Expansion Of Myeloid Cells Research Articles

Modeling Chronic Myeloid Leukemia in Immunodeficient Mice Reveals an Inflammatory State with Expansion of Aberrant Mast Cells and Accumulation of Pre B Cells

Introduction: Chronic myeloid leukemia (CML) is a myeloproliferative neoplasm that if not treated, will progress into blast crisis (BC) of either myeloid or B lymphoid phenotype. The BCR-ABL1 fusion gene, encoding a constitutively active tyrosine kinase, is thought to be sufficient to cause chronic phase (CP) CML, whereas additional genetic lesions are needed for progression into CML BC. To ultimately achieve a cure for CML, there is a need to establish improved disease models allowing increased understanding of disease pathogenesis and to evaluate novel therapeutics.Aim: In this study, we investigated if retroviral expression of BCR-ABL1 in cord blood (CB) CD34+ progenitor cells transplanted into NOD/SCID IL2–receptor gamma deficient (NSG) mice would recapitulate features observed in CP CML.Methods: CD34+ progenitor cells from human CB were transduced with a retroviral vector expressing BCR-ABL1 and subsequently transplanted into NSG mice. Mice were sacrificed at signs of illness and bone marrow (BM) and spleen cells were analyzed using flow cytometry, immunohistochemistry, FISH and RNA sequencing. Secondary transplants were also performed. BM mononuclear cells (MNC) from CP CML patients were analyzed using flow cytometry and FISH.Results: For the majority of the BCR-ABL1 transplanted (BA) mice, signs of disease were evident within 100 days post transplantation. Spleens were enlarged and both control and BA mice showed robust human engraftment in both BM and spleen. Expression of BCR-ABL1 also induced expansion of the transduced cells. In addition to the previously described features of this humanized transplantation model such as a mild expansion of human myeloid cells, BCR-ABL1 expression induced an inflammatory-like state in the BM and spleen of the BA mice as evident by an increase of human macrophages/histiocytes and T cells. In addition, an expansion of aberrant mast cells in BA mice was observed. Secondary transplantations failed to induce a similar disorder as in the primary mice, but long-term engraftment was achieved, showing only mast cells in secondary BA mice. These mast cells aberrantly expressed CD25 (IL2RA), a cell surface receptor normally only expressed on neoplastic mast cells. This was in contrast to secondary control mice where the long-term engraftment was dominated by B cells.Because CP CML can progress into lymphoid BC, we next explored if BCR-ABL1 expression in CB CD34+ cells would show a disturbance also of the B cell lineage. We observed a striking block at the pre B cell stage, resulting in an accumulation of pre B cells in the BM of BA mice. RNA sequencing of sorted BCR-ABL1-expressing pre B cells from BA and control mice revealed that BCR-ABL1 expression results in a significant de-regulation of about 700 genes. Among the up-regulated genes, many were shown to be involved in inflammation and apoptosis. Finally, by sorting BM MNCs from patients with CP CML into different populations of B cell progenitors and performing FISH for BCR-ABL1, we confirmed that a similar block in B cell differentiation is present in CP CML patients.Conclusion: This humanized mouse model of CML reveals previously unexplored features of CP CML, including expansion of aberrant mast cells and a block at the pre B cell stage, and should provide a valuable model for future studies of mechanisms involved in the disease pathogenesis of CML. DisclosuresNo relevant conflicts of interest to declare.

Activation of p53 By Novel MDM2 Antagonist RG7388 Overcomes AML Inherent and Acquired Resistance to Bcl-2 Inhibitor ABT-199 (GDC-0199)

Background: Acute myeloid leukemia (AML) is characterized by the clonal expansion of immature myeloid cells. AML is treated primarily with chemotherapy, but the 5-year survival rate has only marginally increased over the past few decades, highlighting the need for novel therapies to achieve higher cure rates with more acceptable toxicities.Bcl-2 family proteins, together with TP53, are the central regulators of apoptosis. Overexpression of Bcl-2 protein is associated with leukemia progression and chemoresistance. We have observed elevated expression of Bcl-2 in AML and recently demonstrated that Bcl-2 inhibition by ABT-199 effectively induced apoptosis in AML (Pan, et.al., Cancer Discovery, 2014). However, resistance to ABT-199 was observed in cells expressing high levels of Mcl-1 or Bcl-xL. Moreover, a recent study showed heterogeneous but overlapping expression of Bcl-2, Mcl-1, and Bcl-xL proteins in 577 AML patient samples (Bogenberger, et. al., Leukemia, 2014).Although common in solid tumors, p53 mutations are relatively rare in AML. However, p53 functions are diminished by overexpression of MDM2 protein, an E3 ubiquitin ligase of p53 and an inhibitor of p53 transactivation. We previously reported MDM2 overexpression in 53% of primary AMLs (Kojima et al., Blood, 2005). Our group also demonstrated that p53 activation by Nutlins, the prototypical MDM2 inhibitors, induced apoptosis and growth inhibition in AML.Rationale: Since p53 activation by MDM2 inhibitors upregulates pro-apoptotic Bcl-2 proteins like NOXA, PUMA, and Bax, which counteract Mcl-1 and Bcl-xL, we hypothesized that the second-generation MDM2 inhibitor RG7388 could overcome AML resistance to Bcl-2-specific ABT-199, and that the combination could synergistically enhance apoptosis in AML.Results: We first demonstrated that RG7388 induced apoptosis exclusively in p53 wild type (wt) cells. RG7388 was essentially ineffective in p53 mutant or null AML cell lines such as HL-60, KG1 and THP1 (48h IC50s > 5 μM). Nonetheless, it showed high potency against p53 wt cell lines (48h IC50s: MOLM13 = 21.7 nM, MV-4-11 = 29.2 nM). Furthermore, stable knockdown of TP53 rendered the wt cell lines completely resistant to RG7388 (IC50s > 5 μM), confirming TP53-specificity. To study if RG7388 was able to overcome inherent resistance to ABT-199, we tested its efficacy on OCI-AML3 cells, which are inherently resistant to ABT-199, AraC and Idarubicin. As a single agent, RG7388 potently killed OCI-AML3 cells (48h IC50 = 148 nM). Importantly, RG7388 was ~20-fold more effective in OCI-AML3 cells than its predecessors Nutlin-3a and RG7112. We also examined the time- and dose-response of RG7388 in several genetically diverse AML cell lines (p53 wt) and found that 100 nM RG7388 was able to induce apoptosis and inhibit cell growth within 12 h.Next we studied whether RG7388 synergizes with ABT-199 to kill the refractory OCI-AML3 cells. A combination index of 0.35 (Chou-Talalay method) indicated a strong synergy between the two compounds. The combination exhibited higher activity in killing OCI-AML3 cells than either agent alone (48h IC50s: ABT-199 = 1680 nM, RG7388 = 148 nM, ABT+RG = 28 nM). Similar synergy was observed in additional AML cell lines and in primary samples. Next, we generated ABT-199 resistant cells by continuous exposure of initially sensitive AML cells to escalating concentrations of ABT-199. While 1000 nM ABT-199 had no effects on the viability of these cells, additional treatment with 30 nM RG7388 effectively killed them. This finding suggested that RG7388 was able to overcome acquired resistance to ABT-199. The mechanisms underlying this resensitization and its synergism with ABT-199 are under investigation using in vitro and in vivo model systems.Conclusions: The novel MDM2 inhibitor RG7388 induces growth arrest and apoptosis selectively in p53 wt AML cells. Importantly, the combination of RG7388 with ABT-199 synergistically induced apoptosis in AML cell lines and primary patient cells, and RG7388 was able to overcome inherent or acquired resistance to ABT-199. Since both Bcl-2 and MDM2 overexpression are associated with poor prognosis in AML, the proposed combination of the two clinical-stage compounds could have considerable clinical potential. We will report on ongoing experiments with primary AML cells in NSG mice to determine the potential of this combinatorial approach to eliminate AML stem cells. DisclosuresNichols:Roche: Employment, Equity Ownership. Leverson:abbvie: Employment, Equity Ownership. Dangl:Roche: Employment, Patents & Royalties. Konopleva:Abbvie: Research Funding. Andreeff:Roche: Research Funding; Abbvie: Research Funding.

Aberrant Hedgehog Pathway Activity Marks Clinical MDS Progression and Accelerates Leukemic Transformation in Vivo

Introduction:Myelodysplastic Syndromes (MDS) represent a heterogeneous group of hematopoietic stem cell (HSC) disorders with varying clinical outcomes, but prognosis uniformly worsens with transformation to secondary acute myeloid leukemia (AML). Despite recent progress in genomics, the mechanisms responsible for disease progression are not fully understood as most of the somatic mutations defined thus far can be found at early stages of the disease. Previous studies have identified aberrant activation of the Hedgehog (Hh) signaling pathway in a subset of AML patients and the expression of the Hh-regulated transcription factor GLI2 correlated with inferior overall and progression free survival. In solid tumors, Hh pathway activation has been associated with metastatic disease progression, and we examined its role in MDS progression and transformation to AML.Methods/Results: We initially quantified changes in Hh pathway activity in CD34+ cells isolated from serial bone marrow samples collected from MDS patients at the time of diagnosis and following progression to AML. We found that the expression of the Hh target genes GLI1 and PTCH1 was increased in 67% of patients (4/6) suggesting that pathway activation was involved in the development of secondary AML. We also analyzed gene expression in 135 MDS patients and found significantly higher GLI2 expression in high-risk MDS (N=80) compared to low-risk MDS (N=55) (p=0.036). In addition, bone marrow blast percentage was significantly higher in the MDS cohort with higher GLI2 expression (mean±SEM=7.1±0.7%) than with lower expression (5.4±0.5%, p=0.039).In order to mechanistically study the effects of Hh pathway activation on MDS progression, we studied mice expressing the Nup98-HoxD13 (NHD13) fusion gene under the control of the vav promoter that generates progressive cytopenia and, in some animals, progression to AML. We crossed NHD13 mice with mice conditionally expressing the constitutively active mutant of the Hh signal transduction regulator Smoothened (SmoM2) in hematopoietic cells expressing Mx1-Cre. The survival of double transgenic animals (NHD13/SmoM2) was significantly shorter compared to mice expressing NHD13 (median survival of 3 months vs. 12 months, p<0.001, Fig. 1). Similar to previous reports we found that the expression of SmoM2 alone had no significant effect on survival or hematologic phenotype. Compared to NHD13 mice, NHD13/SmoM2 mice had significantly higher peripheral blood WBC (93.9k vs. 5.2k, p<0.001) and splenomegaly (633mg vs. 462mg, p=0.006) at the time of disease progression. Furthermore, an immature population of myeloid cells (Mac1+/Gr1 dim) was widely present in peripheral blood, bone marrow and spleen of NHD13/SmoM2 mice. We also examined mice prior to the onset of disease (4-8 weeks post Smo M2 induction) and found an increased frequency of myeloid cells (Mac1+/Gr1+) within the peripheral blood compared to wild type and NHD13/Smo M2 mice (wild type – 17.2%, NHD13 - 40.5%, NHD13/SmoM2 - 68%, p<0.01).Within the bone marrow, hematopoietic stem/progenitor cells (Lin-Sca1+cKit+) were depleted within both NHD13 and NHD13/SmoM2 mice. To examine the impact of Hh activation on clonogenic growth and self-renewal potential, we plated bone marrow cells in methylcellulose and found that NHD13/SmoM2 cells contained a significantly higher number of CFU compared to NHD13 cells (37 vs. 2, p=0.002) that was sustained with subsequent passages (400 vs. 30, p<0.001). In order to define the compartment enriched for leukemia initiating potential (LIC) we transplanted Lin- and Lin+ leukemic fractions from NHD13/SmoM2 mice into naive recipients and found that Lin+ cells led to death within 3 months suggesting that the Lin+ compartment has acquired self- renewal potential.Conclusions:We found that the Hh signaling pathway may be aberrantly activated in a subset of secondary AML patients progressing from MDS. We also demonstrated that activation of Hh signaling induces fatal AML in a mouse model of MDS characterized by inferior survival and widespread expansion of immature myeloid cells. This disease progression is driven by the acquisition of self-renewal and tumor initiating potential in differentiated Lin+ hematopoietic cells. Our findings suggest that Hh pathway inhibition may be a promising approach for AML arising from MDS. [Display omitted] DisclosuresMaciejewski:Alexion: Speakers Bureau; Celgene: Speakers Bureau.

Next Generation Sequencing Identifies DNA Methylation Patterns Indicative of Disease Progression in Ph+ CML

Ph+ chronic myeloid leukemia (CML) is a stem cell malignancy characterized by the BCR-ABL1 oncoprotein and leukemic expansion of myeloid progenitor cells. In the chronic phase (CP) of CML, clonal cells undergo myeloid differentiation and respond well to BCR-ABL1 inhibitors. In the accelerated phase (AP) and blast phase (BP) of CML, however, neoplastic cells are immature and often resistant against most BCR-ABL1-targeting drugs which is a challenge in clinical hematology. So far, little is known about molecular mechanisms underlying disease progression in CML. Methylation of CG sites around the transcriptional start site of various cancer-related genes including diverse tumor suppressor genes (referred to as methylation) is a frequently occurring epigenetic feature in neoplastic cells resulting in silencing of these genes. Although methylation is considered a critical factor in the pathogenesis of various malignant diseases including myeloid neoplasms, no comprehensive studies on the impact of methylation in the pathogenesis of CML have been conducted so far. We hypothesized that methylation may be an important mechanism regulating the transcriptional gene activity in CML cells during disease progression. Therefore, we investigated the methylome and the transcriptome of neoplastic cells in patients with CML in various phases of the disease (CP, n=15; AP, n=5; BP, n=7). Genome-wide methylation and gene expression patterns were analysed by next generation sequencing approaches using bone marrow (BM) or peripheral blood (PB) mononuclear cells (MNC) obtained from patients with CML and BM or PB MNC from control individuals. Methylation was analysed by reduced representation bisulfite sequencing (RRBS), and mRNA expression was determined by RNA-sequencing (RNA-seq). By comparing the methylome of patients who were initially diagnosed with CP-CML and who relapsed several months later (AP-CML, n=1; BC-CML, n=3), we identified a large number of genes which were methylated around their transcriptional start site in leukemic cells in patients at the time of progression compared to the time of CP-CML (range in the 4 patients: 423-1209 genes, adjusted p<0.05). These methylated genes were found to be less methylated or not methylated in BM or PB MNC of control individuals. When the methylome of all patients in all cohorts was examined and compared, more genes were found to be methylated in AP-CML and BC-CML compared to CP-CML (CP-CML, n=200; AP-CML, n=311; BC-CML, n=570). In addition, we identified several genes that were less methylated or not methylated in AP-CML and BC-CML cells compared to cells in CP-CML samples (range: 16-541 genes). Moreover, we analysed and compared the transcriptome of CP-CML, AP-CML and BC-CML samples and identified a large number of genes whose expression is downregulated in AP-CML and BC-CML samples compared to CP-CML (range: 187-382 genes). By correlating RRBS results and RNA-seq data, we found that expression of >100 of the methylated genes is downregulated in AP-CML/BC-CML compared to CP-CML suggesting that these genes may be regulated by methylation. Expression of the majority of these genes was detected in BM or PB MNC of control individuals. In silico pathway analyses and gene network analyses revealed that some methylated genes are involved in the regulation of apoptosis (e.g. CYP1B1, ZBTB16), negative regulation of cell proliferation (e.g. BTG3, VSX2) or regulation of Wnt signalling (e.g. SFRP1). Currently, a large number of CML patients are analysed gene-specifically for methylation by methylation-sensitive high resolution melting PCR and for expression of selected genes by RT-PCR in order to define prognostic patterns in CML. In summary, our results demonstrate that methylation changes are frequent events accompanying disease progression in patients with CML. These results may contribute to the identification of clinically relevant methylation patterns in CML and thus may improve prognostication. In addition, our data may reveal new epigenetic targets of therapy and may help to develop new treatment strategies for high risk or relapsing patients with CML. DisclosuresValent:Novartis: Honoraria; Pfizer: Honoraria; Ariad: Honoraria; BMS: Honoraria.

KLF4 Regulates Self-Renewal of Leukemic Stem Cells in Chronic Myeloid Leukemia By Repressing Gbl Expression and Altering mTORC2 Activity

Abstract Tyrosine kinase inhibitors (TKIs) are the standard treatment for eradicating BCR-ABL-positive progenitor cells in chronic myeloid leukemia (CML); however, disease often relapses upon drug discontinuation because TKIs do not effectively eliminate leukemic stem cells (LSC). The development of novel strategies aimed at eradicating LSC without harming normal hematopoietic stem cells (HSC) is essential for the cure of CML patients. The generation of LSC-directed therapy relies on the identification of novel molecular pathways that selectively regulate LSC function independent of BCR-ABL. The Krüppel-like factor 4(KLF4) is a transcription factor that can either activate or repress gene transcription acting as an oncogene or a tumor suppressor depending on the cellular context. Analysis of a published dataset from chronic phase CML patients revealed elevated levels of KLF4 in LSC compared to progenitor cells indicating that KLF4 is likely implicated in LSC regulation. To study the role of KLF4 in LSC function, we used a CML mouse model combining somatic deletion of the Klf4 gene and retroviral transduction and transplantation of HSC. In contrast to mice receiving BCR-ABL-transduced Klf4fl/fl HSC that developed and succumbed to CML, mice transplanted with BCR-ABL-transduced Klf4Δ/Δ (Klf4fl/fl Vav-iCre+) HSC showed a progressive loss of leukemia despite an initial expansion of myeloid leukemic cells, which led to increased overall survival. This inability to sustain CML in the absence of KLF4 was caused by attrition of LSC in bone marrow and the spleen. Furthermore, deletion of KLF4 impaired the ability of LSC to recapitulate leukemia in secondary recipients suggesting a loss of self-renewal capacity. In contrast to LSC, KLF4 deletion led to increased self-renewal of normal HSC assessed by serial competitive transplantation. To identify KLF4 target genes involved in LSC self-renewal, we performed a global gene expression analysis using Klf4Δ/Δ LSC purified by cell sorting from leukemic mice. Analysis of gene expression in Klf4Δ/Δ LSC revealed significant upregulation of GβL, a component of mTOR complexes. Finally, we identified that KLF4 binds to GβL promoter by Chip-Seq analysis and that silencing resulted in inhibition of mTORC2 but not mTORC1 activity in 32D-BCR-ABL-positive CML cells. Our findings suggest that KLF4 transcriptionally represses GβL expression in LSC and that mTORC2 inhibition has the potential to completely eradicate LSC and induce treatment-free remission. Disclosures No relevant conflicts of interest to declare.

Dissecting the Molecular Mechanisms of Aneuploidy in Acute Myeloid Leukemia By Next Generation Sequencing

Acute Myeloid Leukemia (AML) is a heterogeneous malignancy characterized by the expansion of myeloid precursor cells with limited or abnormal differentiation capacity. A relatively common event in AML is represented by chromosome gain or loss. Numerical chromosome abnormalities, which define aneuploidy, have a detrimental effect in primary non-malignant cells, since they dramatically reduce cellular fitness. However, evidence suggests that they have a causative role in tumorigenesis and are well tolerated in transformed cells belonging to the myeloid lineage.Aim of the study is to elucidate the pathogenic mechanisms causing and sustaining aneuploidy in AML in order to find novel potential therapeutic targets.A panel of genetic alterations was analyzed on 886 AML cases at Seràgnoli Institute in Bologna between 2002 and 2013. Among them, 31 samples were subjected to whole exome sequencing (WES, Illumina Hiseq2000). Raw data were processed with WES Pipeline web tool for variants detection. Gene expression profiling (GEP, Affymetrix) was performed on bone marrow cells from 49 AML patients at diagnosis with more than 80% blast cells, including 22 aneuploid cases (carrying monosomy, trisomy or a monosomal karyotype) and 27 cases with normal karyotype. The aneuploid status was confirmed by single nucleotide polymorphism (SNP) array.WES analysis of 13 aneuploid and 12 euploid AML cases revealed a significantly higher median value of genetic variants and mutated genes in aneuploid compared with euploid samples (aneuploid vs. euploid: median of variants, 30 vs. 20 (p=0.02) including nonsynonimous single nucleotide variants, frameshift insertions and deletions, stopgains; median of mutated genes, 25 vs. 17 (p=0.05); details will be presented at the meeting). Noticeably, by gene ontology analysis of mutated genes in the aneuploid cohort we observed a strong enrichment in genes regulating cell cycle, including chromosome organization (p=5.4x10-4) and mitotic sister chromatid cohesion (p=6.98x10-4), and chromatin modification (p=1.3x10-4), with most of the variants being not annotated in the COSMIC database. Euploid samples were enriched for mutations affecting genes involved in cytoskeleton (p=1.6x10-3) and metabolic activities (p=1.9x10-3). A number of genes mutated in the aneuploid cases belong to the APCCdc20 complex and localize on chromosomes generally spared by aneuploidy, supporting the key role of the identified aberrations in the molecular mechanisms leading to numerical chromosome abnormalities. Among several mutations predicted as “drivers” by DOTS-Finder tool (CCDC144NL, DNMT3A, GXYLT1, MESP1, TPRX1,TPTE, ZNF717), we defined some candidates involved in cell cycle regulation and DNA replication. Functional analysis are ongoing. Furthermore, a tumor suppressor function was associated with mutated genes involved in the DNA repair process. In our WES analysis, we identified a subgroup of genes linked to DNA damage response, including TP53, which are preferentially mutated in the aneuploid cohort. Since P53 is a limiting-factor in aneuploidy-induced tumorigenesis, we analyzed the mutational status in a larger cohort of AML patients by Next Generation sequencing (NGS) and Sanger sequencing. Interestingly, we identified TP53 mutations in 15/58 aneuploid vs. 1/36 euploid cases (p=3.8x10-3). Finally, differential expression of genes involved in DNA damage and integrity checkpoints was identified by GEP of aneuploid and euploid AML samples.Previous evidence showed that loss of the spindle checkpoint gene BUB1B induces aneuploidy and predisposes to tumorigenesis. Our data, obtained by integrated NGS and GEP approaches, support a causal link between mutations in a panel of genes involved in cell cycle control/chromosome organization and aneuploidy in AML. Genetic and transcriptional alterations of genes regulating DNA damage response were detected in our AML cohort, suggesting novel molecular mechanisms for the acquisition and/or maintenance of the aneuploid condition and consequently, of leukemogenesis. The results indicate that the identified genomic aberrations likely drive chromosome gain and/or loss in AML by cooperating with alterations affecting different pathways, in order to overcome the unfitness barrier induced by aneuploidy.Supported by: FP7 NGS-PTL project, ELN, AIL, AIRC, PRIN, progetto Regione-Università 2010-12 (L. Bolondi). DisclosuresMartinelli:Novartis: Consultancy, Speakers Bureau; BMS: Consultancy, Speakers Bureau; Pfizer: Consultancy; ARIAD: Consultancy.

Epigenetic Silencing of BRCA1 Is Linked to Homologous Recombination Repair Defects and Elevated Mir-155 Expression in Myeloid Neoplasms

Myeloid neoplasms are a heterogeneous group of clonal hematological disorders ranging from chronic myeloproliferative neoplasms (MPNs) to more aggressive mixed myelodysplastic syndrome/myeloproliferative neoplasms (MDS/MPNs) and secondary acute myeloid leukemias (sAMLs). With the exception of BCR-ABL positive chronic myelogenous leukemia (CMLs), these diseases are clinically challenging to diagnose. Due to the clinical heterogeneity and lack of new biologic insights, current therapies fail to eradicate the malignant clone and alter the natural history of the disease. However, these diagnoses share overlapping features in morphologies, cytogenetics and genetic alterations suggesting common alterations in underlying disease pathways.We have previously reported genomic loss of HR genes in MPN patients linked to defective RAD51 foci induction and sensitivity to poly (ADP-ribose) polymerase (PARP) inhibition (McDevitt et al., ASH 2011). In this study, we directly examined homologous recombination (HR) in primary human samples by measuring induction of RAD51 foci after ionizing irradiation. Freshly isolated mononuclear leukocytes from patients diagnosed with MPNs, mixed MDS/MPNs and sAMLs were collected at the Johns Hopkins Hospital from April 2012 to March 2014. 11 of the 22 samples (50%) exhibited impaired RAD51 foci induction. With little evidence for biallelic genetic inactivation of HR genes in previous work, we evaluated promoter CpG island methylation of BRCA1, which we had previously found in a pilot study of myeloid neoplasm samples (McDevitt et al., ASH 2011). Using quantitative methylation-specific PCR (qMSP) with melt curve analysis, we reported BRCA1 promoter hypermethylation in 22 of 104 samples (21.2%), and observed statistically significant down-regulation of BRCA1 transcript in samples with BRCA1 CpG methylation (p<0.05). To determine the relationship between BRCA1 epigenetic silencing and HR status, we categorized BRCA1 gene expression of the samples according to the promoter methylation and HR status. Strikingly, we noted that all BRCA1 methylated samples were defective for HR and 5/12 (41.7%) of HR defective samples had down-regulated BRCA1 levels.We next validated the role of BRCA1 repression in vitro using the AML cell line OCI-AML3 using two independent shRNAs targeting BRCA1. We found that BRCA1-silenced cells have reduced induction of RAD51 foci, recapitulating our observations in primary samples with epigenetic silencing of BRCA1. Given the selective toxicity observed with HR-deficient cancers to PARP inhibition, we treated the BRCA1 knockdown cells with the PARP inhibitor ABT-888 and observed increased drug sensitivity.Finally, we examined additional consequences of BRCA1 loss in myeloid malignancies by investigating its role in repressing miR-155. Frequent up-regulation of miR-155 in leukemia is linked to poor prognosis, and its overexpression in murine models results in myeloproliferative disorders. Our results showed a statistically significant inverse correlation between BRCA1 and miR-155 expression in patient samples (Pearson r:-0.36; p-value<0.005). We also found elevated miR-155 expression in the BRCA1-silenced OCI-AML3 cells that increased during long-term BRCA1 repression.Our results demonstrate that defective HR repair occurs in a significant subset of myeloid malignancies. This provides a rationale for PARP inhibitors in this patient subpopulation who currently lack curative therapy, a strategy currently explored in two clinical studies (J0783, PI: Keith Pratz, MD; J1051, PI: Ivana Gojo, MD). Our finding showing BRCA1 promoter methylation in samples with defective HR highlights a novel mechanism underlying HR defects in myeloid malignancies, and offers a biomarker to identify potential responders to PARP inhibition. We also provide an alternative mechanism in which BRCA1 loss contributes to disease progression, via de-repressing miR-155 linked to aberrant expansion of myeloid cells. DisclosuresNo relevant conflicts of interest to declare.

Tumor-induced myeloid dysfunction and its implications for cancer immunotherapy.

Immune function relies on an appropriate balance of the lymphoid and myeloid responses. In the case of neoplasia, this balance is readily perturbed by the dramatic expansion of immature or dysfunctional myeloid cells accompanied by a reciprocal decline in the quantity/quality of the lymphoid response. In this review, we seek to: (1) define the nature of the atypical myelopoiesis observed in cancer patients and the impact of this perturbation on clinical outcomes; (2) examine the potential mechanisms underlying these clinical manifestations; and (3) explore potential strategies to restore normal myeloid cell differentiation to improve activation of the host antitumor immune response. We posit that fundamental alterations in myeloid homeostasis triggered by the neoplastic process represent critical checkpoints that govern therapeutic efficacy, as well as offer novel cellular-based biomarkers for tracking changes in disease status or relapse.

Role of disease-associated tolerance in infectious superspreaders.

Natural populations show striking heterogeneity in their ability to transmit disease. For example, a minority of infected individuals known as superspreaders carries out the majority of pathogen transmission events. In a mouse model of Salmonella infection, a subset of infected hosts becomes superspreaders, shedding high levels of bacteria (>10(8) cfu per g of feces) but remain asymptomatic with a dampened systemic immune state. Here we show that superspreader hosts remain asymptomatic when they are treated with oral antibiotics. In contrast, nonsuperspreader Salmonella-infected hosts that are treated with oral antibiotics rapidly shed superspreader levels of the pathogen but display signs of morbidity. This morbidity is linked to an increase in inflammatory myeloid cells in the spleen followed by increased production of acute-phase proteins and proinflammatory cytokines. The degree of colonic inflammation is similar in antibiotic-treated superspreader and nonsuperspreader hosts, indicating that the superspreader hosts are tolerant of antibiotic-mediated perturbations in the intestinal tract. Importantly, neutralization of acute-phase proinflammatory cytokines in antibiotic-induced superspreaders suppresses the expansion of inflammatory myeloid cells and reduces morbidity. We describe a unique disease-associated tolerance to oral antibiotics in superspreaders that facilitates continued transmission of the pathogen.

Comparison Analysis of Immune Cells between CT26 Tumor Bearing Mice and Normal Mice

It has well studied that immune cells are strongly related to tumor progression and tumor suppression. To identify the difference of immune cell between tumor bearing mice and normal mice, we examined systemically the immune cell of CT26 tumor bearing mice on 21 days after tumor cell administration. As previously reported, CD4+ and CD8+ T cells population of tumor bearing mice significantly decreased 38% and 30% on day 21 compared to that of normal mice, respectively. All subpopulation of CD4 and CD8+ T cell significantly decreased, except CD49b+ T cell subpopulation. But, myeloid cell population (CD11b high and all Gr-1+ subpopulation) of tumor bearing mice significantly increased on day 21. Especially, all subpopulation of CD11b+Gr-1+ cell of tumor bearing mice significantly increased on day 21. Also, Foxp3+CD25 high CD4 T cell (regulatory T cells) population significantly increased on day 21. These results suggest that tumor can induce the decline of T lymphocyte and the expansion of myeloid cells and regulatory T cells, and provide the basic information for the study of tumor immunology.

Abstract 164: Bone marrow myeloid cells promote multiple myeloma chemoresistance by DNA mediated signaling

Abstract Bone marrow (BM) niche provides a sanctuary for multiple myeloma (MM) cells allowing them to escape chemotherapy. Previous in vitro studies have implicated some accessory cells present in BM microenvironment including stromal cells, macrophages, osteoclasts and plasmacytoid dendritic cells in MM chemoresistance. However, these cells comprise only a small fraction of BM niche. The role of the major cellular component of BM represented by immature myeloid cells, neutrophils, and monocytes in MM survival from chemotherapy remains unknown. To determine whether myeloid cells are involved in regulation of MM growth and chemosensitivity, we initially employed in vivo mouse models that allow for the modulation of the number of myeloid cells in BM. Using the CD11b-DTR model, we demonstrated that depletion of myeloid CD11b-expressing cells from BM led to a significant reduction of MM tumor growth in these mice and improved the antitumor effect of doxorubicin. Conversely, GM-CSF-induced expansion of myeloid cells in the BM of MM-bearing mice resulted in reduced survival and increased resistance to chemotherapy. CD11b+Gr1+ myeloid cells isolated from BM of MM-bearing or control tumor-free mice demonstrated chemoprotective effects on mouse MM cells. Human CD33+CD14-CD11b+ myeloid cells isolated from BM of patients with MM or healthy donors were able to protect human MM cells from doxorubicin and melphalan-induced apoptosis in vitro. The addition of supernatant collected from myeloid cells to MM cells was sufficient to induce chemoprotection in mice and human cells. The protection was completely lost when myeloid cells were separated from MM cells by transwell insert. Our further studies demonstrated that myeloid cells, but not other BM accessory cells, were able to release substantial amount of DNA. Importantly, treatment of these cells with DNAse I abrogated the chemoprotective effect of myeloid cells on MM. The addition of genomic DNA isolated from myeloid cells promoted chemoresistance of MM cells. Further, Stimulation of TLR9 with its ligand CpG oligonucleotides mimicked the chemoprotective effect of DNA on MM cells. Cell-free DNA up-regulated spliced XBP-1, a transcription factor involved in unfolded protein response. Pharmacological inhibition of IRE-1α, a kinase required for XBP-1 splicing, reversed myeloid cell induced MM chemoprotection. Finally, in vivo depletion of myeloid cells in BM of MM-bearing mice resulted in decreased expression of spliced XBP-1 in MM cells and improved response to chemotherapy. Taken together, our data demonstrated a critical role played by myeloid cells in growth and chemosensitivity of MM cells and identified a novel mechanism that mediates this effect. Our results suggest that targeting myeloid cells or TLR9 in MM may reduce tumor growth and improve response to the chemotherapy in this disease. Citation Format: Indu Ramachandran, Thomas Condamine, Dmitry Gabrilovich, Yulia Nefedova. Bone marrow myeloid cells promote multiple myeloma chemoresistance by DNA mediated signaling. [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 164. doi:10.1158/1538-7445.AM2014-164

Abstract 2243: Gene expression signature of aneuploidy in acute myeloid leukemia

Abstract Acute Myeloid Leukemia (AML) is a heterogeneous malignancy characterized by the expansion of myeloid precursor cells with limited or abnormal differentiation capacity. A relatively common event in AML is represented by chromosome gain or loss. Numerical chromosome abnormalities, which define the aneuploid condition, have a detrimental effect in primary non-malignant cells, since they dramatically reduce cellular fitness. However evidence suggests that they have a causative role in tumorigenesis and that they are well tolerated in transformed cells belonging to the myeloid lineage. Aim of the study is to elucidate the pathogenic mechanisms that sustain and contribute to aneuploidy in AML. We have performed gene expression profile (GEP) analysis of bone marrow cells from 42 AML patients, including 19 aneuploid cases and 23 cases with normal karyotype. All samples contained more than 80% blast cells. The aneuploid cohort included AML cases carrying one (or more) monosomy, trisomy or a monosomal karyotype. Our analysis covered more than 245,000 and 40,000 coding and non-coding transcripts, respectively (the latter comprising microRNAs), and a significant number of exon-exon junctions, which allow the analysis of multiple splicing isoforms. Quality controls confirmed that the data show comparable signal values. The gene expression signature of aneuploid samples have been compared to normal karyotype ones. We have identified a set of coding and non-coding transcripts which are differentially expressed between the two groups (p≤0.05, including more than 20 genes with a fold difference ≥2) and defined a gene signature that allows the discrimination between aneuploid and euploid samples in our dataset. The analysis of an increased number of cases will confirm the results and allow the sub-stratification of aneuploid samples according to their GEP. Our data will be further validated by comparing them with published GEP datasets and the gene signature will be characterized by pathway analysis. This study provides novel insights into the molecular mechanism that sustain aneuploidy in AML. The biological validation of genes which are commonly and specifically deregulated in aneuploid AML patients will guide the design of future therapeutic strategies targeting key players in the disease. Acknowledgements: European LeukemiaNet, AIRC, AIL, Prin 2010-2011, FP7 NGS-PTL project. Citation Format: Giorgia Simonetti, Antonella Padella, Viviana Guadagnuolo, Cristina Papayannidis, Francesca Volpato, Emanuela Ottaviani, Serena Formica, Annalisa Astolfi, Ilaria Iacobucci, Giovanni Capranico, Daniel Remondini, Giovanni Martinelli. Gene expression signature of aneuploidy in acute 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 2243. doi:10.1158/1538-7445.AM2014-2243

Abstract 3663: Immune suppressive myeloid cells expansion in vitro requires a simulated tumor microenvironment

Abstract Elevated levels of immune suppressor cells induced by tumor derived factors are associated with inhibition of immune responses. One such group of immune suppressor cells is Myeloid Derived Suppressor Cells (MDSCs). They are phenotypically defined in humans as HLADR low or negative and CD33, CD11b, and CD15 or CD14 positive and functionally by their ability to suppress the activation of other immune cells such as T cells. We hypothesized that MDSCs generated in vitro could be propagated but would lose their suppressive ability on the action of T cells in the absence of a simulated tumor microenvironment. MDSCs were first differentiated in vitro from normal donor peripheral blood mononuclear cells (PBMCs) using a simulated tumor microenvironment consisting of either cytokines (GMCSF and IL-6) or tumor cell lines (pancreatic adenocarcinoma SW-1990 or head and neck cancer CAL-27). The MDSCs generated in vitro suppressed T cell proliferation of autologous T cells using the carboxyfluorescein succinimidyl ester (CFSE) assay (31%, 46% and 75% suppression for cytokines, CAL-27 and SW-1990, respectively). Given the MDSCs obtained from co-culture of PBMCs with SW-1990 were the most suppressive on the proliferation of T cells, we attempted to proliferate these cells in vitro to expand the number of MDSC available for experimentation rather than having to purify MDSC from clinical samples. In vitro generated MDSC were propagated using a Rho-associated kinase (ROCK) inhibitor. Generated MDSC collected via magnetic bead isolation (CD33) were cultured with irradiated fibroblast feeder cells (± 3000 rad) in F medium [3:1 (v/v) F-12 Nutrient Mixture (Ham)-Dulbecco's modified Eagle's medium, 5% fetal bovine serum, 0.4 µg/mL hydrocortisone, 5 µg/mL insulin, 8.4 ng/mL cholera toxin, 10 ng/mL epidermal growth factor (or 10 ng/mL GMCSF), and 24 µg/mL adenine]. The ROCK inhibitor was added at 10 µM and the media was exchanged every 3 days. The generated MDSC did propagate as measured by CFSE staining of the generated MDSC; however, the generated MDSCs completely lost their ability to inhibit T cells by day 11 in culture. In conclusion, generated MDSCs can be propagated in culture but lose their ability to suppress T cells in the absence of a simulated tumor microenvironment and the presence of a Rho kinase inhibitor. Citation Format: Joseph Markowitz, Taylor R. Brooks, William E. Carson. Immune suppressive myeloid cells expansion in vitro requires a simulated tumor microenvironment. [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 3663. doi:10.1158/1538-7445.AM2014-3663

Graft Monocytic Myeloid-Derived Suppressor Cell Content Predicts the Risk of Acute Graft-versus-Host Disease after Allogeneic Transplantation of Granulocyte Colony-Stimulating Factor–Mobilized Peripheral Blood Stem Cells

Myeloid-derived suppressor cells (MDSCs) are powerful immunomodulatory cells that in mice play a role in infectious and inflammatory disorders, including acute graft-versus-host disease (GVHD) after allogeneic hematopoietic stem cell transplantation. Their relevance in clinical acute GVHD is poorly known. We analyzed whether granulocyte colony-stimulating factor (G-CSF) administration, used to mobilize hematopoietic stem cells, affected the frequency of MDSCs in the peripheral blood stem cell grafts of 60 unrelated donors. In addition, we evaluated whether the MDSC content in the peripheral blood stem cell grafts affected the occurrence of acute GVHD in patients undergoing unrelated donor allogeneic stem cell transplantation. Systemic treatment with G-CSF induces an expansion of myeloid cells displaying the phenotype of monocytic MDSCs (Linlow/negHLA-DR−CD11b+CD33+CD14+) with the ability to suppress alloreactive T cells in vitro, therefore meeting the definition of MDSCs. Monocytic MDSC dose was the only graft parameter to predict acute GVHD. The cumulative incidence of acute GVHD at 180 days after transplantation for recipients receiving monocytic MDSC doses below and above the median was 63% and 22%, respectively (P = .02). The number of monocytic MDSCs infused did not impact the relapse rate or the transplant-related mortality rate (P > .05). Although further prospective studies involving larger sample size are needed to validate the exact monocytic MDSC graft dose that protects from acute GVHD, our results strongly suggest the modulation of G-CSF might be used to affect monocytic MDSCs graft cell doses for prevention of acute GVHD.

Crosstalk between FLT3/ITD mutation and the cellular morphogens

FLT3 (fms-like tyrosine kinase 3), also known as FLK2 (fetal liver kinase 2) and STK1 (human stem cell kinase 1), belongs to the class III RTK (receptor tyrosine kinase) family and was originally cloned from hematopoietic progenitor cells. The gain-of-function mutation, FLT3/ITD (internal tandem duplication), occurs in 30% of AML (acute myeloid leukemia) and is associated with inferior clinical prognosis. TKI (tyrosine kinase inhibitor) including sorafenib and AC220 were effective in inducing remission. However, resistance to TKI was common, suggesting the targets are likely to be evolving and warrant further investigation. In this study, we made use of zebrafish embryo model to unveil the mechanism by which FLT3 mutations affect the hematopoiesis and morphogenesis in a whole organism level. Low dosage of FLT3/ITD mRNA (250pg per embryo) injection at 1-2 cell stage induced evident myeloid cells (pu.1 and l-plastin) expansion, but not affecting the erythroid cells (gata1) and hematopoietic progenitor cells (scl) at 18 hpf (hour post fertilization). Intriguingly, high dosage of FLT3/ITD mRNA (450-750pg per embryo) injection resulted in significant dorsalization phenotype (even axis duplication and double-head in a proportion of the embryos), and was effectively ameliorated by AC220 administration. FLT3/ITD embryos showed expanded and radial gsc (goosecoid) expression at shield stage (6 hpf), suggesting that the certain morphogens may be altered by FLT3/ITD. Morphogens regulating the DV (dorsal/ventral) and AP (anterior/posterior) development, such as WNT (wingless), BMP (bone morphogenetic protein), FGF (fibroblast growth factor), TGF-β (transforming growth factor beta), Hedgehog, Retinoic acid, etc., may be implicated in the FLT3 mutated AML and served as valuable therapeutic targets.

Immunosuppressive myeloid cells induced by chemotherapy attenuate antitumor CD4+ T-cell responses through the PD-1-PD-L1 axis.

In recent years, immune-based therapies have become an increasingly attractive treatment option for patients with cancer. Cancer immunotherapy is often used in combination with conventional chemotherapy for synergistic effects. The alkylating agent cyclophosphamide (CTX) has been included in various chemoimmunotherapy regimens because of its well-known immunostimulatory effects. Paradoxically, cyclophosphamide can also induce suppressor cells that inhibit immune responses. However, the identity and biologic relevance of these suppressor cells are poorly defined. Here we report that cyclophosphamide treatment drives the expansion of inflammatory monocytic myeloid cells (CD11b(+)Ly6C(hi)CCR2(hi)) that possess immunosuppressive activities. In mice with advanced lymphoma, adoptive transfer (AT) of tumor-specific CD4(+) T cells following cyclophosphamide treatment (CTX+CD4 AT) provoked a robust initial antitumor immune response, but also resulted in enhanced expansion of monocytic myeloid cells. These therapy-induced monocytes inhibited long-term tumor control and allowed subsequent relapse by mediating functional tolerization of antitumor CD4(+) effector cells through the PD-1-PD-L1 axis. PD-1/PD-L1 blockade after CTX+CD4 AT therapy led to persistence of CD4(+) effector cells and durable antitumor effects. Depleting proliferative monocytes by administering low-dose gemcitabine effectively prevented tumor recurrence after CTX+CD4 AT therapy. Similarly, targeting inflammatory monocytes by disrupting the CCR2 signaling pathway markedly potentiated the efficacy of cyclophosphamide-based therapy. Besides cyclophosphamide, we found that melphalan and doxorubicin can also induce monocytic myeloid suppressor cells. These findings reveal a counter-regulation mechanism elicited by certain chemotherapeutic agents and highlight the importance of overcoming this barrier to prevent late tumor relapse after chemoimmunotherapy.

Functions of flt3 in zebrafish hematopoiesis and its relevance to human acute myeloid leukemia

AbstractFMS-like tyrosine kinase 3 (FLT3) is expressed in human hematopoietic stem and progenitor cells (HSPCs) but its role during embryogenesis is unclear. In acute myeloid leukemia (AML), internal tandem duplication (ITD) of FLT3 at the juxtamembrane (JMD) and tyrosine kinase (TKD) domains (FLT3-ITD+) occurs in 30% of patients and is associated with inferior clinical prognosis. TKD mutations (FLT3-TKD+) occur in 5% of cases. We made use of zebrafish to examine the role of flt3 in developmental hematopoiesis and model human FLT3-ITD+ and FLT3-TKD+ AML. Zebrafish flt3 JMD and TKD were remarkably similar to their mammalian orthologs. Morpholino knockdown significantly reduced the expression of l-plastin (pan-leukocyte), csf1r, and mpeg1 (macrophage) as well as that of c-myb (definitive HSPCs), lck, and rag1 (T-lymphocyte). Expressing human FLT3-ITD in zebrafish embryos resulted in expansion and clustering of myeloid cells (pu.1+, mpo+, and cebpα+) which were ameliorated by AC220 and associated with stat5, erk1/2, and akt phosphorylation. Human FLT3-TKD (D835Y) induced significant, albeit modest, myeloid expansion resistant to AC220. This study provides novel insight into the role of flt3 during hematopoiesis and establishes a zebrafish model of FLT3-ITD+ and FLT3-TKD+ AML that may facilitate high-throughput screening of novel and personalized agents.

Heme oxygenase-1 derived carbon monoxide permits maturation of myeloid cells

Critical functions of the immune system are maintained by the ability of myeloid progenitors to differentiate and mature into macrophages. We hypothesized that the cytoprotective gas molecule carbon monoxide (CO), generated endogenously by heme oxygenases (HO), promotes differentiation of progenitors into functional macrophages. Deletion of HO-1, specifically in the myeloid lineage (Lyz-Cre:Hmox1flfl), attenuated the ability of myeloid progenitors to differentiate toward macrophages and decreased the expression of macrophage markers, CD14 and macrophage colony-stimulating factor receptor (MCSFR). We showed that HO-1 and CO induced CD14 expression and efficiently increased expansion and differentiation of myeloid cells into macrophages. Further, CO sensitized myeloid cells to treatment with MCSF at low doses by increasing MCSFR expression, mediated partially through a PI3K-Akt-dependent mechanism. Exposure of mice to CO in a model of marginal bone marrow transplantation significantly improved donor myeloid cell engraftment efficiency, expansion and differentiation, which corresponded to increased serum levels of GM-CSF, IL-1α and MCP-1. Collectively, we conclude that HO-1 and CO in part are critical for myeloid cell differentiation. CO may prove to be a novel therapeutic agent to improve functional recovery of bone marrow cells in patients undergoing irradiation, chemotherapy and/or bone marrow transplantation.

Loss of wild-type Jak2 allele enhances myeloid cell expansion and accelerates myelofibrosis in Jak2V617F knock-in mice

JAK2V617F is the most common mutation found in Philadelphia chromosome-negative myeloproliferative neoplasms (MPNs). Although a majority of MPN patients carry heterozygous JAK2V617F mutation, loss of heterozygosity (LOH) on chromosome 9p involving the JAK2 locus has been observed in ~30% of MPN patients. JAK2V617F homozygosity via 9pLOH has been associated with more severe MPN phenotype. However, the contribution of 9pLOH in the pathogenesis of MPNs remains unclear. To investigate the roles of wild-type JAK2 (JAK2 WT) and JAK2V617F alleles in the development of MPNs, we have utilized conditional Jak2 knock-out and Jak2V617F knock-in mice and generated heterozygous, hemizygous and homozygous Jak2V617F mice. Whereas heterozygous Jak2V617F expression results in a polycythemia vera-like MPN in mice, loss of Jak2 WT allele in hemizygous or homozygous Jak2V617F mice results in markedly increased white blood cells, neutrophils, reticulocytes and platelets in the peripheral blood, and significantly larger spleen size compared with heterozygous Jak2V617F mice. Hemizygous or homozygous Jak2V617F mice also exhibit accelerated myelofibrosis compared with mice expressing heterozygous Jak2V617F. Together, these results suggest that loss of Jak2 WT allele increases the severity of the MPN. Thus, the Jak2 WT allele functions as a negative regulator of MPN induced by Jak2V617F.

Hes1 upregulation contributes to the development of FIP1L1-PDGRA–positive leukemia in blast crisis

We have previously shown that elevated expression of Hairy enhancer of split 1 (Hes1) contributes to blast crisis transition in Bcr-Abl-positive chronic myelogenous leukemia. Here we investigate whether Hes1 is involved in the development of other myeloid neoplasms. Notably, Hes1 expression was elevated in only a few cases of 65 samples with different types of myeloid neoplasms. Interestingly, elevated expression of Hes1 was found in two of five samples of Fip1-like1 platelet-derived growth factor receptor-α (FIP1L1-PDGFA)-positive myeloid neoplasms associated with eosinophilia. Whereas FIP1L1-PDGFRα alone induced acute T-cell leukemia or myeloproliferative neoplasms in mouse bone marrow transplantation models, mice transplanted with bone marrow cells expressing both Hes1 and FIP1L1-PDGFRα developed acute leukemia characterized by an expansion of myeloid blasts and leukemic cells without eosinophilic granules. FIP1L1-PDGFRα conferred cytokine-independent growth to Hes1-transduced common myeloid progenitors, interleukin-3-dependent cells. Imatinib inhibited the growth of common myeloid progenitors expressing Hes1 with FIP1L1-PDGFRα, but not with imatinib-resistant FIP1L1-PDGFRα mutants harboring T674I or D842V. In contrast, ponatinib efficiently eradicated leukemic cells expressing Hes1 and the imatinib-resistant FLP1L1-PDGFRΑ mutant in vitro and in vivo. Thus, we have established mouse models of FIP1L1-PDGFRA-positive leukemia in myeloid blast crisis, which will help elucidate the pathogenesis of the disease and develop a new treatment for it.