PPAR-delta modulates membrane cholesterol and cytokine signaling in malignant B cells

Leukemia ◽  
2017 ◽  
Vol 32 (1) ◽  
pp. 184-193 ◽  
Author(s):  
L Sun ◽  
Y Shi ◽  
G Wang ◽  
X Wang ◽  
S Zeng ◽  
...  
Keyword(s):  
B Cells ◽  
Cytokine Signaling ◽  
Membrane Cholesterol ◽  
Ppar Delta

The Suppressor of Cytokine Signaling-3 Gene Is Overexpressed in Human De Novo Follicular Lymphomas and Promotes IL-3-Independent Proliferation of the BaF3 Pro-B Cell Line.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 1107-1107
Author(s):  
Jacqueline C. Barrientos ◽  
Sofya Rodov ◽  
Arthur W. Zieske ◽  
K. Gary J. Vanasse
Keyword(s):  
Cell Proliferation ◽  
B Cells ◽  
B Cell ◽  
Cell Line ◽  
Cell Lines ◽  
De Novo ◽  
Myeloid Cells ◽  
Negative Regulator ◽  
Cytokine Signaling ◽  
Socs3 Protein

Abstract The recent generation of mice lacking functional SOCS3 in hepatocytes, macrophages, and neutrophils reveals SOCS3 to be an essential regulator of IL-6 signaling via mediation of gp130-related cellular complexes, as well as a negative regulator of G-CSF signaling in myeloid cells. Although SOCS3 would appear to be a critical physiologic regulator of inflammatory responses, its possible role in hematologic malignancies and the underlying mechanisms which regulate its expression in B cells remain to be clearly defined. We previously showed that CD19+ B cells isolated from Eμ-Bcl-2 transgenic mice express high levels of SOCS3 in addition to overexpression of Bcl-2. Moreover, hematopoietic cell lines transduced to stably overexpress Bcl-2 exhibited marked induction of SOCS3 compared to controls, suggesting Bcl-2-associated pathways may play a role in the induction of SOCS3. In the current study, we describe SOCS3 overexpression limited to neoplastic follicular lymphoma (FL) cells in Bcl-2-associated human de novo FL and show that overexpression of SOCS3 is capable of stimulating cytokine-independent cellular proliferation of the BaF3 pro-B cell line. We measured SOCS3 protein levels by immunohistochemistry in paraffin-embedded biopsies from twelve patients diagnosed with de novo, untreated histologic grade I or II FL which harbored t(14;18) and Bcl-2 overexpression. In 9/12 de novo FL cases examined, immunostaining with two distinct antibodies to SOCS3 revealed marked overexpression of SOCS3 protein that, within the follicular center cell region, was limited to neoplastic FL cells and co-localized with Bcl-2 primarily in the nucleus of positive cells. In contrast, SOCS3 protein was not detected by immunostaining in germinal center follicular B cells from benign hyperplastic tonsil tissue. To further evaluate the role of SOCS3 in B cell biology, the IL-3-dependent BaF3 pro-B cell line was stably transduced with either a retroviral expression construct containing a 675bp human SOCS3 cDNA (BaF3SOCS3) or with vector only control (BaF3Δ). Whereas no SOCS3 protein was detected in control cells, high level expression of SOCS3 in transduced BaF3SOCS3 cells was confirmed by Western analysis using SOCS3 anti-sera. Furthermore, Bcl-2 protein was not detected in either BaF3SOCS3 or control cell lines. 2 x 105 BaF3SOCS3, BaF3Δ, and non-transduced BaF3 cell lines were initially grown in the presence 10% fetal bovine serum (FBS) and 5% WEHI 3B cell-conditioned medium as a source of IL-3. IL-3 was then removed by washing with DMEM/10% FBS. Cell viability was then measured by recording absorbance at 490nm using incorporation of the MTS tetrazolium compound. Interestingly, BaF3SOCS3 cells overexpressing SOCS3 did not undergo apoptosis but were able to proliferate in the absence of IL-3, with percent viable cells approaching 400% at > 96 hours, which represented the final time-point measured. In contrast, BaF3Δ and non-transduced BaF3 cells underwent apoptotic cell death between 8 and 36 hours in response to IL-3 withdrawal. Thus, SOCS3 overexpression confers IL-3-independent cell proliferation to the BaF3 cell line. These data indicate that unlike its negative regulatory effect on G-CSF signaling in myeloid cells, overexpression of SOCS3 in B cells may promote B cell proliferation rather than growth suppression and may play an important role in the pathogenesis of de novo FL in humans.

Prevention Of Minimal Residual Disease In Ph+ ALL

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 1265-1265
Author(s):  
Iris Appelmann ◽  
Elisa DeStanchina ◽  
Gregory Carbonetti ◽  
Chong Chen ◽  
Scott W. Lowe ◽  
...  
Keyword(s):  
B Cells ◽  
Minimal Residual Disease ◽  
Residual Disease ◽  
Ex Vivo ◽  
Lymphoblastic Leukemia ◽  
Philadelphia Chromosome ◽  
Janus Kinase ◽  
Response To Treatment ◽  
Cytokine Signaling ◽  
Minimal Residual

Abstract Aggressive Philadelphia chromosome-positive (Ph+) acute lymphoblastic leukemia (ALL) that genetically and phenotypically mimics the human disease can be induced by the introduction of cultured p185 (BCR-ABL)-expressing Arf-null pre/pro-B cells into healthy, unconditioned syngeneic mice. Only 20 polyclonal donor cells are sufficient to induce lethal ALL within 30 days of their IV administration, indicating that BCR-ABL expression and Arf inactivation are sufficient to guarantee leukemogenesis in healthy recipient animals. Leukemic mice enter transient remission in response to treatment with potent second generation tyrosine kinase inhibitors (TKI) such as dasatinib (SprycelTM). However, like human patients with Ph+ ALL, the continuously treated animals ultimately relapse with the emergence of leukemic clones containing clinically relevant BCR-ABL mutations, the nature of which depends upon the intensity of TKI treatment. Premature withdrawal of dasatinib when animals are in remission results in re-emergence of leukemia; surprisingly, leukemic B cells recovered from these animals lack BCR-ABL mutations and remain sensitive to dasatinib ex vivo. Hence, minimal residual disease depends upon salutary signaling within the hematopoietic microenvironment. In agreement, the response to TKI therapy can be significantly improved by abrogating cytokine signaling through a knockdown of the common gamma chain of the cytokine receptor. Administration of the Janus kinase (JAK) inhibitor ruxolitinib (Jakafi™) mimics this response. Although ruxolitinib demonstrated no anti-leukemic activity of its own, the overall survival of leukemic mice inoculated with 200,000 p185+ Arf-/- pre/pro-B cells was significantly extended after administration of a targeted combination therapy of ruxolitinib and dasatinib in comparison with mice treated with dasatinib alone. Addition of dexamethasone further reduced the leukemic burden, prevented CNS relapse, and led to prolonged survival. This implicates prevention of minimal residual disease and relapse by a non-toxic combination of targeted treatments. These studies have provided a rationale for a Phase I/II clinical trial employing these agents, particularly in older patients who are ineligible for bone marrow transplantation or do not tolerate cytotoxic chemotherapy. Disclosures: Lowe: Blueprint Medicines: Consultancy; Constellation Pharmaceuticals: Consultancy; Mirimus Inc.: Consultancy.

scholarly journals Integrated biochemical and computational approach identifies BCL6 direct target genes controlling multiple pathways in normal germinal center B cells

Blood ◽  
2010 ◽  
Vol 115 (5) ◽  
pp. 975-984 ◽  
Author(s):  
Katia Basso ◽  
Masumichi Saito ◽  
Pavel Sumazin ◽  
Adam A. Margolin ◽  
Kai Wang ◽  
...  
Keyword(s):  
B Cells ◽  
Germinal Center ◽  
Target Genes ◽  
Cytokine Signaling ◽  
Large Set ◽  
Cellular Functions ◽  
Cycle Arrest ◽  
Genome Wide ◽  
Physiologic Function ◽  
Complete Set

Abstract BCL6 is a transcriptional repressor required for mature B-cell germinal center (GC) formation and implicated in lymphomagenesis. BCL6's physiologic function is only partially known because the complete set of its targets in GC B cells has not been identified. To address this issue, we used an integrated biochemical-computational-functional approach to identify BCL6 direct targets in normal GC B cells. This approach includes (1) identification of BCL6-bound promoters by genome-wide chromatin immunoprecipitation, (2) inference of transcriptional relationships by the use of a regulatory network reverse engineering approach (ARACNe), and (3) validation of physiologic relevance of the candidate targets down-regulated in GC B cells. Our approach demonstrated that a large set of promoters (> 4000) is physically bound by BCL6 but that only a fraction of them is repressed in GC B cells. This set of 1207 targets identifies several cellular functions directly controlled by BCL6 during GC development, including activation, survival, DNA-damage response, cell cycle arrest, cytokine signaling, Toll-like receptor signaling, and differentiation. These results define a broad role of BCL6 in preventing centroblasts from responding to signals leading to exit from the GC before they complete the phase of proliferative expansion and of antibody affinity maturaton.

scholarly journals Iκb Kinase α Is Essential for Mature B Cell Development and Function

2001 ◽  
Vol 193 (4) ◽  
pp. 417-426 ◽  
Author(s):  
Tsuneyasu Kaisho ◽  
Kiyoshi Takeda ◽  
Tohru Tsujimura ◽  
Taro Kawai ◽  
Fumiko Nomura ◽  
...  
Keyword(s):  
B Cells ◽  
B Cell ◽  
Fetal Liver ◽  
Cell Development ◽  
B Cell Development ◽  
Cytokine Signaling ◽  
Iκb Kinase ◽  
And Function

IκB kinase (IKK) α and β phosphorylate IκB proteins and activate the transcription factor, nuclear factor (NF)-κB. Although both are highly homologous kinases, gene targeting experiments revealed their differential roles in vivo. IKKα is involved in skin and limb morphogenesis, whereas IKKβ is essential for cytokine signaling. To elucidate in vivo roles of IKKα in hematopoietic cells, we have generated bone marrow chimeras by transferring control and IKKα-deficient fetal liver cells. The mature B cell population was decreased in IKKα−/− chimeras. IKKα−/− chimeras also exhibited a decrease of serum immunoglobulin basal level and impaired antigen-specific immune responses. Histologically, they also manifested marked disruption of germinal center formation and splenic microarchitectures that depend on mature B cells. IKKα−/− B cells not only showed impairment of survival and mitogenic responses in vitro, accompanied by decreased, although inducible, NF-κB activity, but also increased turnover rate in vivo. In addition, transgene expression of bcl-2 could only partially rescue impaired B cell development in IKKα−/− chimeras. Taken together, these results demonstrate that IKKα is critically involved in the prevention of cell death and functional development of mature B cells.

The Hsp90 Inhibitor 17-DMAG Increases SOCS3 and Regulates Cytokine Production, Migration and Cell Death in Chronic Lymphocytic Leukemia

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 1362-1362
Author(s):  
Erin K Hertlein ◽  
Timothy L. Chen ◽  
Rainer Claus ◽  
Christoph Plass ◽  
Amy Lehman ◽  
...  
Keyword(s):  
Gene Expression ◽  
B Cells ◽  
Gene Silencing ◽  
Cell Survival ◽  
Cytokine Production ◽  
Transcriptional Silencing ◽  
Hsp90 Inhibitor ◽  
Lymphocytic Leukemia ◽  
Cytokine Signaling ◽  
Cxcr4 Signaling

Abstract Abstract 1362 Epigenetic or transcriptional silencing of important tumor suppressors has been described to contribute to cell survival and tumorigenesis in chronic lymphocytic leukemia (CLL). We investigated gene silencing in CLL using gene expression microarray analysis, and found that over 2000 genes are repressed more than 2-fold in CLL B cells compared to normal B cells, including genes involved in cell signaling and survival (Jun/Fos signaling, ATF family, cyclin dependent kinases and suppressors of cytokine signaling). In particular, the suppressor of cytokine signaling protein 3 (SOCS3) was decreased 60-fold in CLL B cells compared to peripheral blood B cells from normal donors. Despite this profound effect, few therapeutic approaches have focused on reversing this gene silencing in CLL. NF-κB has been shown to transcriptionally silence gene expression in several types of cancer, and our group has previously identified a similar role for this transcription factor in CLL. In addition, we have previously shown that the Hsp90 inhibitor 17-DMAG targets NF-κB signaling to induce apoptosis in CLL through transcriptional down-regulation of oncogenes such as MCL1 and BCL2. However, our microarray analysis revealed that treatment with 17-DMAG also leads to the re-expression of nearly 200 genes silenced in CLL compared to normal B cells. One of the genes significantly silenced in CLL and re-expressed by 17-DMAG is SOCS3. This increase in SOCS3 was evident as early as 8 hours following treatment with 17-DMAG, and peaking at 16–24 hours just prior to 17-DMAG induced cell death (up-regulated 5.6 fold at 8 hours, 59.8 fold at 16 hours, and 25.7 fold at 24 hours). The magnitude of induction in CLL cells was significantly greater than SOCS3 induction in peripheral blood B cells from normal donors, which correlates with a decreased apoptotic response of normal B cells to 17-DMAG (25.7 fold up-regulated in CLL versus 3.5 fold in normal B cells). While little is known about its regulation or functional impact in CLL, SOCS3 has been shown to be methylated in solid tumors as well as myeloid leukemia, leading to aberrant cytokine production and cell survival. While it is known that promoter hypermethylation and subsequent gene silencing contributes to CLL disease progression, we found that there was no significant methylation of the SOCS3 promoter in CLL compared to normal B lymphocytes, indicating an alternative mechanism of SOCS3 silencing in CLL. In order to further characterize the downstream effect of SOCS3 regulation, we investigated the pathways known to be regulated by this protein, specifically IL-6 and CXCR4 signaling. We found that 17-DMAG prevents phosphorylation of STAT3 induced by IL-6 stimulation, which leads to decreased production of pro-survival cytokines including negative feedback by decreasing IL-6 itself. While STAT3 is a known Hsp90 client protein, the effect on phosphorylation of STAT3 was evident before a decrease in the total protein was observed, indicating a distinct effect on the signaling pathway independent of Hsp90's role as a protein chaperone. SOCS3 has also been shown to prevent phosphorylation of focal adhesion kinase (FAK) and therefore block both integrin and CXCR4 signaling pathways. We found that 17-DMAG prevents constitutive phosphorylation of FAK in primary CLL cells, and subsequently reduces AKT phosphorylation following recombinant SDF-1 stimulation. In order to determine if 17-DMAG inhibits migration of CLL cells towards recombinant SDF-1 as well as the direct signaling through the CXCR4 receptor, we performed transwell migration assays and found that 17-DMAG significantly inhibits migration towards both recombinant SDF-1 and CXCL13 (migration towards CXCL12, 12.4% with Vehicle vs. 8.9% with 17-DMAG, p=0.0061, towards CXCL13, 12.4% with Vehicle vs. 6.1% with 17-DMAG, p<0.0001). Similar results were obtained by over-expression of SOCS3 in a CLL B cell line, suggesting that 17-DMAG inhibits migration through an increase in SOCS3. Based on these results, we suggest that 17-DMAG reverses gene silencing in CLL, and through re-expression SOCS3 inhibits the migration and signaling associated with SDF-1/CXCR4, an important factor in the tumor microenvironment that contributes to CLL cell survival. Therefore Hsp90 inhibitors represent a novel approach to target transcriptional silencing in CLL and other B cell lymphoproliferative disorders. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Constitutive Expression of the B7h Ligand for Inducible Costimulator on Naive B Cells Is Extinguished after Activation by Distinct B Cell Receptor and Interleukin 4 Receptor–mediated Pathways and Can Be Rescued by CD40 Signaling

2002 ◽  
Vol 196 (1) ◽  
pp. 97-108 ◽  
Author(s):  
Linda Liang ◽  
Evelyn M. Porter ◽  
William C. Sha
Keyword(s):  
B Cells ◽  
B Cell ◽  
Constitutive Expression ◽  
Cell Receptor ◽  
B Cell Receptor ◽  
Cytokine Signaling ◽  
Mrna Levels ◽  
Down Regulation ◽  
Inducible Costimulator ◽  
Activated B Cells

The recently described ligand–receptor pair, B7h–inducible costimulator (ICOS), is critical for germinal center formation and antibody responses. In contrast to the induced expression of the related costimulatory ligands B7.1 and B7.2, B7h is constitutively expressed on naive B cells and is surprisingly extinguished after antigen engagement and interleukin (IL)-4 cytokine signaling. Although signaling through both B cell receptor (BCR) and IL-4 receptor (R) converge on the extinction of B7h mRNA levels, BCR down-regulation occurs through Ca2+ mobilization, whereas IL-4R down-regulation occurs through a distinct Stat6-dependent pathway. During antigen-specific B cell activation, costimulation through CD40 signaling can reverse both BCR- and IL-4R–mediated B7h down-regulation. These data suggest that the CD40–CD40 ligand signaling pathway regulates B7h expression on activated B cells and may control whether antigen-activated B cells can express B7h and costimulate cognate antigen–activated T cells through ICOS.

scholarly journals Global Transcriptional States of Follicular Lymphoma B Cells Highlight Distinct Groups of Tumor Identity Associated with Somatic Alterations and Tumor Microenvironment

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 21-22
Author(s):  
Jordan Krull ◽  
Kerstin Wenzl ◽  
Michelle Manske ◽  
Melissa Hopper ◽  
Melissa C. Larson ◽  
...  
Keyword(s):  
B Cells ◽  
B Cell ◽  
Board Of Directors ◽  
Mayo Clinic ◽  
Copy Number ◽  
Research Funding ◽  
Copy Number Variants ◽  
Cytokine Signaling ◽  
Advisory Committees ◽  
Gene Set

Background: Follicular Lymphoma (FL) is the second most common non-Hodgkin lymphoma and presents with significant clinical, cellular, molecular, and genetic heterogeneity. Despite the name and defining microanatomic location, the genetic and molecular identity and pathogenesis of the FL tumor cell is largely uncharacterized. Prior clinical and molecular classifications of FL have been primarily driven by pathologic classification (Grade 1-3b), genetic classification (M7-FLIPI), or gene expression profiling (IR-1/2). Using a unique cohort of 93 FL tumors, we have explored the transcriptomic signature of purified FL B cells, along with their matched whole tumor, and identified unique molecular subsets which are defined by distinct pathway activation, immune content, and genomic signatures identified through whole exome sequencing (WES). Methods: Frozen tumor biopsies from 93 untreated FL (Grade 1-3b) patients enrolled in the University of Iowa/Mayo Clinic Lymphoma SPORE were used for the study. DNA was isolated from whole tumor cell suspensions and RNA was isolated from both whole tumor and B cell enriched cell suspensions. RNA sequencing (RNAseq) and WES was performed in the Mayo Clinic Genome Analysis Core. RNAseq and WES data were processed using the Mayo Clinic standard pipeline and novel driver genes were identified using 20/20+ driver analysis. Copy number variants were identified using GISTIC 2.0. NMF clustering and single sample gene set testing, for B cell lineage and tumor microenvironment (TME) signatures, was performed in R using the NMF and SingScore packages. Results: NMF consensus clustering of FL B cell RNAseq data identified two distinct subsets, C1 (n=32) and C2 (n=57). Clinically, C1 was associated with being FL grade 3 (p&lt;0.001) and more aggressive therapy (e.g. immunochemotherapy) at diagnosis while other clinical factors were similar between the groups. To determine the biologic underpinnings that drove the NMF analysis, the top NMF metagenes were incorporated (n=389 for C1 and n=293 for C2) for GO analysis. Among those genes were AICDA, IRF4, and EZH2 in C1, and BCL2 in C2. Additionally, we identified significant gene set enrichment with NMF metagenes and calculated differential gene set expression comparing C1, C2, and normal B cells (n=5). Together, these analyses suggest C1 displays significantly (p&lt;0.05) enhanced DNA replication and metabolic activity, whereas C2 displays significantly enhanced cytoskeleton rearrangement, repressed inflammatory cytokine signaling, and NF-kB activity. Based on recent scRNAseq data suggesting that FL derives from distinct germinal center B cells, the B lineage difference between C1 and C2 was explored. Both subgroups were heterogeneous for dark (DZ), intermediate (INT), light (LZ) zones, and plasmablast (PBL) gene signatures. When compared to benign B cell samples, C1 was enriched for a pre-PBL (p&lt;0.01), and C2 for an INT phenotype related to a LZ signature (p&lt;0.01). To define if a genomic signature was associated with C1 and C2, mutations and copy number variants (&gt;5% frequency) were examined. TNFAIP3, TP53, and BCL6 alterations were enriched in C1 samples, whereas C2 associated with alterations in BCL2, KMT2D, CREBBP, REL, and MYC. Finally, B cell clusters were analyzed for TME signatures. C1 samples displayed significant enrichment of macrophage, cytotoxic cell, gamma-delta-Tcell, and endothelial cell TME elements (p&lt;0.05) consistent with an inflamed/hot tumor. In contrast, C2 samples exhibited significantly lower signatures (p&lt;0.05) in all entities except for the fibroblast signature, compared to C1 and normal, consistent with an immune desert phenotype. Conclusion: Our results suggest that B cells from FL patients display two distinct transcriptomic signatures. C1 identifies an immunologically active tumor, driven by TNFAIP3 alterations, with pre-PBL characteristics, DNA replication and repair, inflammatory cytokine secretion/signaling, and hyper-metabolic characteristics. C2 identifies an immunologically quiet tumor, driven by alterations in BCL2 and chromatin modifiers, with an intermediate GC phenotype, repressed cytokine signaling, and active cell cycle progression and cytoskeleton rearrangement. This study improves our understanding of the mechanisms driving FL tumors and motivates further investigation into the relationship between tumor intrinsic factors that may influence the TME. Disclosures Maurer: Morphosys: Membership on an entity's Board of Directors or advisory committees; Kite: Membership on an entity's Board of Directors or advisory committees; Pfizer: Membership on an entity's Board of Directors or advisory committees; Nanostring: Research Funding; Celgene / BMS: Research Funding. Ansell:ADC Therapeutics: Research Funding; Trillium: Research Funding; Affimed: Research Funding; Regeneron: Research Funding; AI Therapeutics: Research Funding; Takeda: Research Funding; Seattle Genetics: Research Funding; Bristol Myers Squibb: Research Funding. Cerhan:NanoString: Research Funding; BMS/Celgene: Research Funding. Novak:Celgene/BMS: Research Funding.

Inactivation of the Arf Tumor Suppressor in Mouse BCR-ABL(+) B Cells Greatly Increases the Frequency of Leukemia-Initiating Cells and Confers Imatinib Resistance In Vivo.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 385-385
Author(s):  
Richard T. Williams ◽  
Charles J. Sherr
Keyword(s):  
Bone Marrow ◽  
B Cells ◽  
Tumor Suppressor ◽  
Leukemic Cells ◽  
Cytokine Signaling ◽  
Imatinib Resistance ◽  
Abl Kinase ◽  
Donor Cells ◽  
The Impact

Deletion of the intimately linked INK4A and ARF tumor suppressor genes (CDKN2A) frequently occurs in BCR-ABL-induced [Ph+] acute lymphoblastic leukemia (ALL) and correlates with their poor therapeutic response. Although BCR-ABL cancels the dependence of cultured primary murine pre-B cells on interleukin-7 (IL-7), it efficiently induces an Arf- and p53-dependent checkpoint that limits further cell expansion. In contrast, immortal Arf-null pre-B cells resist BCR-ABL-induced apoptosis. Thus, the combination of BCR-ABL expression and Arf inactivation confers synergistic properties of cytokine independence, resistance to apoptosis, and limitless replicative potential. To determine the impact of Arf inactivation on ALL development, we infected unconditioned bone marrow cells from Arf+/+ or Arf-null mice with retroviral GFP-encoding vectors co-expressing either the p185BCR-ABL or p210BCR-ABL isoforms (hereafter p210 and p185). After short-term culture (8 days) under conditions that select for the outgrowth of pre-B cells, BCR-ABL(+) cells of both Arf genotypes were infused intravenously into immunocompetent syngeneic mice. Only Arf-null donor cells induced lympholeukemias. Remarkably, while 2 ×105Arf+/+ p210+ or p185+ pre-B cells failed to establish leukemias in recipient mice, as few as 20 Arf-null p185+ cells generated lethal lympholeukemias within 30 days. Signs of highly aggressive disease included splenomegaly, significant replacement (60–90%) of bone marrow with GFP(+) cells, and the emergence of numerous circulating GFP(+) lymphoblasts (up to 105 per ul). Leukemic cells recovered from the bone marrow of moribund animals could be continuously cultured in vitro and maintained their pre-B cell immunophenotype and leukemogenic potential when retransplanted. Thus, in comparison to BCR-ABL(+), Arf+/+ donor pre-B cells, their Arf-null counterparts are at least 104-fold enriched in leukemia-initiating cell activity. Cultured BCR-ABL(+), Arf-null pre-B cells are exquisitely sensitive to the cytostatic effects of the BCR-ABL kinase inhibitor, imatinib. However, mice inoculated with as few as 200 BCR-ABL(+), Arf-null donor cells failed oral high-dose imatinib therapy (100 mg/kg bid). Equally surprisingly, tumor cells recovered from treated, moribund mice retained the same sensitivity to the drug (IC50 < 150 nM) as the original donor cells, indicating that imatinib resistance in this setting is tumor cell-extrinsic and does not depend upon mutations or amplification of the BCR-ABL kinase. Imatinib restores the requirement of BCR-ABL(+) cells for IL-7, and conversely, exposure to saturating levels of IL-7 can significantly reduce imatinib sensitivity. Therefore, host-derived factors, such as IL-7, within hematopoietic tissues may rescue Arf-null leukemic cells from imatinib-mediated growth inhibition. Selective inhibition of Janus (JAK) kinases that transduce signals from cytokines like IL-7 can restore imatinib sensitivity in vitro, suggesting that targeted inhibition of cytokine signaling might play an adjunctive role in therapy of imatinib-refractory BCR-ABL-induced ALL.

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