Acute Leukemia Blasts Research Articles

Morphological characteristics of leukemic blasts in acute leukemia in a patient of Down syndrome following transient abnormal myelopoiesis

Morphological characteristics of leukemic blasts in acute leukemia in a patient of Down syndrome following transient abnormal myelopoiesis

A single-chain triplebody with specificity for CD19 and CD33 mediates effective lysis of mixed lineage leukemia cells by dual targeting

A single-chain triplebody (sctb) 33-ds16-ds19 comprising two distal single-chain Fv fragments (scFvs) specific for the lymphoid antigen CD19 and the myeloid antigen CD33 flanking a central scFv specific for CD16, which is the low affinity Fc-receptor (FcγRIII) present on natural killer cells and macrophages, was produced and its properties were investigated. CD33 and CD19 in combina-tion are present on acute leukemiablasts with mixed lineage phenotype, but not on normal human hematopoietic cells. For comparison, two bispecific scFvs (bsscFvs), ds19-ds16 and 33-ds16, with monovalent binding to CD19 and CD33, respectively, were also studied. The sctb 33-ds16-ds19 specifically interacted with all 3 antigens. On the antigen double-positive cell line BV-173, the sctb bound with 2-fold greater avidity than bsscFv ds19-ds16 (KD = 21 vs. 42 nM) and with 1.4-fold greater avidity than bsscFv 33-ds16 (KD = 29 nM). All 3 fusion proteins had similar affinity for CD16 and sufficient thermic stability in human serum. In antibody-dependent cellular cytotoxicity (ADCC) reactions with human mononuclear cells as effectors, the sctb promoted lysis of BV-173 cells at 23-fold lower concentrations than bsscFv ds19-ds16 and at 1.4-fold lower concentrations than bsscFv 33-ds16. The sctb also mediated potent ADCC of the antigen double-positive mixed lineage leukemia cell line SEM, and the half-maximal concentration EC50 for BV-173 cells was 7 pM. Therefore, CD19 and CD33 are present on the surface of these leukemic cell lines such that they can be connected by a single sctb molecule, permitting the recruitment of NK cells via CD16 and tumor cell lysis.

Increased CCL2 and IL‐8 in the bone marrow microenvironment in acute lymphoblastic leukemia

The interactions of acute lymphoblastic leukemia (ALL) blasts with bone marrow (BM) stromal cells have a positive impact on leukemia cell survival. In the present study, we proposed to identify and investigate the role of molecules critically involved in leukemia--microenvironment crosstalk. Gene expression profiling analyses of BM mesenchymal stem cells (BMMSC) were performed following stimulation by ALL cells. CCL2 and IL-8 plasma levels were evaluated from ALL patients and controls. Expression of the CCL2 and IL-8 receptors in ALL was determined by RT-PCR. The biological effects of CCL2, IL-8 or its neutralizing antibodies in primary precursor-B ALL and BMMSC cells were evaluated using in vitro assays. Leukemia stimulation of BMMSC upregulated the expression of several inflammatory chemokines, including CCL2 and IL-8. The BM plasma levels of CCL2 and IL-8 in children at diagnosis were significantly higher than in healthy controls (P < 0.001). Functional studies revealed that CCL2 and IL-8 enhanced the capacity of BMMSC to support adhesion of ALL cells. CCL2 and IL-8 were also found to enhance BMMSC survival and to increase their proliferation. ALL cells were not directly affected by CCL2 or IL-8. The leukemic BM microenvironment had increased levels of CCL2 and IL-8. These chemokines are known to have suppressive effects in normal hematopoiesis. Our data indicate that CCL2 and IL-8 have a positive impact on BMMSC survival, proliferation, and adhesiveness to ALL cells. Leukemia-associated CCL2 and IL-8 upregulation may represent one possible mechanism of microenvironment perversion in favor of ALL cells.

A Pilot Study of the Combination of Nilotinib and Hyper-CVAD for Philadelphia Chromosome Positive Acute Lymphocytic Leukemia and Lymphoid Blast Crisis Chronic Myelogenous Leukemia

AbstractAbstract 2144 Background:Nilotinib is a second generation bcr-abl tyrosine kinase inhibitor (TKI) that has at least 30 times the potency of Imatinib (IM). Nilotinib has been approved by the FDA for the treatment of adults with chronic myeloid leukemia (CML) in chronic phase (CP) in the frontline, and for CP or accelerated phase (AP) who are either resistant or intolerant to prior therapies, including IM. Philadelphia chromosome positive (Ph+) acute lymphocytic leukemia (ALL) and lymphoid blast crisis of CML (Ly-BC) is aggressive and rapidly fatal. Complete remissions rates are low and have short durability. Relapse is inevitable unless patients (pts) receive an allogeneic stem cell transplant (SCT). The use of TKIs against Ph+ ALL or Ly-BC has shown limited durability of response as single agents. The transient nature of response to TKI is because only some of the blasts are still sensitive to bcr-abl inhibition, while the rest of the clones are not and require the addition of chemotherapy. Hyper-CVAD combined with the first generation TKI, IM is one such chemotherapy regimen that has been used in the management of adults with Ph+ ALL or Ly-BC (Thomas D, et al. Blood, 2004). Despite achieving >90% complete remission (CR) during induction phase, some pts have resistant disease or relapse shortly after attaining remission. We hypothesized that combining nilotinib with hyper-CVAD would achieve greater rates of sustained CR which ultimately translate into lower relapse rates, superior disease free survival, long term survival, and potential cure post SCT. The goal of our pilot case series was to study the feasibility of combining the more potent TKI, nilotinib to hyper-CVAD in pts with Ph+ ALL or Ly-BC, so as to improve the efficacy. Methods:Our case series involves 5 pts at our institution diagnosed with either Ph+ ALL or Ly-BC treated with a combination of hyper-CVAD and nilotinib given orally 400mg twice daily (initiated the day after completion of chemotherapy until the start of next cycle). After completion of eight cycles of chemotherapy, pts received either SCT or maintenance POMP in combination with nilotinib. Supportive care included prophylactic antibiotics, granulocyte colony stimulating factor, and transfusions as needed. None of them had central nervous system (CNS) involvement, but received intrathecal prophylaxis. Clinical characteristics at baseline, safety assessments, and efficacy evaluations including morphologic, cytogenetic, and molecular responses were analyzed. Results:All 5 pts (p190 Ph+ ALL-4, p210 Ly-BC-1; all males; Hispanic-2, Caucasian-3) received the combination for a median of 5 months. The median age was 29 (range 22–72) and the median follow-up period was 14 months. The Ly-BC pt was treated upfront with IM during CP and achieved major molecular remission (MMR) for 39 months, however due to noncompliance, relapsed with progression. All 5 patients achieved complete remission (CR), 60% after the first course. All of them achieved complete cytogenetic remission (CCyR) while 4 achieved MMR. Of the 2 Ph+ ALL pts who completed eight cycles, 1 received SCT and is in MMR for 20 months, while the other pt remains in MMR on POMP-nilotinib maintenance for over 19 months. Another pt is in MMR for 5 months. Two patients relapsed and died: the Ly-BC pt who achieved CR (without MMR) for 3 weeks and another pt that was in CCyR and MMR for 11 months. Most common adverse events included pancytopenia, fever, and infections (with no treatment related life threatening events). Conclusions:Our pilot study is the first to demonstrate the feasibility of combining nilotinib with hyper-CVAD. The results thus far are encouraging and will be expanded into a larger investigator initiated phase II study that can potentially prevent the emergence of resistant clones and improve survival in patients with Ph+ ALL or Ly-BC.Table 1ParameterHyper-CVAD + NilotinibNo. treated5Performance, ECOGn (%)04 (80%)1 to 21 (20%)WBC count at diagnosisn (%)30 or higher2 (40%)Platelet count at diagnosisn (%)Lower than 503 (60%)Responsen (%)CR5 (100%)Resistant0 (0%)Death2 (40%)Courses to CRn (%)13 (60%)21 (20%)More than 21 (20%)Adverse Events (Grade)n (%)Pancytopenia (4)5 (100%)FUO (2)2 (40%)Bacteremia (3)2 (40%)PNA (3)1 (20%)Cellulitis (2)1 (20%)Transaminitis (2)2 (40%) Disclosures:Off Label Use: Pilot study of Nilotinib and hyper-CVAD in Ph+ ALL.

Activation of Acute Lymphocytic Leukemia Blasts with CpG ODN, IL-4 and CD40 Ligand Facilitates Enhanced Anti-Leukemic CTL Responses

AbstractAbstract 3907Acute lymphoblastic leukemia (ALL) is the most common pediatric malignancy. Although the vast majority of patients initially respond to up-front chemotherapy, relapses occur in approximately 20% of cases and have a poor prognosis. Thus, novel therapeutic strategies are required to treat minimal residual disease and improve long-term survival. B cell precursor (BCP)-ALL cells express low levels of costimulatory and antigen-presenting molecules and therefore are poorly recognized by the immune system. Previous reports show that CpG oligodeoxynucleotides (CpG) can induce immunogenicity of non-Hodgkin's lymphomas including B-CLL, and that CpG may decrease BCP-ALL tumor burden in a murine xenograft model. In the present study we investigated the effect of various combinations of known potent B cell stimulators including CpG, Interleukin (IL)-2 family cytokines and CD40 ligand (CD40L) on the immunogenicity of both primary BCP-ALL cells and a series of established BCP-ALL cell lines. Using combinations of CpG, IL-4 and CD40L we were able to enhance expression of CD40, CD54, CD86 and MHC class II on BCP-ALL cells, resulting in an increased capacity of BCP-ALL cells to induce proliferative T cell responses and to generate cytotoxic T cells (CTLs). Of note, these CTLs exhibited significantly enhanced anti-leukemic cytotoxicity not only towards treated but also towards untreated BCP-ALL cells, with similar effects found in both allogeneic and syngeneic settings. The strongest effects were observed after exposure of BCP-ALL cells to a combination of CpG, IL-4 and CD40L, whereas untreated control BCP-ALL cells induced minimal T-cell proliferation and cytotoxicity only. Importantly, CpG in combination with IL-4 and CD40L was significantly more effective than CpG alone. Our results demonstrate that combined treatment with CpG, IL-2 family cytokines and CD40L is more efficient than CpG alone in inducing an immunogenic phenotype in BCP-ALL cells. Further studies using a humanized leukemia mouse model may open novel therapeutic approaches in the management of BCP-ALL, for example by using adoptive T cell transfer after in-vitro stimulation of autologous T cells with activated BCP-ALL cells. Disclosures:No relevant conflicts of interest to declare.

Tim-3, a Novel Immune Receptor, Is Constitutively Expressed on Human Natural Killer Cells and Functions as An Activating Coreceptor

AbstractAbstract 106NK cells are an attractive option for immunotherapy as they do not require pre-sensitization for anti-tumor activity and do not induce graft versus host disease (GvHD) in an allogeneic transplant setting. The potential of NK cells in controlling human hematological malignancies has been increasingly recognized in recent years, as the adoptive transfer of alloreactive NK cells in hematopoietic cell transplantation (HCT) clinical trials have demonstrated therapeutic anti-leukemia effects. NK cell function is regulated by the integration of antagonist signals received from cell surface activating and inhibitory receptors. Tim-3 is a novel immune receptor that is a member of the T cell immunoglobulin and mucin-containing domain (TIM) family of glycoproteins. While its role in T cells and antigen presenting cells has been described, little is known about its function in human NK cells. While Tim-3 is present on a variety of immune cells, resting NK cells constitutively express Tim-3 compared to other lymphocyte populations (NK: 73±3%; NKT: 6±1%; T: 1±1%; n=14) and we hypothesized that Tim-3 may be important in mediating NK cell function. The unique subset of cytokine producing CD56Bright NK cells exhibited significantly lower resting Tim-3 expression compared to CD56Dim NK cells (53±3% vs. 75±3%; p<0.001, n=14). Distinct Tim-3 expression patterns were found on resting CD56Dim NK cells and activation with low dose IL-12 (1ng/mL) and IL-18 (10ng/mL), intended to more closely mimic physiologic conditions, resulted in further differentiation of this unique expression pattern dividing NK cells into 4 distinct populations: Tim-3 was homogeneously up-regulated on all CD56Bright NK cells after activation while CD56Dim NK cells were further stratified into 3 defined populations with Tim-3hi, Tim-3lo and Tim-3neg expression. The only identified ligand of Tim-3 is galectin-9 (Gal-9), a β-galactoside binding lectin, which is expressed on a wide range of healthy and malignant cells. To investigate the potential function of Tim-3, an expression vector containing human Gal-9 was transduced into K562 and Raji cells, both without endogenous Gal-9 expression. Resting NK cytotoxicity (51Cr release) was found to be increased in the presence of Gal-9 compared to the non-Gal-9 expressing targets [E:T=0.7:1, K562 vs. K562-Gal-9: 25±3% vs. 33±3% (n=8, p<0.05); E:T=20:1, Raji vs. Raji-Gal-9: 8±1% vs. 17±2% (n=4, p<0.05)]. Analysis of CD107a degranulation showed that resting Tim-3+ CD56Bright cells were more functional against Gal-9 expressing targets than Tim-3− CD56Bright cells, suggesting that Tim-3 might also play a role in IFN-γ production. To further investigate this, resting NK cells were activated with low-dose IL-12/IL-18 overnight and IFN-γ levels were measured in response to soluble rhGal-9 (0, 2.5, 5, 10 and 20nM). Exposure to soluble rhGal-9 alone without IL-12/IL-18 did not induce IFN-γ production. For both the CD56Bright and CD56Dim IL-12/IL-18 activated NK populations, only Tim-3+ NK cells displayed a dose dependent increase in IFN-γ production upon exposure to soluble rhGal-9 compared to Tim-3− NK cells. To understand the relevance of the distinct Tim-3 populations circulating in resting blood, CD56Bright, CD56Dim/Tim-3hi, CD56Dim/Tim-3lo and CD56Dim/Tim-3neg populations were sorted, cultured overnight in IL-12/IL-18 and exposed to soluble rhGal-9. Results showed the Tim-3 expressing populations contain the predominant IFN-γ producing cells that were responsive to rhGal-9 (results for the sorted CD56Dim/Tim-3lo population shown in the figure below). This increase in IFN-γ production within the Tim-3 expressing NK cell populations was abrogated by the addition of β-lactose, a β-galactoside that binds and blocks Gal-9 activity. Lastly, Western blot and immunohistochemistry analysis of human primary acute leukemia blasts revealed high Gal-9 expression. As the presence of ligands for NK cell activating receptors on tumors provide an important prerequisite for NK cell activation and effector function, we show a novel functional role for the receptor Tim-3 in human NK cell biology in the presence of its ligand Gal-9. We, therefore, propose a model where constitutively expressed Tim-3 is up-regulated by NK cell activation and effector function is enhanced by Tim-3/Gal-9 interaction, which may potentiate the elimination of Gal-9 positive tumors by NK cells. [Display omitted] Disclosures:Niki:GalPharma: Membership on an entity's Board of Directors or advisory committees. Hirashima:GalPharma: Membership on an entity's Board of Directors or advisory committees.

Genome Wide Screening Reveals Distinct Signaling Pathways of the ETS Transcription Factor ERG In Primary Acute Leukemia Blasts

AbstractAbstract 4184 Introduction:The Ets related gene, ERG, encodes a transcription factor with a crucial role in hematopoiesis. ERG rearrangements are associated with subtypes of myeloid leukemia, Ewing sarcoma and prostate carcinoma. Overexpression of ERG predicted adverse outcome in patients with acute T-lymphoblastic leukemia (T-ALL) and acute myeloid leukemia (AML). Recently, it was shown that a single ERG missense mutation in mice produced severe defects in embryonic hematopoiesis and adult stem cell maintenance. The significance of ERG signaling contributing to this disadvantageous phenotype in mice as well as in patients with acute leukemia is unknown. Thus, a genome wide screen to identify targets involved in ERG signaling was conducted. Methods:Chromatin immunoprecipitation on chip (ChIP-chip) was carried out in 5 primary AML samples (new diagnosed AML A – E), one primary T-ALL sample and one normal bone marrow sample. ChIP-chip was performed on all samples using an ERG-specific antibody and unspecific IgG antibody. In addition, ChIP-chip was conducted in the HL60 cell line (that lack ERG product) as a negative control. Duplicate ChIP-chips were performed for each immunoprecipitation. Subsequently, the ERG enriched DNA was co-hybridized to total chromatin on a human promoter array. The array represents ∼35.000 human gene loci representing 11.000 independent promoters. Following normalization, a class comparison was carried out between the potential ERG targets against non-targets identified with IgG ChIP-chip. Results:Significantly enriched genes with a P-value ≤ 0.05 included 164 genes from AML A, 1053 genes from AML B, 829 genes from AML C, 4112 genes from AML D, 895 genes from AML E, 1266 genes from T-ALL, and 1941 genes from normal bone marrow ChIP-chips. Functional clustering by gene ontology (DAVID) of significantly ERG enriched gene sets revealed overrepresentation of pathways involved in oncogenesis, proliferation and cell signaling including: the MAPK signaling pathway (KEGG pathway analysis) was enriched in two AML ChIP-chips (AML B, P= 0.007; AML D, P= 0.003) and in the normal bone marrow ChIP-chip (P= 0.02). Moreover, the Ras GTPase protein domain (INTERPRO) was enriched in three of the five AML samples (AML B, P=0.04; AML C, P= 0.02; AML D, P= 0.04). Both, MAPK pathway and Ras GTPase protein domains were not enriched in the HL60 negative control. Furthermore, proliferation assays were performed in an ERG tet-on inducible leukemia cell line system to explore functional consequences of ERG overexpression. In stably transfected cells with potent ERG overexpression a significantly higher proliferation rate was observed compared to non-induced cells. Conclusion:By genome wide ChIP-chip in primary leukemic samples, potential ERG downstream signaling pathways were revealed. Furthermore these data suggest that the ERG induced proliferation induction may be mediated by cellular programs including the MAPK pathway. Further validation of these findings will define the role of ERG involvement in the cellular signaling cascades and provide insights for molecular based treatment approaches to improve outcome for high risk leukemia characterized by high ERG expression. Disclosures:No relevant conflicts of interest to declare.

Bortezomib Interacts Synergistically with Belinostat to Induce Apoptosis In Human Acute Myeloid and Lymphoid Leukemia Cells

AbstractAbstract 3266Previous studies have demonstrated interactions between histone deacetylase (HDAC) and proteasome inhibitors (PIs) in multiple myeloma, NHL, and CLL. However, exploration of this strategy in acute leukemias has been more limited. In this context, we have previously demonstrated that HDACIs activate the cytoprotective NF-κB pathway in acute myeloid leukemia (AML) cells, and that interruption of this process dramatically increases lethality. Such findings raise the possibility that PIs, which block degradation of the NF-κB-inhibitory protein IκBα, may act via an analogous mechanism in acute leukemias. Consequently, interactions between the clinically relevant pan-HDAC inhibitor belinostat (PXD-101) and the FDA-approved proteasome inhibitor bortezomib were evaluated in both continuously cultured cell lines and primary AML and acute lymphoid leukemia (ALL) samples. First, whereas each agent individually displayed only modest toxicity, co-treatment for 24 hr or 48 hr with low concentrations of bortezomib (3 - 5 nM) and belinostat (50 - 300 nM) led to pronounced increases in apoptosis in diverse human acute leukemia cell lines (e.g., AML, U937, HL-60, MV-4-11/Flt3-ITD; T-cell ALL, Jurkat; B-cell ALL, SEM). Interactions between these agents were determined to be synergistic by Median Dose Effect analysis. Significantly, equivalent interactions were observed in multiple primary AML (n = 4) and ALL (n = 3) blast specimens, while largely sparing normal CD34+ hematopoietic cells isolated from umbilical cord blood (n = 4), as determined by annexin V/PI, DiOC6, and/or 7-AAD uptake by flow cytometry. Western blot analysis demonstrated that co-exposure of primary leukemia blasts to bortezomib and belinostat resulted in marked increase in PARP cleavage, compared with each agent administrated alone. In addition, cell morphology exhibited classical features of apoptosis in primary acute leukemia blasts, but not in normal CD34+ cells, following combination treatment. Second, in both cell lines and primary blasts, administration of bortezomib resulted in accumulation of the phosphorylated (S32/S36) form of IκBα, accompanied by diminished belinostat-mediated hyperacetylation (K310) of RelA/p65. Bortezomib also blocked processing of the precursor p100 into the active p52, an event enhanced by co-treatment with belinostat. These results indicate that a regimen combining bortezomib and belinostat interrupts both canonical and non-canonical NF-κB signaling pathways in acute leukemia cells. Moreover, co-exposure to these agents diminished expression of NF-κB-dependent pro-survival proteins including Bcl-xL, XIAP, and SOD2, but not NF-κB-independent anti-apoptotic proteins such as survivin. Third, because the BH3-only Bcl-2 family pro-apoptotic protein Bim plays an important role in the lethality of PIs or HDACIs as single agents, the expression and functional role of Bim in bortezomib/belinostat interactions was examined. Notably, whereas treatment with marginally toxic concentrations of either agent alone clearly increased Bim protein levels, co-exposure of either leukemia cell lines or primary blasts to bortezomib and belinostat led to sharply increased Bim expression (particularly the BimEL isoform). Importantly, shRNA knock-down of Bim substantially attenuated lethality mediated by co-treatment with bortezomib and belinostat in both AML (U937) and ALL (Jurkat) cells, supporting the notion that up-regulation of Bim plays a critical role in anti-leukemic activity of the combination regimen. Lastly, exposure of cultured leukemia cells and primary blasts to belinostat ± bortezomib induced hyperacetylation of a-tubulin, indicating inhibition of HDAC6, a microtubule-associated deacetylase that regulates aggresome formation and cell survival in response to misfolded protein-induced stress. Together, these findings indicate that the regimen combining belinostat and bortezomib is highly active against human AML and ALL cells, including primary leukemic blasts, in association with perturbation in the balance between pro-survival (NF-κB-dependent) and pro-death (e.g., Bim) signals. They also suggest that this strategy warrants further attention in acute leukemias. Accordingly, a Phase I trial of belinostat and bortezomib in patients with refractory acute leukemia or MDS has recently been initiated. Disclosures:Off Label Use: Investigational use of belinostat and bortezomib.

Natural Killer Cell Killing of Acute Myelogenous Leukemia and Acute Lymphoblastic Leukemia Blasts by Killer Cell Immunoglobulin-Like Receptor–Negative Natural Killer Cells after NKG2A and LIR-1 Blockade

Although the study of natural killer (NK) cell alloreactivity has been dominated by studies of killer cell immunoglobulin-like receptors (KIRs), we hypothesized that NKG2A and LIR-1, present on 53% +/- 13% and 36% +/- 18% of normal NK cells, respectively, play roles in the NK cell killing of primary leukemia targets. KIR(-) cells, which compose nearly half of the circulating NK cell population, exhibit tolerance to primary leukemia targets, suggesting signaling through other inhibitory receptors. Both acute myelogenous leukemia and acute lymphoblastic leukemia targets were rendered susceptible to lysis by fresh resting KIR(-) NK cells when inhibitory receptor-major histocompatibility class I interactions were blocked by pan-HLA antibodies, demonstrating that these cells are functionally competent. Blockade of a single inhibitory receptor resulted in slightly increased killing, whereas combined LIR-1 and NKG2A blockade consistently resulted in increased NK cell cytotoxicity. Dual blockade of NKG2A and LIR-1 led to significant killing of targets by resting KIR(-) NK cells, demonstrating that this population is not hyporesponsive. Together these results suggest that alloreactivity of a significant fraction of KIR(-) NK cells is mediated by NKG2A and LIR-1. Thus strategies to interrupt NKG2A and LIR-1 in combination with anti-KIR blockade hold promise for exploiting NK cell therapy in acute leukemias.

IKK inhibitor bay 11‐7082 induces necroptotic cell death in precursor‐B acute lymphoblastic leukaemic blasts

IKK inhibitor bay 11‐7082 induces necroptotic cell death in precursor‐B acute lymphoblastic leukaemic blasts

Autonomous growth potential of leukemia blast cells is associated with poor prognosis in human acute leukemias

We have described a severe combined immunodeficiency (SCID) mouse model that permits the subcutaneous growth of primary human acute leukemia blast cells into a measurable subcutaneous nodule which may be followed by the development of disseminated disease. Utilizing the SCID mouse model, we examined the growth potential of leukemic blasts from 133 patients with acute leukemia, (67 acute lymphoblastic leukemia (ALL) and 66 acute myeloid leukemia (AML)) in the animals after subcutaneous inoculation without conditioning treatment. The blasts displayed three distinct growth patterns: "aggressive", "indolent", or "no tumor growth". Out of 133 leukemias, 45 (33.8%) displayed an aggressive growth pattern, 14 (10.5%) displayed an indolent growth pattern and 74 (55.6%) did not grow in SCID mice. The growth probability of leukemias from relapsed and/or refractory disease was nearly 3 fold higher than that from patients with newly diagnosed disease. Serial observations found that leukemic blasts from the same individual, which did not initiate tumor growth at initial presentation and/or at early relapse, may engraft and grow in the later stages of disease, suggesting that the ability of leukemia cells for engraftment and proliferation was gradually acquired following the process of leukemia progression. Nine autonomous growing leukemia cell lines were established in vitro. These displayed an aggressive proliferation pattern, suggesting a possible correlation between the capacity of human leukemia cells for autonomous proliferation in vitro and an aggressive growth potential in SCID mice. In addition, we demonstrated that patients whose leukemic blasts displayed an aggressive growth and dissemination pattern in SClD mice had a poor clinical outcome in patients with ALL as well as AML. Patients whose leukemic blasts grew indolently or whose leukemia cells failed to induce growth had a significantly longer DFS and more favorable clinical course.

Bortezomib Has No. Ex Vivo Activity in Chronic Myeloid Leukemia: Individual Tumor Response Testing Comparative Study in Pediatric and Adult Acute and Chronic Leukemia.

Abstract 4257 BackgroundResistance to imatinib is one of the most important issues in treatment of CML. Proteasome inhibitor, bortezomib, is known to be effective in therapy of various neoplasms. Preclinical studies demonstrate the ability of bortezomib in chemosensitization and overcoming of chemotherapy resistance. ObjectiveAnalysis of ex vivo drug resistance to bortezomib and another 23 drugs including tyrosine kinase inhibitors (TKI) in CML, in comparison to acute adult and pediatric leukemia. Material and methodsA total number of 241 patients entered the study, including: 106 Ph(-)ALL and 53 AML children (age 0.1-18, median 7 years) and 46 AML and 36 CML adults (age 18-69, median 41 years). All children were diagnosed as de novo leukemia, AML adults as de novo (n=20) or relapsed/refractory (n=26). Due to similar drug sensitivity, all adult AML patients were pooled into one group (Gil et al, Anticancer Res, 2007;27:4021). Among CML patients 19 had advanced disease; 16 were resistant to imatinib and 6 had ABL-kinase domain mutations (M244V, E255K, Y253H, M351T and 2 with F317L). Ex vivo drug resistance profile was studied by the MTT assay with the use of following drugs: prednisolone, vincristine, idarubicin, daunorubicin, doxorubicin, mitoxantrone, etoposide, L-asparaginase, melphalan, cytarabine, fludarabine, cladribine, thiotepa, treosulfan, 4-HOO-cyclophosphamide, thioguanine, bortezomib, topotecan, clofarabine and busulfan. CML patients were also tested for sensitivity to TKI: imatinib, dasatinib and nilotinib. ResultsCML cells were more resistant than AML blasts to following drugs: prednisolone (1.5-fold; p=0.037), vincristine (2.3-fold; p=0.004), doxorubicin (>6.9-fold; p<0.001), etoposide (7.4-fold; p<0.001), melphalan (5.9-fold; p=0.001), cytarabine (12.5-fold; p=0.005), fludarabine (2.6-fold; p=0.008), thiotepa (5.4-fold: p=0.001), 4-HOO-cyclophosphamide (2.3-fold; p=0.015), thioguanine (>4-fold; p<0.001), bortezomib (6.2-fold; p<0.001), topotecan (20-fold; p<0.001), and clofarabine (50-fold; p<0.001). No differences in sensitivity were found for idarubicin, daunorubicin, mitoxantrone, L-asparaginase, cladribine, and treosulfan, while CML cells were 2-fold more sensitive to busulfan (p=0.035). Adult and pediatric AML samples did not differ significantly in ex vivo drug resistance to all tested drugs. Pediatric AML samples were more resistant than pediatric ALL samples to most of tested drugs, however they had comparable sensitivity to cytarabine, thioguanine, bortezomib, and clofarabine. CML patients with mutation had higher ex vivo resistance to: vincristine (3.3-fold; p=0.044), idarubicin (7.9-fold; p=0.031), thiotepa (13.7-fold; p=0.044), and busulfan (21.6-fold; p=0.024). No significant differences were observed with respect to other drugs, including all 3 TKI's. CML patients resistant to imatinib had higher ex vivo resistance to: vincristine (2.5-fold; p=0.016), daunorubicin (3.1-fold; p=0.011), etoposide (2.2-fold; p=0.031), and busulfan (4.5-fold; p=0.032). No significant differences were observed in respect to other drugs, including all 3 TKI's. No significant differences were observed between CML patients with non-advanced and advanced disease to all tested drugs, including TKI's. ConclusionsCML cells are ex vivo more resistant to most drugs than acute leukemia blasts. Bortezomib alone has no ex vivo activity in CML patients. No differences between CML subgroups in sensitivity to 3 various TKI was detected. These findings require further investigations. AcknowledgmentsThis study was supported by grants: EC 2008/2009 ZPORR SPS.IV-3040-UE/217/2009; EFS 9/9/POKL/4.4.1/2008; UMK 09/2009 and MNiSW N407 078 32/2964. Disclosures:No relevant conflicts of interest to declare.

Disease Propagating Blasts in Standard and High Risk Acute Lymphoblastic Leukemia Are Frequent and of Diverse Immunophenotype.

Abstract 1421Poster Board I-444Conflicting results in the field of cancer stem cells have reignited debate regarding the frequency and identity of cells with the ability to self renew and to propagate the complete phenotype of the malignancy. Initially it was suggested by different studies that cancer stem cells represent only a small minority of the malignant population and that the immunophenotypes of these cells resemble a rather immature type in the cell hierarchy. More recent data from our own and other groups have challenged these findings by demonstrating that cells at different maturity levels within the leukemic hierarchy have cancer stem cell abilities and that the frequency of the leukemia maintaining cell is higher than previously thought (Cancer Cell 2008, 14(1), p47-58). We use an in vivo NOD/scid IL2Rγnull (NSG) mouse intra-femoral transplant model to determine the clonogenicity of sorted candidate leukemic stem cell populations, characterized by specific immunophenotypes. We selected the surface markers CD10 and CD20, in order to differentiate between rather immature and more mature cells. Furthermore we carried out limiting dilution experiments on sorted (CD20) and unsorted leukemic blasts to investigate the frequency of the proposed leukemic stem cells. Flow sorted ALL blasts of CD19+CD20low and CD19+CD20high as well as of CD19+CD10low and CD19+CD10high immunophenotype were transplanted into NSG mice. Sorts were performed on primary patient material and on leukemic blasts that had been harvested following prior passage in mice. Different subtypes of ALL were included (high risk: BCR/ABL (t9;22) positive (patients L4967, L4951, L49101, L8849, L2510), high hyperdiploid/MRD positive high risk (L754, L835), intermediate risk: high WBC/MRD negative (L736, L784), age >10 years (L803)). CD20 sorts were performed on primary patient material (L4951, L49101, L754, L835 and L776), on secondary samples harvested from engrafted primary mice (L4967, L4951, L2510, L736 and L754) and on tertiary samples harvested from engrafted secondary mice (L4967 and L736). In total 151 mice were transplanted, with 122 showing engraftment in consecutive bone marrow punctures or in bone marrow harvests. CD10 sorts were performed on primary patient material (L784 and L49101) and on secondary samples harvested from engrafted primary mice (L4951, L8849, L2510 and L803) with 31 out of 52 mice transplanted with sorted material showing engraftment as seen with CD20 sorted cells. Blasts of all selected immunophenotypes were able to engraft the leukemia in unconditioned NSG mice as determined by 5 color flow cytometry. In particular, sorted cells of both fractions were able to reconstitute the complete phenotype of the leukemia. Harvested cells from engrafted mice could then be re-sorted into high and low antigen expressing fractions and successfully re-engrafted on secondary and tertiary mice. Cell purities of transplanted cells were usually higher than 90% (range 67-100%). The ability of all populations to serially engraft mice demonstrates long-term self-renewal capacity. Two additional patients were used in the limiting dilution assays (high WBC/t(4;11) high risk (L826); low WBC/MRD negative low risk (L792)) and experiments were performed on primary unsorted and secondary sorted material. Cell numbers necessary for ALL engraftment differed between individual leukemias but as little as 100 cells proved to be sufficient in one unsorted and in both the CD19+CD20low and CD19+CD20high fractions (Table 1). Mice transplanted with 10 cells only are still under observation.Table 1PatientTransplantPopulationCell doseMice engrafted/transplantedL4951SecondaryCD20 high5003/3CD20 low3/3CD20 high1003/3CD20 low3/3L2510SecondaryCD20 high3,0002/4CD20 low4/4CD20 high3000/4CD20 low1/4L49101PrimaryUnsorted5003/41000/4L792PrimaryUnsorted1,0005/51001/5L826PrimaryUnsorted1,0003/41000/4In conclusion we present strong evidence that leukemia-propagating cells are much more prevalent than previously thought and that blasts of diverse immunophenotype are able to serially reconstitute the complete leukemia in immune-deficient mice. Disclosures:No relevant conflicts of interest to declare.

Artificial Antigen Presenting Cells (aAPC) Expanded NK-Mediated Enhanced Lysis of Allogeneic Tumor Cells Regardless of HLA Class I/KIR Mismatch and Its Implication of Use in Eradicating Acute Lymphoblastic Leukemia (ALL).

Abstract 3023Poster Board II-999NK Killer cell immunoglobulin-like receptors (KIRs) and their human leukocyte antigen (HLA) ligands play critical roles in maintaining natural killer (NK) cell tolerance, while providing surveillance against pathogens and malignant transformation. Natural killer (NK) cells have been explored as tools for adoptive anti-tumor or leukemia immunotherapy and current models hold that a mismatch or absence of KIR ligands on target cells is essential for efficient NK cell mediated cytolysis. However, new approaches are now available to activate NK cells and the role for KIR mediated signaling in regulating cytotoxicity of activated NK cells has not been well studied. In this study, aAPCs comprising IL15Ra+K562 cells engineered to express 4-1BBL activated and expanded peripheral NK cells in the presence of exogenous IL15 up to 1000-fold in 3 weeks. Compared to resting NK cells, 4-1BBL/IL15-activated NK cells upregulated TRAIL and NKp30, 44, 46 expression, and showed significantly enhanced cytotoxicity against a multitude of tumor targets including K562, Daudi, Ewing's tumors, osteosarcoma, as well as autologous tumors (50%-90% killing vs. 0%-8% with non-activated NK cells). Meanwhile we could detect little to no influence of KIR signaling in regulating cytotoxicity by aAPC activated NK cells, since sorted CD158a+ and CD158b+ activated NK cells showed similar killing of tumor cells expressing HLA group C1 (CD158b ligand) and/or C2 (CD158a ligand) antigens. In contrast, killer activating receptors (KARs) were indispensable for the cytolysis of solid pediatric tumors by aAPC-activated NK cells, since the killing was significantly inhibited by fusion proteins binding to the ligands of NKG2D, NK p30, p44, p46, p80 (KARs). About 20-40% inhibition of the killing was accomplished when all four activating receptors were blocked, though other activating receptors have not been well defined. Although acute lymphoblastic leukemia (ALL) blasts were refractory to fresh NK cytotoxicity, 4-1BBL/IL15 activated NK cells demonstrated higher lytic activities (20%-50%) against ALL blasts from either patients or cell lines. ALL blast lysis could be completely or partially inhibited by KAR-blocking fusion proteins, indicating that expression levels of KAR ligands vary among ALL cases and other solid tumors. We conclude that KIR ligand mismatch or absence is not essential for effective NK cytotoxicities on either solid tumors or ALL when fully activated NK cells are utilized. This suggests that adoptive therapy with autologous aAPC-activated NK cells may prove effective in some clinical settings, such as ALL, AML, or certain solid tumors. Further studies to assess the impact of KAR ligand expression on aAPC-activated NK killing of ALL blasts are in progress.Percentage of Activated NK Killings vs. Fresh NK's with/without KAR-Ig Fusion ProteinsActivated NK (E:T=2.5:1)Fresh NK (E:T=25:1)-KAR-Ig Fc+KAR-Ig FcSB tumor (Ewing's)48%30%0.5%HOS (Osteo sarcoma)63%36%0.7%Daudi (B. lymphoma)78%46%0.2%REH (ALL)54%8%3% DisclosuresNo relevant conflicts of interest to declare.

Combination of the Hypercvad Regimen with Dasatinib Is Effective in Patients with Relapsed Philadelphia Chromosome (Ph) Positive Acute Lymphoblastic Leukemia (ALL) and Lymphoid Blast Phase Chronic Myeloid Leukemia (CML-LB).

Abstract 2043Poster Board II-20 Background:The dual Src and Abl inhibitor dasatinib is ∼325 times more potent than imatinib in vitro against the kinase activity of BCR-ABL and has significant clinical activity in patients with imatinib-resistant lymphoid blast phase CML (CML-LB) and Ph+ ALL. Prognosis of patients with relapsed Ph+ ALL and CML-LB is poor with few effective options other than allogeneic stem cell transplant. Aim:To investigate the feasibility of and response to a combination of chemotherapy and dasatinib to patients with relapsed Ph+ ALL or CML-LB. Methods:Patients with relapsed Ph+ ALL or CML-LB received dasatinib 50 mg po bid (or 100 mg daily) for the first 14 days of each of 8 cycles of alternating hyperCVAD and high dose cytarabine and methotrexate. Patients in CR continued to receive maintenance with dasatinib 50 mg po bid (or 100 mg daily) and vincristine and prednisone monthly for 2 years followed by dasatinib indefinitely. Result:To date, 23 patients with relapsed Ph+ ALL (n=14) or CML-LB (n=9) have received a median of 3 cycles of this regimen (range 1-8 cycles). Median age was 49 years (range 21-69); 10 (43%) patients were older than 50 years. Median WBC at start of treatment was 9.6 × 109/L (range, 0.4 – 295.5 × 109/L). Median bone marrow blast percentage was 80% (range 0-98%). Six patients had CNS involvement. Pre-treatment ABL mutations included: 1 patient with F317L, 1 M351T, 1 T315I, 1 Y253F, 1 Y253H, and 1 with Y253H, F359V, and E459K). Median number of previous therapies was 1 (range, 1-4); these include hyperCVAD plus imatinib (n=8), other combination chemotherapy regimens (n=8), monotherapy with tyrosine kinase inhibitors other than dasatinib (n=7), investigational agents (n=2) and allogeneic stem cell transplantation (n=2). All patients were evaluable for response. 15 patients (65%) achieved CR, 6 patients (26%) achieved CR with incomplete platelets recovery (CRp), 1 patient (4%) did not have day 21 bone marrow to assess response, and 1 patient (4%) died during induction. Nineteen of 21 (90%) patients have achieved a major cytogenetic response after 1 cycle of therapy, complete in 17, 1 (5%) had insufficient metaphases, and 1 (5%) had no response (still had 95% Ph+ metaphases after 1 cycle). Overall, so far 15 (65%) patients had achieved a major molecular response, complete in 10. Median time to neutrophil and platelet recovery for the first cycle was 18 and 22 days, and for subsequent cycles, 18 and 26 days, respectively. Nine patients have relapsed after median response duration of 26 weeks (range, 8-49); six of them had acquired ABL kinase domain mutations (1 T315I and E450G; 3 T315I; 1 F317L, and 1 G303fs). Three patients proceeded to an allogeneic transplantation and one previously transplanted patient received a donor lymphocyte infusion. Grade 3 and 4 adverse events included bleeding (GI, GU, and subdural hematomas)(9), pleural effusions (5), pericardial effusions (2), as well as infections, diarrhea, hypophosphatemia, hypocalcemia, elevated transaminases, and hyperbilirubinemia. With a median follow up of 13 months (range, 5-52 months), 10 patients are alive; 8 are in CR/CRp. Seven patients died after disease relapse, 1 died post transplant, 4 died in CR/CRp from infections, and 1 died of unknown causes. The median survival was 39 weeks (range, 2 to 226 weeks) with 42% of patients alive at 1 year. Conclusion:The combination of the hyperCVAD regimen with dasatinib is feasible and effective in patients with relapsed Ph-positive ALL and CML-LB and can provide a bridge to an allogeneic stem cell transplant. Disclosures:Ravandi:Bristol Myers Squibb: Honoraria, Research Funding. Kantarjian:Bristol Myers Squibb: Research Funding. Cortes:Bristol Myers Squibb: Research Funding. O'Brien:Bristol Myers Squibb: Research Funding.

Aberrant Underexpression of CD81 in Precursor B-Cell Acute Lymphoblastic Leukemia

We studied CD81 expression by flow cytometry (FC) on benign precursor B cells (hematogones) and leukemic blasts in precursor B-cell acute lymphoblastic leukemia (pre-B-ALL) and established its usefulness in minimal residual disease (MRD) assays. Hematogones showed uniformly bright CD81 expression. In 98 pre-B-ALLs at diagnosis or overt relapse, 80 (82%) showed aberrantly decreased CD81 intensity. We used hematogones in 139 MRD- specimens to set a lower threshold for normal CD81 expression. In 133 specimens positive for residual pre-B-ALL, 87.2% showed increased CD81-dim immature B cells (>10%) and/or a discrete cluster of CD81-dim cells in a background of hematogones. Only 1 of 139 MRD- specimens showed more than 10% CD81-dim cells. Decreased CD81 expression was maintained in 91% of aberrant cases analyzed before and after chemotherapy. Decreased CD81 expression is a sensitive and specific marker for residual pre-B-ALL, even in a background of hematogones, making CD81 a useful addition to a panel for MRD detection by FC.

Bcl-2 in Combination to Myeloid Antigen Expression in Adult Acute Lymphoblastic Leukemia and Prognostic Outcome

The present study was performed to find the importance of two myeloid (CD13 and CD33) antigens aberrantly expressed on the blasts of acute lymphoblastic leukemia (ALL) patients and Bcl-2 expression in relation to clinical and biological features and treatment outcome. Bone marrow or peripheral blood samples of 50 patients were assessed for the expression of markers by immunostaining methods. Twenty-one patients (42%) showed more than 20% positivity for Bcl-2. Aberrant expression of myeloid antigens was found in 14% of cases. The expression of Bcl-2 was associated with shorter survival (p = 0.009). A significant correlation between expression of myeloid antigens (MY) and survival and complete remission duration was found. The mean survival was 656 + 301 days for MY+ cases and 1009 +/- 230 days for MY- patients (p < 0.0001). Expression of Bcl-2 in combination to myeloid antigens was associated with a poorer outcome. Survival of MY+ patients expressing Bcl-2 was shorter than MY- Bcl-2+ and MY+ Bcl-2- ALL cases (p = 0.038). In conclusion, results of this study indicated the prognostic value of Bcl-2 and myeloid antigen expression in ALL patients. Presence of these markers together on the leukemic cells was associated with a poorer response to therapy and may implicate modified therapeutic strategies in the patients.

Targeting C-type lectin-like molecule-1 for antibody-mediated immunotherapy in acute myeloid leukemia

C-type lectin-like molecule-1 is a transmembrane receptor expressed on myeloid cells, acute myeloid leukemia blasts and leukemic stem cells. To validate the potential of this receptor as a therapeutic target in acute myeloid leukemia, we generated a series of monoclonal antibodies against the extracellular domain of C-type lectin-like molecule-1 and used them to extend the expression profile analysis of acute myeloid leukemia cells and to select cytotoxic monoclonal antibodies against acute myeloid leukemia cells in preclinical models. C-type lectin-like molecule-1 expression was analyzed in acute myeloid leukemia cell lines, and in myeloid derived cells from patients with acute myeloid leukemia and healthy donors. Anti-C-type lectin-like molecule-1 antibody-mediated in vitro cytotoxic activity against acute myeloid leukemia blasts/cell lines and in vivo anti-cancer activity in a mouse xenograft model were assessed. Internalization of C-type lectin-like molecule-1 monoclonal antibodies upon receptor ligation was also investigated. C-type lectin-like molecule-1 was expressed in 86.5% (45/52) of cases of acute myeloid leukemia, in 54.5% (12/22) of acute myeloid leukemia CD34(+)/CD38(-) stem cells, but not in acute lymphoblastic leukemia blasts (n=5). Selected anti-C-type lectin-like molecule-1 monoclonal antibodies mediated dose-dependent complement-dependent cytotoxicity and antibody-dependent cellular cytotoxicity specifically against acute myeloid leukemia-derived cell lines. Exogenous expression of the transmembrane receptor in HEK293 cells rendered the cells susceptible to antibody-mediated killing by monoclonal antibodies to the receptor. Furthermore, these monoclonal antibodies demonstrated strong complement-dependent cytotoxicity against freshly isolated acute myeloid leukemia blasts (15/16 cases; 94%). The monoclonal antibodies were efficiently internalized upon binding to C-type lectin-like molecule-1 in HL-60 cells. Moreover, a lead chimeric C-type lectin-like molecule-1 monoclonal antibody reduced the tumor size in xenograft mice implanted with HL-60 cells. Conclusions Our results demonstrate that targeting C-type lectin-like molecule-1 with specific cytotoxic monoclonal antibodies is an attractive approach which could lead to novel therapies for acute myeloid leukemia.

Molecular target therapy – towards curative regimen: a 20-year experience in the treatment of acute promyelocytic leukemia (APL) in the Shanghai Institute of Hematology

Since the first description of acute promyelocytic leukemia (APL) in 1957 as the most malignant form of acute leukemia, several developments have paved the way to make this disease the most curable leukemia in adults and change the paradigm of cancer treatment. Therapy of APL was pioneered by Bernard et al in 1973 demonstrating a striking sensitivity to daunorubicin, probably related to significantly lower P-glycoprotein expression observed in APL cells compared to other subtypes of acute myeloid leukemia (AML). The incorporation of ATRA, a noncytotoxic differentiating agent that is regarded as the first differentiation therapy has changed dramatically the management, outcome, and prognosis of APL. ATRA was first introduced to clinical use for the treatment of APL in 1986. Since then, randomized studies in many centers around the world document a rising CR rate, a decrease in severe adverse effects, and a prolongation of remission duration. ATRA combined with anthracycline-based chemotherapy can achieve CR in 90–95% of patients with APL and cure the disease in 70–75% of the cases. Combination therapy with ATRA and chemotherapeutic agents should now be considered as a standard treatment of APL. Over the last decade, tremendous efforts have been made to elucidate the molecular genesis of APL, as well as the mechanism of action of ATRA. The mechanism of action of ATRA can be summarized as follows: 1. The binding of ATRA to RAR receptors causes degradation of PML-RARα protein through the ubiquitin-proteosome and caspase system, leading to restoration of terminal differentiation of promyelocytes; 2. Exposure of APL cells to ATRA in vitro or in vivo induces relocalization of PML and restores the normal structure of PODs; and 3. Under the action of ATRA, CoR is dissociated from the repressive complex, whereas CoA (coactivator) is recruited to the complex. As a result, the repression of transcriptional activation of target genes is relieved and the differentiation of promyelocytes is restored. Treatment of APL by arsenic compounds represents a successful example of apoptosis induction therapy of acute leukemia. As2O3 exerts dual effects on APL cells. Studies in vitro with NB4 cells showed that a higher concentration of As2O3 (0.5–1.0 μM) induced apoptosis with typical morphological changes, DNA laddering on agarose gel electrophoresis, appearance of an apoptotic peak on flow cytometric analysis, and increased expression of annexin V on the cell surface membrane. At lower concentrations, As2O3 can induce APL cells to partially differentiate along the granulocytic pathway. Synergistic effect of ATRA and As2O3 was confirmed by several research works in vivo and in vitro. The first clinical trial was completed by SIH in 2001. CR rate was same as ATRA or As2O3 alone, but the median day to CR was very short, only 26 days, and OS and DFS were much better than ATRA or As2O3 alone. Now timing and dose of As2O3 combined with ATRA in newly diagnosed APL patients need to be confirmed. Recently, several genetic and phenotypic characteristics of acute promyelocytic leukemia (APL) blasts have been demonstrated. These include the PML/RARα fusion and the transcription co-repressor complex recruited at the promoter of target genes by the hybrid protein, the intense and homogeneous expression of the CD33 antigen, and absence of multidrug resistance-related phenotype, a frequently mutated and constitutively activated FLT3 receptor. Such genotypic and phenotypic features are targeted by agents currently in use in front-line therapy or at relapse (i.e., retinoids, As2O3, anthracyclines and anti-CD33 monoclonal antibodies), and by novel agents that many find a place in future treatments such as histone deacetylase and FLT3 inhibitors.

Functional involvement of RINF, retinoid-inducible nuclear factor (CXXC5), in normal and tumoral human myelopoiesis

AbstractRetinoids triggers differentiation of acute promyelocytic leukemia (APL) blasts by transcriptional regulation of myeloid regulatory genes. Using a microarray approach, we have identified a novel retinoid-responsive gene (CXXC5) encoding a nuclear factor, retinoid-inducible nuclear factor (RINF), that contains a CXXC-type zinc-finger motif. RINF expression correlates with retinoid-induced differentiation of leukemic cells and with cytokine-induced myelopoiesis of normal CD34+ progenitors. Furthermore, short hairpin RNA (shRNA) interference suggests for this gene a regulatory function in both normal and tumoral myelopoiesis. Interestingly, RINF localizes to 5q31.3, a small region often deleted in myeloid leukemia (acute myeloid leukemia [AML]/myelodysplasia [MDS]) and suspected to harbor one or several tumor suppressor gene.