CML Patient Samples Research Articles

Wnt and Notch Pathways Regulate Stemness of Myeloid Leukemia Cells Via IL33/ST2 Signaling

Therapy-resistant leukemic stem cells (LSCs) are a main cause of relapse in chronic and acute myeloid leukemia (CML and AML) patients. Stemness, a cellular state of LSCs characterized by their quiescence, pluripotency, and long-term self-renewal capacity, is thought to have major implications on therapy resistance and outcome in myeloid leukemias. However, signaling pathways that induce stemness are often not specific for LSCs and shared with normal hematopoietic stem cells (HSCs). Therefore, LSCs are difficult to be targeted therapeutically. IL-33/ST2 signaling may be a potential target to eradicate CML and AML LSCs. To investigate the ST2 gene (IL1RL1) expression in different hematological malignancies, we used the MILE gene expression dataset containing 2096 samples. The IL1RL1 high expressing samples (n=62) were exclusively represented by myeloid leukemias (CML and AML), and 50% of the IL1RL1 high samples harbored either the BCR/ABL1 (CML) or the AML1/ETO (AML) oncogenes. We confirmed the result from the MILE database by analyzing the ST2 protein expression on FACS-sorted CD34+ LSCs from AML and CML patient samples. Indeed, AML1/ETO and BCR/ABL1 harboring LSCs expressed ST2, while healthy hematopoietic stem cells (HSCs) and LSCs harboring other oncogenes were ST2-negative. Transfecting the ST2-negative cell line Ramos with the AML1/ETO and BCR/ABL1 oncogenes induced ST2-expression. Next, we performed a gene set enrichment analysis (GSEA), comparing the IL1RL1 high vs. IL1RL1 low samples in the MILE dataset. Genes involved in stemness (p<0.01), Wnt- (p<0.001), and NF-ΚB (p=0.054) signaling were upregulated, while the Notch-pathway (p<0.05) was downregulated. Since the NF-ΚB is a known downstream pathway of ST2, we hypothesized that Wnt and/or Notch signaling pathways regulate ST2 expression. Therefore, we treated FACS-sorted CD34+ AML1/ETO expressing LSCs with the Wnt pathway inhibitor XAV939 or HES1 and NOTCH1 shRNAs, silencing the Notch pathway. Treatment with XAV939 led to a decrease in ST2 expression in these cells, while silencing HES1 and NOTCH1 enhanced ST2-expression. To analyze the molecular mechanisms induced by IL-33/ST2 signaling, we performed a transcriptomic analysis using FACS-sorted CD34+ AML1/ETO LSCs upon treatment with or without IL-33. In total, 377 genes were up- and 139 genes downregulated after IL-33 stimulation. IL-33-induced genes were involved in the positive regulation of NF-ΚB (p<0.05) and canonical Wnt (p<0.001) signaling pathways, and the regulation of stem cell division and maintenance (p<0.001). To functionally test whether IL-33/ST2 signaling triggers stemness in myeloid leukemia, we performed a colony forming assay using CD34+ AML1/ETO or BCR/ABL1 expressing LSCs from leukemia patient samples. The colony numbers increased 1.5-3fold upon IL-33 treatment in three consecutive platings, indicating an increase in stem cell numbers. IL-33 had no effect on colony numbers of HSCs and LSCs harboring other oncogenes. To test the in vivo relevance of IL-33/ST2 signaling in leukemia development, we used a congenic AML1/ETO9a (AE9a) or BCR/ABL1 (CML) mouse model. Briefly, we transduced ST2-negative HSCs originating from BL/6 or ST2-knockout (ST2-/-) mice with AE9a-GFP or BCR/ABL1-GFP and transplanted those cells into recipients. In both models, the LSCs strongly expressed ST2, but IL-33/ST2 signaling had no influence on the disease development. Stemness supports resistance against standard therapy in both, AML and CML. Since we were able to show that IL-33/ST2 signaling promotes stemness in AML1/ETO and BCR/ABL1 LSCs, we hypothesized that IL-33 addition protects LSCs from treatment with standard therapeutics. Indeed, IL-33 rescued the reduced colony forming capacity of LSCs that were either treated with the chemotherapeutic agent cytarabine (AML1/ETO LSCs) or the tyrosine kinase inhibitor Nilotinib (BCR/ABL1 LSCs). Furthermore, ST2-deficiency led to a reduced LSC count in CML mice compared to control mice when treated with Nilotinib (p=0.047). We showed that ST2 is expressed on AML1/ETO and BCR/ABL1 harboring LSCs and that activated IL-33/ST2 signaling induced stemness in these LSCs. Targeting IL-33/ST2 signaling in these specific subtypes of AML and CML may eliminate LSCs and overcome resistance towards chemotherapy or TKIs. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

LncRNA Hmrhl regulates expression of cancer related genes in chronic myelogenous leukemia through chromatin association.

Long non-coding RNA has emerged as a key regulator of myriad gene functions. One such lncRNA mrhl, reported by our group, was found to have important role in spermatogenesis and embryonic development in mouse. Recently, its human homolog, Hmrhl was shown to have differential expression in several type of cancers. In the present study, we further characterize molecular features of Hmrhl and gain insight into its functional role in leukemia by gene silencing and transcriptome-based studies. Results indicate its high expression in CML patient samples as well as in K562 cell line. Silencing experiments suggest role of Hmrhl in cell proliferation, migration & invasion. RNA-seq and ChiRP-seq data analysis further revealed its association with important biological processes, including perturbed expression of crucial TFs and cancer-related genes. Among them ZIC1, PDGRFβ and TP53 were identified as regulatory targets, with high possibility of triplex formation by Hmrhl at their promoter site. Further, overexpression of PDGRFβ in Hmrhl silenced cells resulted in rescue effect of cancer associated cellular phenotypes. In addition, we also found TAL-1 to be a potential regulator of Hmrhl expression in K562 cells. Thus, we hypothesize that Hmrhl lncRNA may play a significant role in the pathobiology of CML.

Allosteric Bcr-Abl Inhibitor ABL001 Overcomes Bcr-Abl Tyrosine Kinase Inhibitor Resistance and Enhances MDM2 Inhibitor CGM097 Activity in Blast Crisis CML Cells

Despite Bcr-Abl tyrosine kinase inhibitors (TKIs) are highly effective in CML, development of TKI-resistance leading to blast crisis (BC) progression is still a challenge in CML therapy. This has motivated the generation of novel agents and combination strategies to overcome the resistance. ABL001 is a small molecule allosteric Bcr-Abl kinase inhibitor that occupies the myristoyl pocket, leading to a kinase autoinhibition conformation. ABL001 has entered phase I clinical trials to treat patients with chronic, accelerated, or blast phase CML that relapsed from TKIs (Hughes et al., ASH 2016). Its activity in BC CML and ability to enhance the traditional TKIs that target ATP binding site are under investigation. TP53, a key tumor suppressor gene plays a crucial role in mediating cell death in part through regulating the expression or activity of Bcl-2 family proteins. Although highly mutated in solid tumors, the mutation rate of TP53 is relatively low in CML. However, p53 antagonist, MDM2 can be regulated by Bcr-Abl signaling and ARF, which inhibits MDM2 is frequently deleted in BC CML suggesting therapeutic potentials of inhibition of MDM2 in BC CML. Indeed, activation of p53 by MDM2 inhibition has been shown to enhance the toxicity of TKIs in BC CML (Carter et al., Oncotarget 2015, 6(31):30487). In this study, we examined the activity of ABL001 in CML cells and investigated if ABL001 enhances the activity of TKIs and synergizes with MDM2 inhibitor CGM097 in samples from heavily treated TKI resistant BC CML patients.BaF3 cells expressing BCR-ABL with or without mutations, human CML cell lines (KBM5 and KBM5-STI-T315I), and peripheral blood mononuclear cells (PBMC) isolated from primary BC CML patient samples were treated with various doses of ABL001, CGM097 or Nilotinib alone or in combination. Apoptosis was determined by Annexin V/7-AAD staining in cell lines and CD45+ (bulk), CD34+CD38+ and CD34+CD38- cells of CML patient PBMC samples. EC50 was calculated using CompuSyn or Graphpad. Protein levels were examined by Western blotting.We found that ABL001 induced apoptosis in cell lines expressing BCR-ABL (BaF3p210 and KBM5) or mutant BCR-ABL (BaF3p210E255K and KBM5-STI-T315I) but had limited activity in BaF3p210T315I cells. Importantly, ABL001 induced apoptosis in bulk, CD34+CD38+, or/and CD34+CD38- cell populations at low nM concentrations in 5 out of 6 BC CML patient samples tested. Patient samples that responded to ABL001 had either T315I and/or E255 mutations in BCR-ABL kinase domain. ABL001 inhibited Bcr-Abl signaling as measured by the levels of p-Bcr-Abl/p-Stat5/p-Crkl in BCR-ABL positive cells with or without BCR-ABL mutations. ABL001 showed higher activity than Nilotinib in 4 out of 5 responding patient samples. Nilotinib did not significantly enhance ABL001-induced apoptosis in the tested BC CML patient samples and CML cell lines. Inhibition of MDM2 with CGM097 activated p53 and induced apoptosis in BC CML patient samples. The activity of ABL001 was enhanced by CGM097 in the majority of patient samples which did not have p53 mutations. ABL001 combined with CGM097 was more effective than the combination of nilotinib with CGM097. The combinations of TKI and CGM097 were more effective in killing leukemia cells than each agent alone even when leukemia cells were co-cultured with MSCs. Neither ABL001 or CGM097 alone, nor the combination showed toxicity on normal bone marrow stem/progenitor cells.In conclusion, our results demonstrate that the allosteric Bcr-Abl Inhibitor ABL001 overcomes TKI-resistance and enhances MDM2 inhibitor CGM097 activity in BC CML cells. The combination of ABL001 and CGM097 warrants clinical evaluation. DisclosuresCarter:novartis: Research Funding; Daiichi Sankyo: Research Funding.

Optimal Whole Blood Storage Conditions for Measuring BCR-ABL1 Accurately for Xpert® BCR-ABL Ultra

Abstract Reliably quantifying BCR-ABL1 and ABL1 transcripts in whole blood for patients on TKI therapy is essential, but it can be challenging because intracellular RNA may rapidly degrade during storage. The purpose of the studies is to evaluate the effect of blood storage conditions with increased time interval on BCR-ABL1 and ABL1 gene expression, and demonstrate the validity of results in %BCR-ABL/ABL (IS) generated from Xpert® BCR-ABL Ultra. The whole blood stability was evaluated in contrived and CML patient samples. Contrived samples were prepared by spiking CML+ cells into normal EDTA or PAXgene blood at ~0.1% (IS). In EDTA, it was stable for 48 hours at room temperature (RT) and up to 3 days at 2-8°C. In PAXgene, it was stable for 5 days at RT or 2-8°C, and up to 25 months at -80°C. The CML EDTA specimens evaluated at ~10%, ~1%, ~0.1% and 0.01% (IS) were stable at RT for 48 hours and up to 4 days at 2-8°C. To evaluate long term storage for whole blood, contrived samples at ~0.1% (IS) and CML EDTA specimens at various % IS were spotted onto a Dried Blood Spot (DBS) paper template. The DBS stability at RT was demonstrated for more than 40 days from the CML clinical specimens and up to 12 months on the contrived samples. In addition, the long term stability was evaluated on frozen lysates prepared from contrived and CML EDTA specimens using Xpert® BCR-ABL Ultra lysis buffer containing Guanidine Hydrochloride. The frozen contrived blood lysates were stable for 48 hours at RT and 2-8°C and up to 3.5 months at -20°C with ≤2-fold difference from the original testing time-point. The frozen CML blood lysates were stable up to 4 years at -80°C with ≤2-fold difference at ≥ 0.01% (IS) and ≤2.5-fold below 0.01% (IS). In summary, the presented studies demonstrated that the fresh blood can be stored at RT for 48 hours and at refrigerated temperature for more 3 days without compromising the Xpert® BCR-ABL Ultra assay sensitive, precision and accuracy. The long term storage can be achieved on DBS at RT for up to 1 year and in PAXgene and frozen lysate at -80oC for more than 2 years and 4 years, respectively. DBS, the spotted blood method, can potentially benefit the CML patients in resource-poor areas, who can be monitored more frequently during TKI therapy by reducing the sample transport cost significantly. Disclosures Day: Cepheid: Employment. Levitas: Cepheid: Employment. Shridhar: Cepheid: Employment. Huang: Cepheid: Employment. Rhees: Cepheid: Employment. Bates: Cepheid: Employment. Wong: Cepheid: Employment. Radich: Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding; BMS: Membership on an entity's Board of Directors or advisory committees; ARIAD: Membership on an entity's Board of Directors or advisory committees; Pfizer: Membership on an entity's Board of Directors or advisory committees; Giliad: Membership on an entity's Board of Directors or advisory committees; AMGEN: Membership on an entity's Board of Directors or advisory committees.

ID3008 Differential expression of circadian genes in leukemia and a possible role for Sirt1 in restoring the circadian clock in chronic myeloid leukemia

&#x0D; Dis-regulation of genes making up the mammalian circadian clock has been associated with different forms of cancer. This study initially aimed to address how the circadian clock genes behave over the course of treatment for both the acute and chronic forms of leukemia and whether any could be used as potential biomarkers as a read-out for therapeutic efficacy. Expression profiling for both core and peripheral clock genes revealed that the majority of clock genes are down-regulated in AML patients apart from Cry2 which is up-regulated towards the end of treatment. A similar observation was seen in ALL patients however here, Cry2 expression came back up towards control levels upon treatment completion. In addition, all of the core clock genes are down-regulated in both chronic forms of leukemia (CML and CLL) apart from Cry2 which is not affected when the disease is diagnosed. Furthermore, the NAD(+) – dependent protein deacetylase Sirt1 has been proposed to have a dual role in both control of circadian clock circuitry as well as promotion of cell survival by inhibiting apoptotic pathways in cancer. We used a pharmacological-based approach to see whether Sirt1 played a role in regulating the circadian clock circuitry in both Acute and Chronic forms of leukemia. Our results suggest that interfering with Sirt1 leads to a partial restoration of BMAL1 oscillation in CML patient samples. Furthermore, interfering with Sirt1 activity leads to both the induction and repression of circadian clock genes in both Acute and chronic forms of leukemia and therefore makes it a potential therapeutic target to either augment existing therapies for chronic leukemia or to act as a means of facilitating chronotherapy in order to maximize both the effectiveness of existing therapies and to minimize therapy-associated toxicity.

Abstract 3513: Decoding the role of myocyte enhancer factor 2C in chronic myeloid leukemia disease progression

Abstract Chronic myeloid leukemia (CML) is a myeloproliferative disorder of hematopoietic stem cell and is marked by the presence of the Philadelphia chromosome, a reciprocal translocation occurring between the long arm of chromosome 9 and 22 resulting in the formation of a fusion BCR-ABL1+ oncogene which encodes a constitutively active non-receptor tyrosine kinase. Clinically, the disease progresses through three phases: an initial chronic phase, accelerated phase and terminal blast phase, which is marked by increased production of immature myeloid precursor cells. Earlier we have shown that BCR-ABL mediated repression of miR-223 results in the activation of mef2C and ptbp2 in chronic myeloid leukemia. Transcription factor MEF2C, required for cell survival and proliferation, is important for lymphopoiesis, and its ectopic expression in myeloid progenitors antagonizes myelopoiesis. We reported that MEF2C expression increases as the disease progresses from chronic phase to blast phase and by knocking down MEF2C in a CML cell line we showed that it negatively correlates with the CEBPA expression, a granulocytic transcription factor. Imatinib, the drug of choice for CML, abrogates MEF2C expression and reverses CEBPA repression both in the cell line and the primary cells. In the present study we observed that when the cells were treated with anisomycin it degraded MEF2C expression in 16hrs which imatinib can achieve after 24hrs. Investigators have demonstrated that anisomycin acts as a potent signaling agonist, synergizing with growth factors and phorbol esters to superinduce immediate-early genes such as c-fos, fosB, c-jun, JunB, and JunD. However here blocking the MAPK kinases with inhibitors didn't reverse the inhibition suggesting that these pathways may not be involved in the anisomycin mediated MEF2C degradation in CML. Mef2c gene consists of three alternative exons, α1/α2, skipping exon β and 3'splice site γ. In order to characterize the role of mef2c gene in CML, first we investigated the different isoforms of mef2c in CML cell line (KCL22) by amplification of the whole mef2c gene and cloning the same in a TOPO vector. Several clones were checked and sequenced. Two different types of isoforms (α1+ β- γ+ and α1+ β- γ-) were found to coexist in KCL22 cells along with an exclusion of 9 bases (AACACAGGT) from the splice junction of exon 4 and 5, which was not reported earlier. However, K562 cells along with KYO-1 and LAMA-84 showed the presence of 9 bases. Nine base pair deletion was also observed in CML patient samples (both CP and BC). Interestingly sequencing of the mef2C genomic region from the cell lines and the same patient samples showed the presence of 9 bases. The different isoforms were transduced in mouse lineage negative bone marrow cells and also transplanted in mice either alone or in combination with BCR-ABL to study the effect of the isoforms in disease progression, the results of which will be discussed. Citation Format: Soumen Chakraborty. Decoding the role of myocyte enhancer factor 2C in chronic myeloid leukemia disease progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3513. doi:10.1158/1538-7445.AM2017-3513

Increasing the Sensitivity of BCR-ABL Monitoring By Digital PCR By Reducing the Background Noise

BackgroundDroplet digital PCR (dPCR) is a potentially appealing technology for quantitative detection of specific mutations with simultaneous measurement of the reference gene and without the need for standard curves. This makes dPCR particularly suitable for minimal residual disease (MRD) diagnostics in chronic myeloid leukaemia. However, a limitation of the dPCR assays compared to standard quantitative PCR (qPCR) is the background (termed lower limit of blank, LoB) of 1 or 2 positive droplets for BCR-ABL (Franke et al, ASH 2015, Cross et al, Leukemia 2016). The resulting false positive rate (FPR) limits the sensitivity and the ability of the test to detect deep molecular remissions.AimsThe aim was to establish BCR-ABL (b2/a2 and/or b3/a2 transcript) dPCR assays with a sensitivity at least equivalent to that of qPCR, in which <5% of negative controls generate a single positive drop (FPR<5%, LoB=0).MethodsOne assay (Assay 1) was developed in Leipzig and another (Assay 2) by Bio-Rad laboratories. Sample preparation was identical for both assays up to the stage of reverse transcription (RT). Assay 1 used routine local RT procedure followed by a duplex BCR-ABL / ABL dPCR reaction modified from the M-BCR-ABL PCR published by Jennings et al. 2014. We tested ABL primer combinations from exons 4, 8, 10, and 11 generating a range of product lengths and optimised quenchers and PCR conditions to maximise the separation between positive and negative droplets. Assay 2 consisted of a reverse transcriptase reaction followed by a duplex PCR specific for ABL and both the b2/a2 and b3/a2 transcripts of BCR-ABL. Digital droplet PCR was performed in both cases using the Bio-Rad QX200 system.Background noise and cross hybridisation were assessed in non-template controls (NTC), BCR-ABL negative cell lines and healthy wild-type donor samples. In addition, assay 2 was tested on 40 CML patient samples with MRD levels ranging from BCR-ABL negative to 1% BCR-ABL/ABL as defined by standard qPCR.ResultsAssay 1 yielded a FPR of 4% (4/94) in NTC with 1 positive droplet for BCR-ABL and a FPR of 3% for ABL (n = 3/94), resulting in a specificity of >95% and a LOB of 0. The BCR-ABL FPR using wild type target (HEK 293T cell line and healthy donors) was 5% (5/92 and 2/37, respectively).Assay2: The specificity was >95% for both BCR-ABL and ABL in NTC and wild type samples. Extensive NTC analysis yielded no false positives for BCR-ABL PCR (n = 0/176; LoB = 0) and 1% false positives in ABL PCR (n = 2/176; 1-2 positive droplets, LoB = 0). The BCR-ABL negative cell line was confirmed negative for BCR-ABL PCR (n = 44), while the BCR-ABL assays of healthy donors were positive in 2% (n = 5/234) with 1 positive droplet per positive sample. The BCR-ABL copy numbers of the 40 CML samples were lower by dPCR than by RT-PCR (median 3/6) with similar ABL transcripts (median 55500/54949). This resulted in lower ratios by dPCR (mean 0.0313 vs. 0.0461 by qPCR). However, after conversion of the qPCR results with our lab specific conversion factor (CF) <1, the dPCR ratios were higher (mean 0.0313 vs. 0.0134). The MR classes were concordant in 60% of the samples, but 30% had more MRD while 10% had less MRD by dPCR than by qPCR, confirming our previous results using the EAC protocol (Franke, ASH 2015). More samples were MRD positive by dPCR than by qPCR (58% vs 50%).ConclusionsAn assay with a FPR of <5% and a LoB = 0 for the detection of BCR-ABL was developed. The BIO-RAD QX 200-Dx BCR-ABL Kit is fully functional and has a high specificity for BCR-ABL and ABL with a LoB of 0. The high specificity and low FPR/LoB of both assays enables sensitive and accurate monitoring of deep molecular remissions in CML. DisclosuresFranke:Novartis: Honoraria, Research Funding. Tzonev:Bio-Rad: Employment, Equity Ownership. Shelton:Bio-Rad: Employment. Niederwieser:Amgen: Speakers Bureau; Novartis Oncology Europe: Research Funding, Speakers Bureau. Lange:Novartis: Research Funding.

Integrative Analysis of RNA-Interactome and Translatome Reveal Functional Targets of MSI2 in Myeloid Leukemia

Musashi 2 (MSI2) is an RNA binding protein (RBP) that regulates asymmetric cell division and cell fate of several cell types including hematopoietic stem and progenitor cells. MSI2 was recently shown to be transcriptionally up-regulated in aggressive myeloid leukemia including chronic myelogenous leukemia in blast crisis (CML-BC) and acute myeloid leukemia (AML). Loss-of-function studies have confirmed an important role for MSI2 in the aggressive biology of these leukemia, but the precise molecular mechanisms remain undefined. MSI2 is thought to bind to the 3' untranslated region (3'UTR) of target mRNA in a sequence-specific fashion and inhibit their translation, but functional targets of MSI2 are not known. We implemented two transcriptome-wide techniques to define these targets: iCLIP (individual nucleotide-resolution cross-link and immunoprecipitation) and polysome profiling. We first defined the RNA interactome of MSI2 in a CML-BC cell line (K562) by iCLIP of FLAG-tagged MSI2. iCLIP relies on the ability of ultraviolet (UV) radiation to cross-link RBPs to interacting RNA allowing for stringent immunoprecipitation of RBP-RNA complexes and subsequent analysis by next generation sequencing. iCLIP analysis revealed direct binding of MSI2 to transcripts of 5036 genes, specifically enriched in the 3'UTR. These iCLIP peaks were also enriched in UAG, the minimal sequence motif of MSI2 previously defined through biochemical assays. Given that MSI2 is ubiquitously expressed, but has very specific cellular functions, we hypothesized that only a small fraction of the direct targets demonstrable by iCLIP are functionally relevant. To determine those target mRNA, we performed translatome profiling of K562 cells (MSI2 knock-down and control cells) with polysome profiling. Polyribosomes or polysomes are aggregates of ribosomes assembled on efficiently translated transcripts. Comparing change in abundance of polysome-associated transcripts (polysome profiling) allows determination of quantitative changes to translation. Using polysome profiling, we found that transcripts from only 413 genes underwent translational up-regulation (without a corresponding change in total transcript abundance) in response to MSI2 knock-down, of which 319 were deemed to be direct MSI2 targets from iCLIP analysis. Translational targets thus defined were highly enriched in biological pathways such as differentiation, cell-cycling, DNA damage response and apoptosis. Specific transcripts thus identified included transcription factors critical to myeloid and leukemia biology (cJUN, MYC and SP1) and cell-cycle regulators (CDK1, CDKN1B, RAD21 and RB1). Using expression profiles of 91 CML patient samples (reported in Radich JP et al PNAS 2006 Feb 21;103(8)) we tested if changes in gene-expression during disease progression (from chronic phase to blast crisis) reflect the change in translation of transcription factors as predicted by the translatome analysis. Accordingly, we found that transcripts down-regulated in CML-BC were highly enriched in targets of JUN and SP1, confirming a primary role for MSI2 targets in progression of CML. In summary, our novel approach that integrates two transcriptome-wide techniques show that while thousands of transcripts are directly bound by MSI2, only a small proportion of these undergo translational repression. Functional targets thus identified include transcription factors and cell-cycle regulators critical to progression of myeloid leukemia. Our results will form the basis of further studies to explore therapeutic targeting of MSI2 in myeloid leukemia. DisclosuresNo relevant conflicts of interest to declare.

Celecoxib suppresses autophagy and enhances cytotoxicity of imatinib in imatinib-resistant chronic myeloid leukemia cells.

BackgroundChronic myelogenous leukemia (CML) is a hematological stem cell disorder. Tyrosine kinase inhibitors (TKIs) are the standard treatments for CML, but a number of patients fail to respond effectively due to gene mutations. Celecoxib, a cyclooxygenase-2 (COX-2) inhibitor, has been shown to have anti-tumor effect on solid tumor whereas the anti-CML effect and its underlying mechanism have not been completely elucidated.MethodsThe cytotoxic effects of celecoxib and/or imatinib were evaluated by MTT assay. Cell cycle distribution was examined by propidium iodide (PI) assay. Apoptosis or necrosis was analyzed by Annexin-V/PI, Hoechst 33342 staining and Western blot assays. Autophagy suppression effect of celecoxib was examined by Western blot and LysoTracker probe labelling. Lysosensor probe labelling was used to detect the effect of celecoxib on the lysosomal function.ResultsIn this study, we found that celecoxib had therapy efficacy in KBM5 and imatinib-resistant KBM5-T315I CML cell lines. Celecoxib caused significant cytotoxic effect in both cell lines, especially in KBM5-T315I cells exposed to celecoxib for 72 h. Moreover, celecoxib induced necrosis and apoptosis while inhibited autophagy in CML cell lines and patient samples. Furthermore, this study demonstrated that celecoxib prevented the autophagic flux by inhibiting lysosome function. Celecoxib was tested in combination with imatinib, demonstrating that celecoxib could strengthen the cytotoxicity of imatinib in imatinib-resistant CML cells.ConclusionsThese findings showed that celecoxib had therapy efficacy on CML cells. And it is first time to demonstrate that celecoxib is an autophagy suppresser and a combination of celecoxib and imatinib might be a promising new therapeutic strategy for imatinib-resistant CML cells.Electronic supplementary materialThe online version of this article (doi:10.1186/s12967-016-1012-8) contains supplementary material, which is available to authorized users.

Abstract 676: Axitinib targets gatekeeper-mutant BCR-ABL1(T315I)-driven leukemia in a distinct and selective fashion

Abstract The T315I gatekeeper mutation confers resistance to majority of approved ABL1 inhibitors, with only ponatinib demonstrating efficacy in BCR-ABL1(T315I)-driven disease. However, vascular adverse events and selection of resistant compound mutations limit its clinical utility. Hence, there is an unmet need for novel therapies for patients with gatekeeper-mutated Ph+ leukemia. In this study we integrated comprehensive drug sensitivity testing with structural analysis to characterize the tyrosine kinase inhibitor axitinib as a putative novel therapy for BCR-ABL1(T315I)-driven leukemias. To address this we profiled BCR-ABL1(T315I)-driven CML/Ph+ ALL patient samples against 300 anti-cancer compounds (approved and investigational drugs). Ex vivo drug sensitivity testing of primary cells derived from a Ph+ ALL patient revealed a marked and cancer-selective response to the VEGFR inhibitor axitinib. Strikingly, axitinib exhibited higher sensitivity in T315I positive Ph+ patient samples in comparison to T315I negative CML and ALL patient samples. In line with the ex vivo drug response data, axitinib inhibited the kinase activity of ABL1(T315I) with similar potency as its primary target VEGFR2, while the potency to non-mutated ABL1 was 30-fold lower. Analogously, in engineered Ba/F3 cells, axitinib showed a 10-fold higher inhibition of T315I than non-mutated ABL1-driven autophosphorylation and cell growth. To better understand the molecular mechanisms of the BCR-ABL1(T315I) selectivity, we solved the crystal structure of axitinib in complex with ABL1(T315). The structure revealed that axitinib bound to a mutation induced active conformation of ABL1(T315I), different than the binding mode in non-mutated ABL1, likely explaining the increased potency towards ABL1(T315I). Moreover, axitinib occupied a distinct binding space than all approved ABL1 inhibitors, signifying that axitinib will have a unique mutation vulnerability profile. Compassionate two week treatment of a CML patient harboring the T315I mutation with axitinib, resulted in a 5-fold reduction of T315I transcript levels in the bone marrow, further suggesting that axitinib can produce specific and effective responses in patients with BCR-ABL1(T315I)-driven leukemia. In conclusion, we demonstrate that axitinib potently inhibits BCR-ABL1(T315I) via a gatekeeper mutant-selective mechanism. Since axitinib is in clinical use for treatment of refractory renal cell carcinoma with a manageable safety profile, our data provide a sound basis for readily repurposing axitinib for BCR-ABL1(T315I)-driven leukemia. Finally, the distinct mechanism of inhibition by axitinib serves as an exemplar for development of even more effective gatekeeper-mutant selective inhibitors targeting ABL1 as well as other clinically important kinases, such as EGFR and KIT. Citation Format: Tea Pemovska, Eric Johnson, Mika Kontro, Gretchen A. Repasky, Jeffrey Chen, Peter Wells, Ciarán N. Cronin, Michele McTigue, Olli Kallioniemi, Kimmo Porkka, Brion W. Murray, Krister Wennerberg. Axitinib targets gatekeeper-mutant BCR-ABL1(T315I)-driven leukemia in a distinct and selective fashion. [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 676. doi:10.1158/1538-7445.AM2015-676

Clonal distribution of BCR-ABL1 mutations and splice isoforms by single-molecule long-read RNA sequencing.

BackgroundThe evolution of mutations in the BCR-ABL1 fusion gene transcript renders CML patients resistant to tyrosine kinase inhibitor (TKI) based therapy. Thus screening for BCR-ABL1 mutations is recommended particularly in patients experiencing poor response to treatment. Herein we describe a novel approach for the detection and surveillance of BCR-ABL1 mutations in CML patients.MethodsTo detect mutations in the BCR-ABL1 transcript we developed an assay based on the Pacific Biosciences (PacBio) sequencing technology, which allows for single-molecule long-read sequencing of BCR-ABL1 fusion transcript molecules. Samples from six patients with poor response to therapy were analyzed both at diagnosis and follow-up. cDNA was generated from total RNA and a 1,6 kb fragment encompassing the BCR-ABL1 transcript was amplified using long range PCR. To estimate the sensitivity of the assay, a serial dilution experiment was performed.ResultsOver 10,000 full-length BCR-ABL1 sequences were obtained for all samples studied. Through the serial dilution analysis, mutations in CML patient samples could be detected down to a level of at least 1%. Notably, the assay was determined to be sufficiently sensitive even in patients harboring a low abundance of BCR-ABL1 levels. The PacBio sequencing successfully identified all mutations seen by standard methods. Importantly, we identified several mutations that escaped detection by the clinical routine analysis. Resistance mutations were found in all but one of the patients. Due to the long reads afforded by PacBio sequencing, compound mutations present in the same molecule were readily distinguished from independent alterations arising in different molecules. Moreover, several transcript isoforms of the BCR-ABL1 transcript were identified in two of the CML patients. Finally, our assay allowed for a quick turn around time allowing samples to be reported upon within 2 days.ConclusionsIn summary the PacBio sequencing assay can be applied to detect BCR-ABL1 resistance mutations in both diagnostic and follow-up CML patient samples using a simple protocol applicable to routine diagnosis. The method besides its sensitivity, gives a complete view of the clonal distribution of mutations, which is of importance when making therapy decisions.

Connective Tissue Growth Factor Induces Anti-Apoptotic Factors in Chronic Myeloid Leukemia Stem Cells

Abstract Chronic Myeloid Leukemia (CML) is a progressive hematopoietic malignancy where expression of the oncogenic fusion protein BCR-ABL1 in leukemia stem cells (LSCs) prevents the proper differentiation of myeloid progenitor populations, leading to accumulation of undifferentiated blasts. Current treatments target BCR-ABL1 with tyrosine kinase inhibitors (TKIs). Though effective if administered continuously, TKIs generally fail to eradicate the bone marrow niche-residing LSCs responsible for patient relapse or progression of CML to its terminal stage, Blast Crisis (BC), as evidenced by the high molecular relapse rate following TKI discontinuation. Previous studies performed by ourselves and others show that BC progenitors (CD34+CD38+Lin-) exhibit stem-like behaviors, such as quiescence, self-renewal, and induction of pro-survival gene expression through alternative splicing of BCL2 family members, and thus behave like LSCs. Notably, BC CML LSCs co-cultured on LSC (SL/M2) supportive stroma are resistant to TKIs compared to culturing the cells alone, indicating a role of the extracellular matrix (ECM) in promoting LSC survival. We performed RNA-seq and qRT-PCR of CD34+CD38+Lin- progenitor cells in CML patient samples and found a significant increase in CTGF (Connective Tissue Growth Factor) expression in BC CML versus chronic phase (CP). Interestingly, CTGF is an ECM protein that enhances cell adhesion and has been shown to predict therapeutic resistance in cancers, such as acute lymphoblastic leukemia (ALL). Lentiviral overexpression of CTGF in a CML cell line K562 and CD34+ CP CML patient samples caused proliferation and a decrease in apoptosis markers (cleaved caspase-3), as measured by FACS analysis. Moreover, qRT-PCR analysis of mRNA indicated an increase in pro-survival BCL2 family gene expression. These changes were not observed in normal CD34+ cord blood cells. Currently, lentiviral CTGF transduction of CP CML followed by transplantation into RAG2-/-gc-/- and NSG-S mice will be used to determine the effects of CTGF on LSC maintenance in vivo. In conclusion, CTGF promotes CML LSC survival in vitro and thus could be a key factor in BC transformation and TKI resistance. Disclosures Jamieson: J&amp;J, Roche: Research Funding.

Regulation of Self-Renewal through Modulation of Let-7 Stem Cell Regulatory Family of microRNAs in Chronic Myeloid Leukemia Stem Cells

IntroductionChronic myeloid leukemia (CML) was one of the first malignancies shown to be initiated in hematopoietic stem cells by the BCR-ABL1 oncogene and sustained in blast crisis (BC) by progenitor cells that co-opt stem cell properties and behave like leukemia stem cells (BC LSCs). The BCR-ABL fusion oncogene encodes a constitutively active tyrosine kinase BCR-ABL. Although tyrosine kinase inhibitor (TKI) therapy targeting BCR-ABL suppresses CML during the chronic phase (CP), progenitors undergo expansion as a consequence of subsequent genetic and epigenetic alterations that fuel blast crisis transformation, BC LSC generation and TKI resistance. Self-renewing human BC LSCs harbor increased expression of Inflammation responsive adenosine deaminase acting on RNA (ADAR1), which can alter transcript as well as microRNA (miRNA) maturation, splicing and translation by Adenosine (A)-to-Inosine (I) editing of double stranded RNA. miRNAs are a family of small non-coding RNA molecules that regulate gene expression at a post-transcriptional level by inhibiting protein translation and/or reducing mRNA stability. Eukaryotic cells employ miRNAs in diverse biological processes including cell proliferation, differentiation, pluripotency and self-renewal. The stem cell pluripotency RNA binding protein LIN28B plays critical roles in BC transformation of CML. In this study we sought to characterize CML related-oncogenes, such as BCR-ABL, JAK2 and ADAR1, alone or in stromal co-culture in terms of their ability to regulate LSC self-renewal through modulation of let-7 /LIN28B stem cell transcriptional regulatory axis.MethodsMiRNAs were extracted from purified CD34+ cells derived from CP and BC CML patient samples as well as cord blood by RNeasy microKit (QIAGEN) and let-7 expression was evaluated by qRT-PCR using miScript Primer assay (QIAGEN). CD34+ cord blood (n=3) were transduced with lentiviral human BCR-ABL, JAK2, let-7a, wild type ADAR1 and ADAR1 mutant, which lacks a functional deaminase domain. Then, 72 hours after transduction, lentivirally transduced cells were plated on irradiated SL/M2 cells. After 5 days of culture, cells were collected for RNA and microRNA extraction. Transduction efficiency and LIN28B levels were evaluated by qRT-PCR and let-7 expression was quantified by qRT-PCR using miScript primer assay. Hematopoietic Progenitor and Replating assaywere performed on lenti-let-7a-overexpressing CB cells to assess differentiation, survival and self-renewal capacity.ResultsLentiviral overexpression of human BCR-ABL in CD34+ CB did not induce any significant change in let-7 family members and LIN28B expression in absence of stromal co-culture. However, stromal co-culture of BCR-ABL overexpressing CB led to the significant downregulation of members of the let-7 family as well as to upregulation of their target gene LIN28B, thus suggesting that extrinsic microenvironmental cues are necessary for modulating let-7 family levels in presence of BCR-ABL. Notably, qRT-PCR of CB transduced with JAK2 showed significant upregulation of ADAR1 in the absence of stroma, thus suggesting that JAK2 might be a mediator of inflammatory cytokine-driven ADAR1 activation. Lentiviral overexpression of both human JAK2 and ADAR1 significantly reduced the expression of let-7 family members and induced up-regulation of LIN28B. Interestingly, lentiviral overexpression of ADAR1 mutant did not induce any significant change in most let-7 family members. Finally, lentiviral overexpression of let-7a induced significant reduction in survival and self-renewal.ConclusionThese finding suggest that BCR-ABL requires extrinsic signals from the niche to modulate self-renewal of BC LSCs. Conversely, lentiviral JAK2 overexpression induces activation of aberrant RNA editing and subsequent reduction of let-7 family members in the absence of the niche. Interestingly, experiments with ADAR1 mutant, suggest that ADAR1 downregulates most of the let-7 family members in a RNA–editing dependent way manner. In summary these findings suggest a novel mechanism for BC LSC generation that may have utility in prognostication and selective LSCs targeting. DisclosuresJamieson:J&J: Research Funding; Sanofi: Research Funding, Travel Support, Travel Support Other; Roche: Research Funding.

Synergism between bosutinib (SKI-606) and the Chk1 inhibitor (PF-00477736) in highly imatinib-resistant BCR/ABL+ leukemia cells

Interactions between the dual BCR/ABL and Src inhibitor bosutinib and the Chk1 inhibitor PF-00477736 were examined in BCR/ABL+ leukemia cells, particularly imatinib-resistant cells, including those with the T315I mutation. Bosutinib blocked PF-00477736-induced ERK1/2 activation and sharply increased apoptosis in association with Mcl-1 inhibition, p34(cdc2) dephosphorylation, BimEL up-regulation, and DNA damage in imatinib-resistant CML or Ph+ ALL cell lines. Inhibition of Src or MEK1 by shRNA significantly enhanced PF-0047736 lethality. Bosutinib/PF-00477736 co-treatment also potentiated cell death in CD34+ CML patient samples, including dasatinib-resistant blast crisis cells exhibiting both T315I and E355G mutations, but was minimally toxic to normal CD34+ cells. Finally, combined in vivo treatment significantly suppressed BaF3/T315I tumor growth and prolonged survival in an allogeneic mouse model. Together, these findings suggest that this targeted combination strategy warrants attention in IM-resistant CML or Ph+ ALL.

Abstract 2760: CSF3R ligand-independent mutations found in chronic neutrophilic leukemia have altered O-linked glycosylation

Abstract We have recently discovered that a large percentage (59%) of patients with chronic neutrophilic leukemia (CNL) or atypical chronic myeloid leukemia (aCML) have mutations in Colony Stimulating Factor 3 Receptor (CSF3R, aka GCSFR) (Maxson et al, NEJM 2013). These mutations are either point mutations in the extracellular domain (T618I, T615A) or truncations of the cytoplasmic domain. The most frequent mutation is T618I (aka T595I). The two classes of CSF3R mutations operate through distinct mechanisms that result in differential sensitivity to inhibition of tyrosine kinases downstream of CSF3R. While the mechanism of action of CSF3R truncation mutations has been well characterized-they have increased cell surface expression due to loss of negative regulatory motifs-the way in which the CSF3R point mutations activate the receptor is unclear. Consistent with a previous report, the CSF3R T618I mutation is ligand independent in both mouse bone marrow colony assays and the Ba/F3 pro-B cell line. WT CSF3R consists of two molecular weight species suggestive of differential posttranslational modification. Analysis of CSF3R point mutations T618I and T615A revealed an alteration of banding pattern of these mutations. Since extracellular threonine residues are capable of being O-glycosylated, we employed endoglycosidase digestion to reveal that the larger CSF3R species is O-glycosylated. Furthermore the T618I and T615A mutations reduce O-glycosylation of the receptor. This analysis was confirmed by direct labeling of O-glycosylation using azide-modified galactosamine, which labeled WT CSF3R, but had minimal labeling of CSF3R T618I. Receptor dimerization studies showed increased dimerization of the CSF3R T618I mutant relative to WT. Sequencing of CSF3R from an atypical CML patient samples revealed an additional mutation not previously observed in this disease, T640N. The T640N mutation (aka T617N) is a ligand-independent mutation originally found in a family with hereditary chronic neutrophilia (Plo et al, JEM 2009). Plo et al found that this mutation resides within the transmembrane domain and predicted by molecular modeling that it increased receptor dimerization through interaction of the transmembrane helices. In mouse bone marrow colony assays we confirmed the ligand-independence of the mutation and also found that it had similar potency as T618I in colony and cytokine-independent growth assays. Furthermore, like T618I, the T640N mutation conferred sensitivity to a JAK kinase inhibitor in colony assays. Surprisingly, the T640N mutation had an alteration of banding pattern very similar to T618I, suggesting that it may also have altered glycosylation status, even though the mutation resides within the transmembrane domain. Our studies demonstrate that O-glycosylation is critical for regulation of CSF3R dimerization and that the T618I and T640N mutations can activate the receptor to promote oncogenesis. Citation Format: Julia Maxson, Samuel Luty, Jason MacManiman, Samantha Savage, Melissa Abel, Swaleh Bahamadi, Brian Druker, Jeffrey Tyner. CSF3R ligand-independent mutations found in chronic neutrophilic leukemia have altered O-linked glycosylation. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2760. doi:10.1158/1538-7445.AM2014-2760

Abstract 1912: ADAR1-mediated microRNA regulation and blast crisis leukemia stem cell generation in chronic myeloid leukemia

Abstract Introduction Chronic myeloid leukemia (CML) was one of the first malignancies shown to be initiated in hematopoietic stem cells by the BCR-ABL1 oncogene and sustained in blast crisis (BC) by progenitor cells that co-opt stem cell properties and behave like leukemia stem cells (BC LSC). The BCR-ABL fusion oncogene encodes a constitutively active tyrosine kinase BCR-ABL. Although tyrosine kinase inhibitor (TKI) therapy targeting BCR-ABL suppresses CML during the chronic phase (CP), progenitors undergo expansion as a consequence of subsequent genetic and epigenetic alterations that fuel blast crisis (BC) transformation, BC LSC generation and TKI resistance. We have recently shown that adenosine to insoine (A-to-I) RNA editing activity, mediated by the adenosine deaminase acting on dsRNAs ADAR1, is enhanced in BC CML and drives leukemic progression by inducing myeloid progenitor reprogramming and enhancing self-renewal. ADAR1 targets double-stranded RNA hairpin-containing loop structures, such as microRNAs (miRNAs). MiRNAs, by operating through base-pairing with complementary sequences within a mRNA molecule lead to mRNA degradation and gene silencing. Emerging evidence suggests miRNAs alterations introduced at the pri-miRNA or pre-miRNA level by A-to-I editing by ADAR1 may be responsible for broad changes in the transcriptome that drives LSC maintenance. However, the overall change in edited miRNA profiles in CP CML compared with BC progenitors is unclear. In this study we aimed to investigate the role of ADAR1 in driving malignant progression by focusing on its regulation of miRNA expression in CML. Methods and Results MicroRNA expression profiles were evaluated in CD34+ cells derived from CP and BC CML patient samples as well as cord blood. MiRNA PCR array (Qiagen) allowed us to identify differentially expressed miRNAs that were subsequently validated by q-PCR with specific primers targeting individual miRNA. We observed the downregulation of 13 miRNA in CML BC compared to CML CP progenitors. Lentiviral overexpression of ADAR1 in CD34+ cord blood cells led to significant downregulation of 17 miRNAs, including members of the let-7 family, and other miRNAs involved in the regulation of self-renewal. Interestingly, BC CD34+ cells showed a miRNA profile similar to that of lentiviral ADAR1 transduced cord blood progenitors. Lentiviral overexpression of ADAR1 in CD34+ chronic phase CML cells, led to a significant downregulation of 14 miRNAs compared with their lentiviral backbone transduced counterparts. Conclusion Our data suggest that ADAR1 contributes to BC LSC generation by downregulating miRNAs that target stem cell regulatory gene products. A further characterization of ADAR-editing related miRNA signatures may enable BC LSC detection and early intervention with combined BCR-ABL1 and ADAR1-targeting strategies that may obviate CML relapse and progression. Citation Format: Maria Zipeto, Qingfei Jiang, Leslie Crews Robertson, Catriona HM Jamieson. ADAR1-mediated microRNA regulation and blast crisis leukemia stem cell generation in chronic myeloid leukemia. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1912. doi:10.1158/1538-7445.AM2014-1912

Mapping of Apoptin-interaction with BCR-ABL1, and development of apoptin-based targeted therapy

Majority of chronic myeloid leukemia patients experience an adequate therapeutic effect from imatinib however, 26-37% of patients discontinue imatinib therapy due to a suboptimal response or intolerance. Here we investigated derivatives of apoptin, a chicken anemia viral protein with selective toxicity towards cancer cells, which can be directed towards inhibiting multiple hyperactive kinases including BCR-ABL1. Our earlier studies revealed that a proline-rich segment of apoptin interacts with the SH3 domain of fusion protein BCR-ABL1 (p210) and acts as a negative regulator of BCR-ABL1 kinase and its downstream targets. In this study we show for the first time, the therapeutic potential of apoptin-derived decapeptide for the treatment of CML by establishing the minimal region of apoptin interaction domain with BCR-ABL1. We further show that the apoptin decapeptide is able to inhibit BCR-ABL1 down stream target c-Myc with a comparable efficacy to full-length apoptin and Imatinib. The synthetic apoptin is able to inhibit cell proliferation in murine (32Dp210), human cell line (K562), and ex vivo in both imatinib-resistant and imatinib sensitive CML patient samples. The apoptin based single or combination therapy may be an additional option in CML treatment and eventually be feasible as curative therapy.

MiR-29b suppresses CML cell proliferation and induces apoptosis via regulation of BCR/ABL1 protein

MicroRNAs (miRNAs) are small RNAs that regulate gene expression posttranscriptionally and are critical for many cellular pathways. Recent evidence has shown that aberrant miRNA expression profiles and unique miRNA signaling pathways are present in many cancers. Here, we demonstrate that miR-29b is markedly lower expressed in CML patient samples. Bioinformatics analysis reveals a conserved target site for miR-29b in the 3′-untranslated region (UTR) of ABL1. miR-29b significantly suppresses the activity of a luciferase reporter containing ABL1-3′UTR and this activity is not observed in cells transfected with mutated ABL1-3′UTR. Enforced expression of miR-29b in K562 cells inhibits cell growth and colony formation ability thereby inducing apoptosis through cleavage of procaspase 3 and PARP. Furthermore, K562 cells transfected with a siRNA targeting ABL1 show similar growth and apoptosis phenotypes as cells overexpression of miR-29b. Collectively, our results suggest that miR-29b may function as a tumor suppressor by targeting ABL1 and BCR/ABL1.

Imatinib mesylate and nilotinib affect MHC-class I presentation by modulating the proteasomal processing of antigenic peptides.

Imatinib (IM) has been described to modulate the function of dendritic cells and T lymphocytes and to affect the expression of antigen in CML cells. In our study, we investigated the effect of the tyrosine kinase inhibitors IM and nilotinib (NI) on antigen presentation and processing by analyzing the proteasomal activity in CML cell lines and patient samples. We used a biotinylated active site-directed probe, which covalently binds to the proteasomally active beta-subunits in an activity-dependent fashion. Additionally, we analyzed the cleavage and processing of HLA-A3/11- and HLA-B8-binding peptides derived from BCR-ABL by IM- or NI-treated isolated 20S immunoproteasomes using mass spectrometry. We found that IM treatment leads to a reduction in MHC-class I expression which is in line with the inhibition of proteasomal activity. This process is independent of BCR-ABL or apoptosis induction. In vitro digestion experiments using purified proteasomes showed that generation of epitope-precursor peptides was significantly altered in the presence of NI and IM. Treatment of the immunoproteasome with these compounds resulted in an almost complete reduction in the generation of long precursor peptides for the HLA-A3/A11 and -B8 epitopes while processing of the short peptide sequences increased. Treatment of isolated 20S proteasomes with serine-/threonine- and tyrosine-specific phosphatases induced a significant downregulation of the proteasomal activity further indicating that phosphorylation of the proteasome regulates its function and antigen processing. Our results demonstrate that IM and NI can affect the immunogenicity of malignant cells by modulating proteasomal degradation and the repertoire of processed T cell epitopes.

Combination Targeted Therapy to Impair Self-Renewal Capacity of Human Blast Crisis Leukemia Stem Cells

Abstract 1693The aim of this study is to develop clinical strategies that will HALT progression of CML by reducing leukemia stem cell (LSC) burden using a clinical grade JAK2 inhibitor, SAR302503 (SAR503, Sanofi, Cambridge, MA), alone or in combination with a potent BCR-ABL inhibitor, dasatinib.For this, CML patient samples in blast crisis phase (BC CML) were subjected to immunomagnetic bead CD34 selection or FACS Aria ll sorted to obtain leukemic progenitors (LSC/CD34+CD38+Lin−). Malignant progenitors were then transplanted into neonatal RAG2−/−gc−/− mice, and 8 weeks post-transplant, mice were treated with SAR503, dasatinib and vehicle for 14 days. Following treatment, hematopoietic tissues were analyzed for human engraftment by FACS analysis.Our results revealed that single agent experiments with SAR503 had a cytostatic rather than a cytoreductive effect on BC LSC. The treatment alone (60 mg/kg twice daily administered by oral gavage) did not significantly reduce leukemic progenitor burden in the liver, spleen, bone marrow and peripheral blood. Conversely, combination therapy with SAR503 and dasatinib (50mg/kg/day) significantly reduced LSC progenitors in all tissues examined.Interestingly, we observed that dasatinib alone therapy reduced the LSC burden in the liver, spleen, and peripheral blood, but the bone marrow retained a significant population of BC LSC. Also we found that the GMP population, previously shown to be enriched for BC LSC (Jamieson et al NEJM 2004; Abrahamsson et al PNAS 2009), was preferentially localized in the bone marrow. As shown by our laboratory and others, LSC therapeutic resistance may be influenced by extrinsic cues provided by the niche (e.g. promoting quiescence). Because quiescence has been implicated in driving tyrosine kinase inhibitor resistance and LSC survival and because the bone marrow retains a resistant population, we decide to perform secondary transplantation experiments to determine relapse potential (self-renewal). LSC progenitors were isolated by immunomagnetic bead selection of human CD34+ cells from marrows and spleens of treated mice. After serially transplanting an equal number of this cells into secondary recipients, we observed a significant reduction in LSC serial transplantation only following combination treatment, suggesting that the combination therapy can abolish LSC self-renewal capacity and thereby potentially prevent relapse.To validate drug exposure, we have been performing both genomic and nanoproteomic analysis. Regarding the proteomics validation studies, we analyzed sorted LSC derived from spleen (pooled 5 mice per group) that were treated with vehicle or SAR503 for 14 days. The analysis was performed to detect status of p-JAK2, JAK2, p-STAT5 and B2-microglobulin (loading control). We observed a down regulation on the levels of p-JAk2 (active site Tyr 1007–08) and p-Stat5 (active site Tyr 694) (35% and 42% respectively), while no changes are observed for total JAK2 protein or B2M between both conditions.The full transcriptome sequencing, on sorted LSC treated with SAR503 alone and in combination with dasatinib, identified specific isoform changes in the JAK/STAT pathway that could be used as biomarkers of response and could explain the synergistic effect of the combination therapy. We have also characterized, at an isoform level, biomarkers of resistance that could explain relapse of disease after single agent therapy and we are currently validating these findings. Disclosures:No relevant conflicts of interest to declare.