Dipeptidylpeptidase IV (CD26) defines leukemic stem cells (LSC) in chronic myeloid leukemia

Chronic myeloid leukemia (CML) is a stem cell (SC) neoplasm characterized by the BCR/ABL1 oncogene. Although mechanisms of BCR/ABL1-induced transformation are well-defined, little is known about effector-molecules contributing to malignant expansion and the extramedullary spread of leukemic SC (LSC) in CML. We have identified the cytokine-targeting surface enzyme dipeptidylpeptidase-IV (DPPIV/CD26) as a novel, specific and pathogenetically relevant biomarker of CD34+/CD38─ CML LSC. In functional assays, CD26 was identified as target enzyme disrupting the SDF-1-CXCR4-axis by cleaving SDF-1, a chemotaxin recruiting CXCR4+ SC. CD26 was not detected on normal SC or LSC in other hematopoietic malignancies. Correspondingly, CD26+ LSC decreased to low or undetectable levels during successful treatment with imatinib. CD26+ CML LSC engrafted NOD-SCID-IL-2Rγ−/− (NSG) mice with BCR/ABL1+ cells, whereas CD26─ SC from the same patients produced multilineage BCR/ABL1– engraftment. Finally, targeting of CD26 by gliptins suppressed the expansion of BCR/ABL1+ cells. Together, CD26 is a new biomarker and target of CML LSC. CD26 expression may explain the abnormal extramedullary spread of CML LSC, and inhibition of CD26 may revert abnormal LSC function and support curative treatment approaches in this malignancy

Overexpression of primary microRNA 221/222 in acute myeloid leukemia

BACKGROUND: Acute myeloid leukemia (AML) is a hematopoietic malignancy with a dismal outcome in the majority of cases. A detailed understanding of the genetic alterations and gene expression changes that contribute to its pathogenesis is important to improve prognostication, disease monitoring, and therapy. In this context, leukemia-associated misexpression of microRNAs (miRNAs) has been studied, but no coherent picture has emerged yet, thus warranting further investigations. METHODS: The expression of 636 human miRNAs was compared between samples from 52 patients with AML and 13 healthy individuals by highly specific locked nucleic acid (LNA) based microarray technology. The levels of individual mature miRNAs and of primary miRNAs (pri-miRs) were determined by quantitative reverse transcriptase (qRT) PCR. Transfections and infections of human cell lines were performed using standard procedures. RESULTS: 64 miRNAs were significantly differentially expressed between AML and controls. Further studies on the clustered miRNAs 221 and 222, already known to act as oncogenes in other tumor types, revealed a deficiency of human myeloid cell lines to process vector derived precursor transcripts. Moreover, endogenous pri-miR-221/222 was overexpressed to a substantially higher extent than its mature products in most primary AML samples, indicating that its transcription was enhanced, but processing was rate limiting, in these cells. Comparison of samples from the times of diagnosis, remission, and relapse of AML demonstrated that pri-miR-221/222 levels faithfully reflected the stage of disease. CONCLUSIONS: Expression of some miRNAs is strongly regulated at the posttranscriptional level in AML. Pri-miR-221/222 represents a novel molecular marker and putative oncogene in this disease

Small-molecule inhibition of BRD4 as a new potent approach to eliminate leukemic stem- and progenitor cells in acute myeloid leukemia (AML)

Acute myeloid leukemia (AML) is a life-threatening stem cell disease characterized by uncontrolled proliferation and accumulation of myeloblasts. Using an advanced RNAi screen-approach in an AML mouse model we have recently identified the epigenetic ‘reader’ BRD4 as a promising target in AML. In the current study, we asked whether inhibition of BRD4 by a small-molecule inhibitor, JQ1, leads to growth-inhibition and apoptosis in primary human AML stem- and progenitor cells. Primary cell samples were obtained from 37 patients with freshly diagnosed AML (n=23) or refractory AML (n=14). BRD4 was found to be expressed at the mRNA and protein level in unfractionated AML cells as well as in highly enriched CD34+/CD38− and CD34+/CD38+ stem- and progenitor cells in all patients examined. In unfractionated leukemic cells, submicromolar concentrations of JQ1 induced major growth-inhibitory effects (IC50 0.05-0.5 μM) in most samples, including cells derived from relapsed or refractory patients. In addition, JQ1 was found to induce apoptosis in CD34+/CD38− and CD34+/CD38+ stem- and progenitor cells in all donors examined as evidenced by combined surface/Annexin-V staining. Moreover, we were able to show that JQ1 synergizes with ARA-C in inducing growth inhibition in AML cells. Together, the BRD4-targeting drug JQ1 exerts major anti-leukemic effects in a broad range of human AML subtypes, including relapsed and refractory patients and all relevant stem- and progenitor cell compartments, including CD34+/CD38− and CD34+/CD38+ AML cells. These results characterize BRD4-inhibition as a promising new therapeutic approach in AML which should be further investigated in clinical trials

Interleukin-9 (IL-9) and NPM-ALK each generate mast cell hyperplasia as single ‘hit’ and cooperate in producing a mastocytosis-like disease in mice

Mast cell neoplasms are characterized by abnormal growth and focal accumulation of mast cells (MC) in one or more organs. Although several cytokines, including stem cell factor (SCF) and interleukin-9 (IL-9) have been implicated in growth of normal MC, little is known about pro-oncogenic molecules and conditions triggering differentiation and growth of MC far enough to lead to the histopathological picture of overt mastocytosis. The anaplastic lymphoma kinase (ALK) has recently been implicated in growth of neoplastic cells in malignant lymphomas. Here, we describe that transplantation of NPM-ALK-transplanted mouse bone marrow progenitors into lethally irradiated IL-9 transgenic mice not only results in lymphoma-formation, but also in the development of a neoplastic disease exhibiting histopathological features of systemic mastocytosis, including multifocal dense MC-infiltrates, occasionally with devastating growth in visceral organs. Transplantation of NPM-ALK-transduced progenitors into normal mice or maintaintence of IL-9-transgenic mice without NPM-ALK each resulted in MC hyperplasia, but not in mastocytosis. Neoplastic MC in mice not only displayed IL-9, but also the IL-9 receptor, and the same was found to hold true for human neoplastic MC. Together, our data show that neoplastic MC express IL-9 rececptors, that IL-9 and NPM-ALK upregulate MC-production in vivo, and that both ‘hits’ act in concert to induce a mastocytosis-like disease in mice. These data may have pathogenetic and clinical implications and fit well with the observation that neoplastic MC in advanced SM strongly express NPM and multiple “lymphoid” antigens including CD25 and CD30

Revealing Six Phases of CML Stem Cell Development to Explain Clinical Phenomena Seen in TKI-Treated Patients.

Abstract Abstract 4263 Chronic myeloid leukemia (CML) is a stem cell (SC) disease defined by the BCR/ABL oncoprotein that is considered essential for abnormal growth and accumulation of neoplastic cells. Based on in vitro studies and mathematical models, CML clones are considered to be organized hierarchically similar to normal hematopoiesis. More recent data suggest, that CML cells grow in subclones that usually exhibit SC function but vary in their leukemia-initiating potential. The situation is even more complex in patients treated with TKI. In these patients, intrinsic as well as acquired resistance against TKI have been described and recognized as an emerging problem and challenge in practice and research. Most SC concepts focus on imatinib-resistant mutants of BCR/ABL, that are detectable in subclones. However, several questions and phenomena that occur in TKI-treated patients remain to be solved. Based on laboratory and clinical observations, we propose the existence of 6 distinct phases of CML SC development (SCD): a) a Ph-negative phase, b) an early Ph-positive preleukemic SCD-phase in which subclones are (very) small and usually undetectable, c) a pre/leukemic SCD-phase in which one or more subclones expand(s) and replace(s) normal myelopoiesis but still produce(s) normal WBC, d) chronic phase (CP), e) accelerated phase (AP), and f) a blast phase (BP). The latency period until progression into a next SCD phase is variable and may depend on several different factors including subclone inhibition by chalones like lipocalin and other factors, different growth kinetics produced by various BCR/ABL mutants, and TKI-induced subclone-selection. Phase a) may explain the rare occurrence of Ph-negative subclones (+OCA) during TKI treatment. Phase b) may explain why BCR/ABL mutants are not detectable before TKI therapy is initiated, why mutant- and ACA subclones “appear” in CCyR patients after a certain latency period, and why one patient can develop two or more BCR/ABL mutants in different subclones. Phase b) may also explain the rare detection of very small quantities of BCR/ABL in healthy individuals and constant low level-MRD in a few CML patients in whom therapy was stopped. Phase c) can explain early CML patients in whom WBC are normal in repeated tests; and explain relapsed TKI-resistant patients in whom under TKI therapy normal hematopoiesis is replaced by the mutant subclone but WBC remain normal for weeks to several months. The SCD phases a) through c) are not accessible in any of the conventional xenotransplant models available. Even in SCD phases d) through f), it is quite difficult to demonstrate stable long term engraftment in xenotransplant mouse models, although stem cell subclones obtained from patients in f) may grow in NOG- or NSG mice similar to AML SC. However, even if this may be a reproducible approach, analysis of all relevant subclones in these patients will probably not be achievable unless additional oncogenes are introduced in these subclones. In summary, our new concept extends the hydra model of cancer stem cell development for CML by introducing a step wise progression-model with 6 defined phases of SCD. This model may have theoretical and clinical implications for patients with freshly diagnosed and TKI-resistant CML and for cancer stem cell research in general. Disclosures: No relevant conflicts of interest to declare. </jats:sec

Expression of the CAMPATH-1 Antigen (CD52) on CD34+/CD38- Progenitor Cells in Patients with 5q- MDS and in a Subset of AML

Abstract Abstract 1728 The target antigen CAMPATH-1 (CD52) is widely expressed in various hematopoietic lineages inlcuding lymphocytes, basophils, and blood monocytes. The anti-CD52 antibody Alemtuzumab is used successfully to treat patients with chemotherapy-refractory chronic lymphocytic leukemia. Based on its strong immunosuppressive effects, Alemtuzumab has also been considered for patients with aplastic anemia and hypoplastic myelodysplastic syndromes (MDS). Indeed, more recently, Alemtuzumab was found to induce major hematologic responses in a group of patients with MDS. Although the immunosuppressive effect was considered to play a role, the exact mechanisms underlying this drug effect remained speculative. In the current study, we asked whether CD34+ bone marrow (BM) progenitor cells in MDS and acute myeloid leukemia (AML) express the CAMPATH-1 antigen. Twelve patients with MDS (5 females, 7 males; median age: 70 years), 25 patients with AML (16 females, 9 males; median age: 62 years), and 34 control cases (normal reactive BM, n=12; idiopathic cytopenia of unknown significance, n=11; chronic myeloid leukemia, CML, n=4; chronic myelomonocytic leukemia, CMML, n=3; JAK2 V617F+ myeloproliferative neoplasms, MPN, n=4) were examined. Surface expression of CD52 on CD34+/CD38+ and CD34+/CD38- BM progenitor cells was analyzed by monoclonal antibodies and multicolor flow cytometry. In the group of MDS, CD52 was detectable on CD34+/CD38- stem cells in 3/4 patients with isolated 5q-. In most of the other MDS patients, CD52 was weakly expressed or not detectable on CD34+/CD38- cells. In AML, CD34+/CD38- cells displayed CD52 in 12/25 patients, namely 3 with complex karyotype including 5q-, 2 with inv(3), one with t(8;21), one with inv(16), one with del13q, one with trisomy 8, one with monosomy 7, and 2 with normal karyotype. Expression of CD52 mRNA in CD34+/CD38- AML stem cells was confirmed by qPCR in all patients tested (n=14). In addition, a good correlation was found between surface CD52 expression and CD52 mRNA expression in AML progenitor fractions. In patients with normal hematopoiesis (n=12) or idiopathic cytopenia (n=11), CD34+/CD38- cells stained weakly positive or negative for CD52. Almost in all cases tested, blood monocytes and blood basophils stained positive for CD52. Together, our data suggest that the target antigen CAMPATH-1 (CD52) is expressed on primitive CD34+/CD38- progenitor cells in MDS, preferentially in 5q- patients, and in a subset of patients with AML. These observations may have clinical implications and explain recently described effects of Alemtuzumab in patients with MDS. Our data also suggest that Alemtuzumab may be an interesting targeted drug in patients with refractory or relapsed AML in whom neoplastic stem cells express the target antigen CD52. Disclosures: No relevant conflicts of interest to declare. </jats:sec

Identification of Campath-1 Antigen (CD52) As a Novel Therapeutic Target in Advanced Systemic Mastocytosis.

Abstract Abstract 2866 Systemic mastocytosis (SM) is a neoplasm of mast cells (MC) and MC progenitor cells. The clinical picture in SM ranges from an indolent course to highly aggressive cases with short survival time. In a majority of all patients with SM, the KIT mutation D816V is detectable. In addition, activating RAS mutations have recently been identified in patients with advanced SM. So far, no curative therapy for advanced SM is available. To identify molecular targets in neoplastic MC, we have generated novel human MC lines by lentiviral immortalization of cord blood-derived MC progenitors with RAS G12V, SV40 large T antigen, and hTERT. These cell lines, designated MCPV1 through MCPV4, display a morphology and ultrastructure resembling immature MC progenitors. MCPV cells also express a number of MC differentiation antigens, such as KIT and tryptase, and produce an aggressive MC disease when injected into NOD-SCID IL-2RG−/− (NSG) mice. As assessed by flow cytometry, MCPV cells were found to express a number of different surface antigens, including CD13, CD30, CD33, CD44, CD52, CD54, CD63, and CD95. In consecutive analyses, we studied the cell surface target CD52, also known as CAMPATH-1 antigen. As assessed by FACS, CD52 was found to be expressed on primary neoplastic MC in patients with aggressive SM (ASM, n=3), whereas in all patients with indolent SM (ISM, n=6), neoplastic MC stained negative or only slightly positive for CD52. Normal MC stained negative for CD52 in these experiments. Expression of CD52 in neoplastic MC was also demonstrable by immunohistochemical staining of bone marrow biopsy sections. We then asked whether the putative stem cells in ISM and ASM would express CD52. Indeed, in all patients examined, including cases with ISM and ASM, the immature CD34+/CD38- cells were found to express CD52. Next, we studied the regulation of expression of CD52 in neoplastic MC. Since activating RAS mutants have been described to be expressed in neoplastic MC in patients with advanced SM, we asked whether oncogenic RAS would promote expression of CD52 in MC. Indeed, lentiviral-mediated expression of activated RAS mutants was found to upregulate mRNA expression and to promote surface expression of CD52 in the human MC line HMC-1 as well as in various other myeloid cell lines. To validate CD52 as a potential target in neoplastic MC, we applied the CD52-targeting drug alemtuzumab. In these experiments, alemtuzumab exerted only minimal direct effects on viability of MCPV cells after IgG-mediated crosslinking. However, incubation of MCPV cells with alemtuzumab induced rapid and dose-dependent cell death via complement-dependent cytotoxicity (Figure 1). Furthermore, alemtuzumab was found to induce complement-dependent cytotoxicity in CD52+ primary neoplastic MC obtained from patients with advanced SM (n=2). In conclusion, we have established a new powerful model for studying the biology of advanced SM. Moreover, our study has identified CD52 as a novel promising therapeutic target in neoplastic MC and MC progenitor cells. Whether targeting of neoplastic (stem) cells in advanced SM using alemtuzumab will improve therapy by eradicating malignant cells remains to be determined in clinical trials. Figure 1. Alemtuzumab induces complement-dependent cytotoxicity in MCPV cells. MCPV cells were incubated with alemtuzmab and human serum as a source of complement (black bars). Heat-inactivation of the serum (white bars) abolished the cytotoxic effect. Figure 1. Alemtuzumab induces complement-dependent cytotoxicity in MCPV cells. MCPV cells were incubated with alemtuzmab and human serum as a source of complement (black bars). Heat-inactivation of the serum (white bars) abolished the cytotoxic effect. Disclosures: Valent: Phadia: Research Funding. </jats:sec

Identification of Oncostatin M as a Novel KIT D816V-Dependent Cytokine in Neoplastic Human Mast Cells.

Abstract Abstract 213 Systemic mastocytosis (SM) is a neoplastic disease of mast cells (MC) and their bone marrow-derived progenitors. The clinical picture in SM is variable ranging from an indolent course to highly aggressive variants with short survival time. The pathologic hallmark in SM is the multifocal dense infiltrate of MC in the bone marrow. Other typical features of SM include alterations of the bone marrow microenvironment such as increased angiogenesis and fibrosis. In a majority of patients, MC display the KIT mutation D816V which affects the activation loop at the entrance to the enzymatic pocket of the KIT kinase. As a consequence, KIT D816V exhibits constitutive tyrosine kinase activity and promotes cytokine-independent differentiation of MC. However, so far, little is known about KIT D816V-dependent expression of pathogenetically relevant molecules in neoplastic MC. Oncostatin M (OSM) is a pleiotropic cytokine of the interleukin-6 family which is produced mainly by activated T cells and monocytes. OSM has been shown to inhibit cell growth in cell lines derived from solid tumors but to stimulate proliferation of fibroblasts and endothelial cells. Recently, it has been reported that OSM produced by activated MC promotes growth of human dermal fibroblasts. Moreover, it has been suggested that OSM stimulates growth of murine bone marrow-derived mast cells in a mast cell/fibroblast coculture. However, expression of OSM in neoplastic MC or a potential pathogenetic role of OSM in SM have not been examined so far. The aim of the present study was to analyze expression of OSM in neoplastic human MC and to determine the role of KIT D816V in OSM expression. As assessed by immunohistochemistry performed on bone marrow sections of patients with SM, typical spindle-shaped neoplastic MC were found to express OSM. Serial section-staining confirmed that tryptase-positive MC co-express OSM. Expression of OSM was found in neoplastic MC in all patients investigated (n=15) and in all variants of SM (indolent SM as well as aggressive variants) with comparable staining intensities. Preincubation of anti-OSM antibody with a specific blocking peptide resulted in a negative stain. In Ba/F3 cells, doxycycline-inducible expression of KIT D816V led to a substantial upregulation of OSM mRNA and OSM protein, whereas expression of wild type KIT did not affect expression of OSM. In addition, the KIT D816V-positive HMC-1.2 mast cell line was found to express OSM at high levels, whereas the KIT D816V-negative HMC-1.1 subclone expressed only baseline levels of OSM. Correspondingly, the KIT D816V-targeting drug midostaurine (PKC412) decreased the expression of OSM in HMC-1.2 cells as well as in KIT D816V-expressing Ba/F3 cells in a dose-dependent manner. To investigate signaling pathways involved in KIT D816V-dependent expression of OSM, we applied pharmacologic inhibitors and dominant negative-acting signaling molecules. We found that KIT D816V-dependent expression of OSM is inhibited by the mitogen-activated protein-kinase/extracellular signal-regulated kinase (MEK) inhibitor, PD98059, but not by the phosphoinositide 3-kinase inhibitor, LY294002. Expression of dominant negative mutants of signal transducer and activator of transcription 5 (STAT5) did not affect expression of OSM in KIT D816V-expressing cells. In summary, our data identify OSM as a novel cytokine expressed in neoplastic MC in patients with SM and show that KIT D816V directly promotes expression of OSM through activation of the mitogen-activated protein-kinase pathway. OSM may be an important KIT D816V-dependent effector promoting angiogenesis and fibrogenesis/sclerosis in patients with SM. Disclosures: No relevant conflicts of interest to declare. </jats:sec