BCR-ABL target. Treatment of primary cells from newly diagnosed CML patients in chronic phase as well as BCR-ABL(+) cell lines with imatinib increased IRF-8 transcription. Furthermore, IRF-8 expression in cell line models was necessary for imatinib-induced antitumor responses. We have demonstrated that IRF-8 is a direct target of STAT5 and that silencing of STAT5 induced IRF-8 expression. Conversely, activating STAT5 suppressed IRF-8 transcription. Finally, we showed that STAT5 blockade using a recently discovered antagonist increased IRF-8 expression in patient samples. These data reveal a previously unrecognized BCR-ABL-STAT5-IRF-8 network, which widens the repertoire of potentially new anti-CML targets.

Nilotinib for the Frontline Treatment of Chronic Myeloid Leukemia Carrying the p230 Transcript: Dream or Reality?

OPINION article Front. Oncol., 03 February 2014Sec. Hematologic Malignancies https://doi.org/10.3389/fonc.2014.00017

Imatinib causing drug rash with eosinophilia and systemic symptoms: A rare cutaneous reaction.

Imatinib causing drug rash with eosinophilia and systemic symptoms: A rare cutaneous reaction.

Dual Specific Inhibitors Of The BCR-ABL and MNK Kinases As Potential Therapeutics For Blast Crisis Chronic Myeloid Leukemia

Pharmacologic inhibition of BCR-ABL by tyrosine kinase inhibitors (TKI) provides effective therapy for chronic phase but not blast crisis (BC) CML. The inability of TKIs to control BC CML is due to the reactivation of BCR-ABL through TKI resistance-conferring mutations, as well as the activation of alternative oncogenic pathways that mediate acquired leukemia stem cell (LSC) function and differentiation block in leukemic progenitors. Accordingly, the development of effective BC therapeutics will require reversing multiple contributors to the BC phenotype, and indicates that targeting BCR-ABL alone will not be sufficient to control BC CML. Indeed, despite the ability of third-generation TKIs to effectively inhibit the majority of clinically-relevant BCR-ABL kinase domain mutations, most patients with BC treated with these agents continue to relapse despite initial responses. Because our recent work has highlighted the importance of the MNK kinase-eIF4E axis in b-catenin-associated BC self-renewal (Lim et al., PNAS18;110(25):E2298-307, 2013), we set out to develop compounds capable of targeting BCR-ABL, TKI-resistant BCR-ABL mutants, as well as the MNK kinases simultaneously. Here, we report the discovery and characterization of two lead compounds ETC-219 and ETC-027 that inhibit clinically relevant BCR-ABL isoforms (E255K, T315I) as well as the MNK1/2 kinases at nanomolar potency (Table 1).Table 1In vitro kinase assays for ETC-219 and ETC-027.Compound IDIC50 MNK1 (µM)IC50 MNK2 (µM)IC50 ABL (µM)IC50 ABL (T315I) (µM)IC50 ABL (E255K) (µM)ETC-2190.2540.0150.0140.0130.148ETC-0275.0150.0240.2140.0913.19Using a panel of 104 kinases, we showed that ETC-219 and ETC-027 inhibited 90% of the activity of 15 and 12 kinases respectively when tested at 1uM. ETC-219 and ETC-027 interact with only 27 and 20 of off-target kinases respectively, and offer significant selectivity over kinases outside of the MNK and BCR-ABL families.ETC-219 and ETC-027 have favorable pharmacokinetic properties, and include the following ADME characteristics (Table 2).Table 2ADME characteristics of ETC-219 and ETC-027.ETC-219ETC-027ParametersUnitIVPOIVPODosemg/kg1050550C0ng/ml21241714Cmaxng/ml12122490Tmaxh12T1/2h22.21.82.1CLL/h/kg39.72.83.5VzL/kg8.831.37.410.8VssL/kg7.25.8AUC0-t(last)ng.h/ml32905128179514337AUC0-infng.h/ml33025133179814349Extrapolated area for AUC0-inf%0.37000.11000.16000.0800F%3180Regarding targeting of BCR-ABL-dependent cellular proliferation and survival, we found that ETC-219 and ETC-027 exhibited potent anti-proliferative activity against a panel of BC CML cell lines (K562, BV-173, EM-2, KCL-22, JURL-MK1), as well as Ba/F3-BCR-ABL cells containing the T315I mutation. In an in vivo xenograft model, employing K562-eIF4E cell lines in NOD-SCID mice, we found that both compounds inhibited tumor growth in a dose-dependent manner, and a dose-dependent decrease in eIF4E phosphorylation in tumor explants were also observed. Tumor regression was observed at 100 mg/kg for ETC-219. Importantly, mice given ETC-219 and ETC-027 at 100 mg/kg and 200 mg/kg doses did not exhibit significant toxicity.With respect to MNK-eIF4E-b-catenin axis inhibition, ETC-219 and ETC-027 inhibited eIF4E phosphorylation at IC50s of 0.186 nM and 0.28 nM respectively in HeLa cells. In parallel, both compounds inhibited Wnt/b-catenin reporter activity in BC CML cell lines, as well as the expression of a panel of Wnt-regulated genes. Using the serial replating assay as a readout for LSC function and self-renewal capacity, we found that ETC-219 and ETC-027 were effective at 1nM at preventing serial replating using primary CD34+ cells from patients in BC. Furthermore, ETC-219 and ETC-027 inhibited the serial replating efficacy of BC cells from patients with clinical resistance to the third generation TKI, ponatinib. ConclusionOur results demonstrate that: i) small molecule compounds with favorable ADME properties can be developed to potently and specifically inhibit the BCR-ABL and MNK kinases simultaneously; ii) Dual specific MNK and BCR-ABL inhibitors are effective at abrogating BCR-ABL-driven growth and proliferation, as well as the MNK-eIF4E-dependent self-renewal function of BC LSCs. We conclude that dual specific BCR-ABL and MNK inhibitors may warrant testing in patients with BC CML. Disclosures:Chuah:Novartis: Honoraria; BMS: Honoraria.

GADD45a Is a Tumor Suppressor In BCR-ABL-Driven Leukemogenesis

Abstract The BCR-ABL fusion oncogene which encodes a fused deregulated tyrosine kinase causes chronic myelogenous leukemia (CML) in humans. Imatinib, a small molecule ABL kinase inhibitor has been highly effective in treating chronic phase (CP) CML patients. However, a substantial number of patients undergo relapse due to development of resistance to imatinib therapy that leads to blast crisis (BC-CML), which is invariably fatal within weeks to months. Additional genetic aberrations assist in progression and identification of key players that are responsible for transformation is of utmost importance from a therapeutic point of view. Growth arrest DNA damage 45a (Gadd45a) gene, a member in the gadd45 family of genes including Gadd45b &amp; Gadd45g, was identified as a myeloid differentiation primary response gene. There is evidence consistent with it’s involvement in G2/M cell cycle arrest and apoptosis in response to multiple stressors, including genotoxic and oncogenic stress. To investigate the effect of Gadd45a in the development of CML, adaptive bone marrow transplantation experiments with either wild type or Gadd45a null myeloid progenitors expressing 210-kD BCR-ABL fusion oncoprotein revealed that loss of Gadd45a accelerated BCR-ABL driven CML resulting in the development of a more aggressive AML/BC like disease. Recent newly obtained data indicate that number of Gadd45a deficient Leukemic stem cells (LSC) harboring BCR-ABL increased as disease progressed confirming Gadd45a as a crucial tumor suppressor in CML. Recent data also indicate, that transformed Gadd45a deficient progenitors exhibit increased proliferation and decreased apoptosis, associated with enhanced PI3K-AKT-mTOR-4E-BP1 signaling and upregulated oncogenic p30C/EBPα. More importantly, newly obtained data indicate that Gadd45a transcript levels in peripheral blood of human blast crisis (BC-CML) samples was found to be reduced compared to accelerated phase (AP-CML), chronic phase (CP-CML) and normal controls, assessed by Quantitative real time PCR analysis. Collectivly these data strongly suggest that Gadd45a expression is a novel prognostic indicator of CML progression, implicating Gadd45a as a downregulated target of BCR-ABL associated with progression to more aggressive stages. To conclude, our findings provide novel evidence that Gadd45a functions as a suppressor of BCR/ABL driven myeloid leukemogenesis, &amp; that suppresion of Gadd45a is associated with CML progression. These data provide the impetus to further elucidate the role Gadd45a plays in suppressing the development of CML, and explore how its loss contributes to the progression of CML to more aggressive leukemic phenotypes. Disclosures: No relevant conflicts of interest to declare.

Cytokine-STATs Signalings Upregulate C/EBPβ In BCR-ABL+ Leukemic Cells Independently From BCR-ABL/JAK-STAT Pathway

Abstract Since residual chronic myelogenous leukemia (CML) stem cells may be a cause of relapse after Imatinib (IM) cessation, targeting these IM-resistant stem cells is mandatory for complete cure of this disease. CCAAT/enhancer binding protein β (C/EBPβ), a leucine zipper transcription factor, promotes both cell cycle progression and differentiation toward granulocytes in hematopoietic stem/progenitor cells under stress conditions such as infection. We have recently reported that C/EBPβ was upregulated in leukemic stem/progenitor cells derived from patients in chronic phase of CML (CP-CML) through STAT5, a major downstream target of BCR-ABL. In CML mouse model, C/EBPβ enhanced exhaustion of CML stem cells through promoting their differentiation (Hayashi Y et al, Leukemia 2013). In spite of the upregulation of C/EBPβ by BCR-ABL, CML stem cells will not be exhausted spontaneously in patients with CP-CMP. Therefore, we hypothesized that quiescent CML stem cells maintain their immature status in the bone marrow niche by suppressing C/EBPβ expression or function induced by BCR-ABL and that induction of C/EBPβ expression in CML stem cells through BCR-ABL-independent pathways may be a novel therapeutic strategy targeting CML stem cells. The aim of this study is to propose a novel therapeutic strategy that can stimulate quiescent CML stem cells with cytokine-STATs signalings and induce their exhaustion through C/EBPβ-mediated differentiation. STATs are family members of molecules which convey signals from various kinds of cytokine receptors to nucleus. In order to investigate whether STAT molecules can induce C/EBPβ expression, we first examined the effects of constitutive active (CA) form of STAT1, STAT3 and STAT5 on C/EBPβ expression in a murine hematopoietic stem cell line, EML cells. Retroviral transduction of CA-STAT5 significantly upregulated C/EBPβ mRNA and protein in EML cells. EML cells begun to differentiate toward CD11b+ myeloid lineage upon introduction of CA-STAT5. CA-STAT1 and CA-STAT3 also upregulated C/EBPβ mRNA when they were retrovirally transduced into EML cells (Figure 1). These results suggest that signaling mediated by various kinds of STATs can upregulate C/EBPβ. Consensus binding sites for STATs were not found in the proximal (∼ 4 kb) promoter region of C/EBPβ and we are currently identifying the cis-regulatory elements responsible for the STATs-dependent activation of C/EBPβ.Figure 1Figure 1. Interferon-α (IFNα) exerts STATs-mediated signaling in hematopoietic stem cells and has been used for therapy of CML. Therefore we investigated the possible involvement of C/EBPβ in efficacy of IFNα in CML treatments. Stimulation of EML cells with 500 U/ml IFNα upregulated C/EBPβ mRNA (Figure 2). Higher levels of phosphorylation of STAT1 than those of STAT3 or STAT5 were observed after stimulation with IFNα, suggesting that STAT1 mediated activation of C/EBPβ was induced by IFNα. As previously reported, C/EBPβ was upregulated in EML cells transduced with BCR-ABL (EMLBCR-ABL). Treatment of EMLBCR-ABL cells with IFNα augmented this effect significantly. IM effectively inhibited phosphorylation of STAT5 and blunted the upregulation of C/EBPβ in EMLBCR-ABL cells. Simultaneous treatment of EMLBCR-ABL cells with IFNα and IM resulted in maintained upregulation of C/EBPβ with increased phosphorylation of STAT1 and decreased phosphorylation of STAT5. These data suggested that IFNα treatment can upregulate C/EBPβ independently of signals mediated by BCR-ABL.Figure 2Figure 2. In conclusion, cytokine-STATs signalings can induce C/EBPβ expression in BCR-ABL+ leukemic cells independently from BCR-ABL/JAK-STAT pathway. Stimulations of dormant CML stem cells with cytokines might be a novel treatment strategy to eliminate these populations, leading to complete cure of CML. We are currently evaluating the in vivo effects of IFNα treatment on CML stem cells in mice models. Disclosures: No relevant conflicts of interest to declare.