Survival Of CD34 Research Articles

R-2-HG in AML… friend or foe?

R-2-HG in AML… friend or foe?

Distinct profile of CD34+ cells and plasma-derived extracellular vesicles from triple-negative patients with Myelofibrosis reveals potential markers of aggressive disease

BackgroundMyelofibrosis (MF) is a clonal disorder of hemopoietic stem/progenitor cells (HSPCs) with high prevalence in elderly patients and mutations in three driver genes (JAK2, MPL, or CALR). Around 10–15% of patients are triple-negative (TN) for the three driver mutations and display significantly worse survival. Circulating extracellular vesicles (EVs) play a role in intercellular signaling and are increased in inflammation and cancer. To identify a biomolecular signature of TN patients, we comparatively evaluated the circulating HSPCs and their functional interplay with the microenvironment focusing on EV analysis.MethodsPeripheral blood was collected from MF patients (n = 29; JAK2V617F mutation, n = 23; TN, n = 6) and healthy donors (HD, n = 10). Immunomagnetically isolated CD34+ cells were characterized by gene expression profiling analysis (GEP), survival, migration, and clonogenic ability. EVs were purified from platelet-poor plasma by ultracentrifugation, quantified using the Nanosight technology and phenotypically characterized by flow cytometry together with microRNA expression. Migration and survival of CD34+ cells from patients were also analyzed after in vitro treatments with selected inflammatory factors, i.e. (Interleukin (IL)-1β, Tumor Necrosis Factor (TNF)-α, IL6) or after co-culture with EVs from MF patients/HD.ResultsThe absolute numbers of circulating CD34+ cells were massively increased in TN patients. We found that TN CD34+ cells show in vitro defective functions and are unresponsive to the inflammatory microenvironment. Of note, the plasma levels of crucial inflammatory cytokines are mostly within the normal range in TN patients. Compared to JAK2V617F-mutated patients, the GEP of TN CD34+ cells revealed distinct signatures in key pathways such as survival, cell adhesion, and inflammation. Importantly, we observed the presence of mitochondrial components within plasma EVs and a distinct phenotype in TN-derived EVs compared to the JAK2V617F-mutated MF patients and HD counterparts. Notably, TN EVs promoted the survival of TN CD34+ cells. Along with a specific microRNA signature, the circulating EVs from TN patients are enriched with miR-361-5p.ConclusionsDistinct EV-driven signals from the microenvironment are capable to promote the TN malignant hemopoiesis and their further investigation paves the way toward novel therapeutic approaches for rare MF.

PS971 AT1413 ANTIBODY DERIVED FROM A CURED AML PATIENT RECOGNIZES A UNIQUE CD43 EPITOPE SHARED BY AML, MDS AND MELANOMA AND HAS IN VIVO ACTIVITY AS UNMODIFIED ANTIBODY AND AS BISPECIFIC T CELL ENGAGER

Background: Nuclear mislocalization of proteins can interfere with normal cellular function and cooperatively drive tumor development. To understand how this process mediates AML development, we analyzed the nuclear proteome of AML blasts in comparison with normal human CD34+ cells to identify misregulated nuclear proteins. In a preliminary study, we identified S100A4 to be over-expressed in 73% (11/15) AML patients and mislocalized to the nucleus in AML blasts. S100A4 belongs to the S100 multigene family of calcium-binding proteins of the EF-hand type and has been implicated in tumor progression and metastasis in many solid tumours but little is known of its role in hematological malignancy. Aims: To validate S100A4 overexpression and mislocalization in primary AML blasts. To determine the functional role of S100A4 on hematopoietic cell development, growth and survival in normal human CD34+ cells and AML cell lines. Methods: S100A4 expression was determined by western blot. Lentiviral vectors were used to ectopically express S100A4 and shRNA was used to knock down S100A4 expression. Effects on cell growth, survival and differentiation were determined by flow cytometry. To determine S100A4 interacting proteins, S100A4 was co-immunoprecipitated with its binding partners using Ca+2 enriched conditions from nuclear extracts of ME-1 cells followed by LC-MS/MS analysis. Results: Using western blotting, S100A4 protein expression was observed in the nucleus of AML blasts (73%; 24/33) whilst normal CD34+ or CD14+ differentiated monocytic controls have shown only cytosolic expression of S100A4. Upregulated S100A4 is also supported by transcriptome analysis indicating that overexpression arises at least partly at a transcriptional level. An independent dataset (TCGA) supports the overexpression of S100A4 mRNA in AML and suggests that overexpression may confer a poor prognosis (p = 0.0118). To determine whether ectopic expression of nuclear S100A4 can affect the growth and survival of CD34+ cells, we attempted to overexpress nuclear-targeted S100A4 in normal human hematopoietic cells. Overexpression of nuclear S100A4 could not be demonstrated in transduced CD34+ or in normal differentiated cells (probably due to rapid degradation of ectopically expressed S100A4 in these cells). To examine the importance of S100A4 expression in normal and leukaemic cells we next knocked-down S100A4 expression. In CD34+ cells we observed no significant effect on the growth or lineage development of these cells suggesting S100A4 is not required for normal hematopoiesis. We next examined the consequences of knocking down S100A4 expression in AML cell lines (NOMO-1, TF-1, THP-1, and OCI-AML2). S100A4 knockdown severely impaired the growth TF-1 and OCI-AML2 (p < 0.05). Significant apoptosis was observed in OCI-AML-2, TF-1, and NOMO-1 (p < 0.05). Using co-immunoprecipitation coupled with LC/MS to identify binding partners of S100A4, we identified heterogeneous nuclear Ribonucleoprotein M (hnRNPM) as novel binding partner of S100A4 AML. Interestingly, hnRNPM has been implicated in metastasis in breast cancer through mediating alternative splicing. Summary/Conclusion: We found that S100A4 is mislocalized to the nucleus in AML blasts compared to normal hematopoeitic cells and is essential for AML cells growth and survival. HnRNPM emerged as novel binding partner to S100A4 and we hypothesise that S100A4 mediates its effect through forming a splicing complex with known leukemic splicing factors such as SF3B1 and SRSF2 (also been pulled down with S100A4 co-IP).

PS970 ACUTE MYELOID LEUKEMIA CELLS ARE DEPENDENT ON S100A4 FOR GROWTH AND SURVIVAL

Background:Nuclear mislocalization of proteins can interfere with normal cellular function and cooperatively drive tumor development. To understand how this process mediates AML development, we analyzed the nuclear proteome of AML blasts in comparison with normal human CD34+ cells to identify misregulated nuclear proteins. In a preliminary study, we identified S100A4 to be over‐expressed in 73% (11/15) AML patients and mislocalized to the nucleus in AML blasts. S100A4 belongs to the S100 multigene family of calcium‐binding proteins of the EF‐hand type and has been implicated in tumor progression and metastasis in many solid tumours but little is known of its role in hematological malignancy.Aims:To validate S100A4 overexpression and mislocalization in primary AML blasts. To determine the functional role of S100A4 on hematopoietic cell development, growth and survival in normal human CD34+ cells and AML cell lines.Methods:S100A4 expression was determined by western blot. Lentiviral vectors were used to ectopically express S100A4 and shRNA was used to knock down S100A4 expression. Effects on cell growth, survival and differentiation were determined by flow cytometry. To determine S100A4 interacting proteins, S100A4 was co‐immunoprecipitated with its binding partners using Ca+2 enriched conditions from nuclear extracts of ME‐1 cells followed by LC‐MS/MS analysis.Results:Using western blotting, S100A4 protein expression was observed in the nucleus of AML blasts (73%; 24/33) whilst normal CD34+ or CD14+ differentiated monocytic controls have shown only cytosolic expression of S100A4. Upregulated S100A4 is also supported by transcriptome analysis indicating that overexpression arises at least partly at a transcriptional level. An independent dataset (TCGA) supports the overexpression of S100A4 mRNA in AML and suggests that overexpression may confer a poor prognosis (p = 0.0118). To determine whether ectopic expression of nuclear S100A4 can affect the growth and survival of CD34+ cells, we attempted to overexpress nuclear‐targeted S100A4 in normal human hematopoietic cells. Overexpression of nuclear S100A4 could not be demonstrated in transduced CD34+ or in normal differentiated cells (probably due to rapid degradation of ectopically expressed S100A4 in these cells). To examine the importance of S100A4 expression in normal and leukaemic cells we next knocked‐down S100A4 expression. In CD34+ cells we observed no significant effect on the growth or lineage development of these cells suggesting S100A4 is not required for normal hematopoiesis. We next examined the consequences of knocking down S100A4 expression in AML cell lines (NOMO‐1, TF‐1, THP‐1, and OCI‐AML2). S100A4 knockdown severely impaired the growth TF‐1 and OCI‐AML2 (p &lt; 0.05). Significant apoptosis was observed in OCI‐AML‐2, TF‐1, and NOMO‐1 (p &lt; 0.05). Using co‐immunoprecipitation coupled with LC/MS to identify binding partners of S100A4, we identified heterogeneous nuclear Ribonucleoprotein M (hnRNPM) as novel binding partner of S100A4 AML. Interestingly, hnRNPM has been implicated in metastasis in breast cancer through mediating alternative splicing.Summary/Conclusion:We found that S100A4 is mislocalized to the nucleus in AML blasts compared to normal hematopoeitic cells and is essential for AML cells growth and survival. HnRNPM emerged as novel binding partner to S100A4 and we hypothesise that S100A4 mediates its effect through forming a splicing complex with known leukemic splicing factors such as SF3B1 and SRSF2 (also been pulled down with S100A4 co‐IP).

Combined targeting of STAT3 and STAT5: a novel approach to overcome drug resistance in chronic myeloid leukemia.

In chronic myeloid leukemia, resistance against BCR-ABL1 tyrosine kinase inhibitors can develop because of BCR-ABL1 mutations, activation of additional pro-oncogenic pathways, and stem cell resistance. Drug combinations covering a broad range of targets may overcome resistance. CDDO-Me (bardoxolone methyl) is a drug that inhibits the survival of leukemic cells by targeting different pro-survival molecules, including STAT3. We found that CDDO-Me inhibits proliferation and survival of tyrosine kinase inhibitor-resistant BCR-ABL1+ cell lines and primary leukemic cells, including cells harboring BCR-ABL1T315I or T315I+ compound mutations. Furthermore, CDDO-Me was found to block growth and survival of CD34+/CD38− leukemic stem cells (LSC). Moreover, CDDO-Me was found to produce synergistic growth-inhibitory effects when combined with BCR-ABL1 tyrosine kinase inhibitors. These drug-combinations were found to block multiple signaling cascades and molecules, including STAT3 and STAT5. Furthermore, combined targeting of STAT3 and STAT5 by shRNA and STAT5-targeting drugs also resulted in synergistic growth-inhibition, pointing to a new efficient concept of combinatorial STAT3 and STAT5 inhibition. However, CDDO-Me was also found to increase the expression of heme-oxygenase-1, a heat-shock-protein that triggers drug resistance and cell survival. We therefore combined CDDO-Me with the heme-oxygenase-1 inhibitor SMA-ZnPP, which also resulted in synergistic growth-inhibitory effects. Moreover, SMA-ZnPP was found to sensitize BCR-ABL1+ cells against the combination ‘CDDO-Me+ tyrosine kinase inhibitor’. Together, combined targeting of STAT3, STAT5, and heme-oxygenase-1 overcomes resistance in BCR-ABL1+ cells, including stem cells and highly resistant sub-clones expressing BCR-ABL1T315I or T315I-compound mutations. Whether such drug-combinations are effective in tyrosine kinase inhibitor-resistant patients with chronic myeloid leukemia remains to be elucidated.

Involvement of urokinase receptor in the cross-talk between human hematopoietic stem cells and bone marrow microenvironment.

Hematopoietic stem cells (HSCs) reside in bone marrow (BM) and can be induced to mobilize into the circulation for transplantation. Homing and lodgement into BM of transplanted HSCs are the first critical steps in their engraftment and involve multiple interactions between HSCs and the BM microenvironment.uPAR is a three domain receptor (DIDIIDIII) which binds urokinase, vitronectin, integrins. uPAR can be cleaved and shed from the cell surface generating full-length and cleaved soluble forms (suPAR and DIIDIII-suPAR). DIIDIII-suPAR can bind fMLF receptors through the SRSRY sequence (residues 88-92).We previously reported the involvement of soluble uPAR in HSC mobilization. We now investigate its possible role in HSC homing and engraftment.We show similar levels of circulating full-length suPAR in healthy donors and in acute myeloid leukemia (AML) patients before and after the pre-transplant conditioning regimen. By contrast, levels of circulating DIIDIII-suPAR in AML patients are higher as compared to controls and significantly decrease after the conditioning.We found that suPAR and uPAR84-95, a uPAR-derived peptide which mimics active DIIDIII-suPAR, induce a significant increase in Long Term Culture (LTC)-Initiating Cells (ICs) and in the release of clonogenic progenitors from LTCs of CD34+ HSCs. Further, suPAR increases adhesion and survival of CD34+ KG1 AML cells, whereas uPAR84-95 increases their proliferation.Thus, circulating DIIDIII-suPAR, strongly increased in HSC mobilization, is indeed down-regulated by pre-transplant conditioning, probably to favour HSC homing. BM full-length suPAR and DIIDIII-suPAR may be involved in HSC lodgement within the BM by contributing to a suitable microenvironment.

Chlorpromazine, an Inhibitor of Intracellular Trafficking of FLT3-ITD and KIT D816V, Shows Prominent Anti-Leukemic Activities Against AML Cells and AML Stem Cells in Vitro and in Vivo.

Activating mutations of receptor tyrosine kinases (RTKs) are involved in the pathophysiology of various types of cancers. Among them, FLT3 internal tandem duplications (ITD) and a KIT point mutation at D816V are frequently found in acute myeloid leukemia (AML) and associated with poor prognosis. Although a number of clinical trials were conducted, an effective RTK inhibitor with acceptable toxicity hasn’t been developed.Recent studies have demonstrated these oncogenic RTKs are mislocalized in the cytoplasm, where they transmit aberrant leukemogenic signals to downstream molecules in a compartment-specific manner. So, we speculated that manipulation of their intracellular trafficking between intracellular compartments might be promising as a new therapeutic approach for AML with mutant RTKs. In this study, we evaluated the in vitro and in vivo anti-leukemic effects of chlorpromazine (CPZ) as an inhibitor of membrane trafficking, which is a well-established antipsychotic drug. All of the protocols were approved by the Institutional Review Board of Kinki University Faculty of Medicine, and we obtained AML samples after the written informed consent was given from all the patients.First, to examine the effect of CPZ on the intracellular trafficking of RTK mutants, we introduced murine FLT3-ITD and KIT-D814V (corresponding to human KIT D816V) into murine embryonic fibroblasts (MEFs) isolated from E14.5 fetuses, respectively. Both FLT3-ITD and KIT-D814V were distributed in cytoplasm with a punctate pattern, both of which were mainly located close to endoplasmic reticulum (ER) and Golgi. In contrast, when treated with CPZ, both RTK mutants were predominantly localized in the compartment apart from ER and Golgi in a diffused pattern, indicating that the intracellular trafficking system that retains mutated RTKs at ER or Golgi is disrupted by CPZ treatment.Next, we evaluated the effect of CPZ on cell growth and survival using several human leukemia cell lines and primary AML cells with or without mutant RTK. As a result, CPZ severely inhibited the growth and survival of leukemia cell lines and primary AML cells with mutant RTK in a dose-dependent manner (up to 95% inhibition; IC50: between 1.5 μM and 10.7 μM). In addition, CPZ inhibited the growth of the cells without mutant RTK, whereas the IC50 levels were 3- to 4-fold higher than those seen in the cells with mutant RTK. We also examined the effects of CPZ on the growth and survival of CD34+38- leukemic stem cells (LSCs) derived from AML patients (n=4), and found that CPZ effectively inhibited the growth and/or survival of LSCs, especially in LSCs with mutant RTK (IC50=7.5 μM). As for this mechanism, we found that CPZ significantly suppressed the activities of mutant RTKs and their downstream molecules such as Stat5 in all tested FLT3-ITD(+) LSCs. These results indicate that CPZ inhibits cell growth and/or survival of AML cells and LSCs harboring mutant RTKs. Next, we evaluated the in vivo anti-leukemic effect of CPZ with a xenograft model. Three days after the transplantation of human AML cells with mutant RTKs (n=8), NOD/Scid/IL2Rg-KO (NOG) mice were treated with normal saline or 10 mg/kg CPZ intraperitoneally on everyday schedule. At first, we confirmed that plasma concentration levels of CPZ on this schedule were within the non-toxic range as previously reported (0.03 to 0.71 μM, n=3) and that CPZ doesn’t affect normal hematopoiesis (n=3). Eight to ten weeks after the transplantation, the frequencies of human CD45+ AML cells and CD34+38- LSCs were significantly reduced by CPZ treatment in the bone marrow (BM) of the recipient mice compared with normal saline-treated control (ctrl) mice (CD45+ 58.2% vs 8.47%, p<.001; LSCs 0.51% vs 0.07%, p<.005), indicating that CPZ can inhibit the growth and survival of both AML cells and LSCs in vivo. We further tested the anti-leukemic activities of CPZ on established human AML in a zenograft model. After confirming the engraftment of human AML cells by BM sampling six weeks after transplantation of FLT3-ITD(+) AML cells (n=3), these mice were treated with CPZ or normal saline. In all cases tested, CPZ treatment for 4 weeks significantly reduced human AML fraction in the BM. (18.4% vs 1.99%, p<.005)Together, these results suggest that CPZ, an inhibitor of the intracellular trafficking of leukemogenic RTKs, would be a promising therapeutic drug to eradicate AML cells with an established safety profile. DisclosuresRai:Shionogi & Co., Ltd. : Research Funding. Tanaka:Shionogi & Co., Ltd. : Research Funding. Tanimura:Shionogi & Co., Ltd. : Research Funding. Matsui:Shionogi & Co., Ltd. : Research Funding. Kanakura:Shionogi & Co., Ltd. : Research Funding. Matsumura:Shionogi & Co., Ltd. : Research Funding.

CD82 regulates STAT5/IL‐10 and supports survival of acute myelogenous leukemia cells

We recently reported that adhesion molecule CD82 is aberrantly expressed in CD34(+) /CD38(-) leukemia stem cells (LSCs). Here, we report the results of a functional analysis of CD82 in CD34(+) /CD38(-) acute myelogenous leukemia (AML) cells. Short hairpin (sh)RNA-mediated downregulation of CD82 resulted in a decrease in the level of IL-10. In contrast, forced expression of CD82 in CD34(+)/CD38(+) AML cells by transduction with CD82-expressing lentiviral particles resulted in an increase in the levels of IL-10. Notably, exposure of CD34(+)/CD38(-) AML cells to IL-10 stimulated clonogenic growth of these cells. Moreover, downregulation of CD82 by a shRNA dephosphorylated STAT5 in CD34(+)/CD38(-) AML cells. On the other hand, forced expression of CD82 resulted in increase in the levels of p-STAT5 in CD34(+)/CD38(+) AML cells. Chromatin immunoprecipitation (ChIP) assay results indicated that STAT5A binds to the promoter region of the IL-10 gene, while reporter gene assay results indicated stimulation of IL-10 expression at the transcriptional level. These results suggest that CD82 positively regulates the STAT5/IL-10 signaling pathway. Moreover, shRNA-mediated downregulation of CD82 expression in CD34(+)/CD38(-) AML cells dephosphorylated STAT5 in immunodeficient mice. Taken together, our data suggest that the CD82/STAT5/IL-10 signaling pathway is involved in the survival of CD34(+)/CD38(-) AML cells and may thus be a promising therapeutic target for eradication of AML LSCs.

Bortezomib sensitivity of acute myeloid leukemia CD34+ cells can be enhanced by targeting the persisting activity of NF-κB and the accumulation of MCL-1

Sustained NF-κB activation is often observed in acute myeloid leukemia (AML); therefore, proteasome inhibition has been proposed to efficiently target AML cells. In this study, we questioned whether leukemic stem cell-enriched CD34(+) cells are sensitive to the proteasome inhibitor bortezomib. Surprisingly, we observed in short-term and long-term culture assays that CD34(-) AML cells were more sensitive to bortezomib treatment compared with the CD34(+) AML cells at a clinical relevant dosage. Cotreatment with the apoptosis-inducing cytokine TRAIL did not enhance cell death in CD34(+) AML cells, in contrast to the effects in AML cell lines. The better survival of CD34(+) AML cells upon bortezomib treatment was due to a persisting NF-κB activity that could be overcome by the IKK inhibitor BMS-345541. This difference in sensitivity might be related to differences in NF-κB activation in AML CD34(+) versus CD34(-) cells, as suggested by a gene expression profiling study. Besides NF-κB, MCL-1 strongly determines the effectiveness of bortezomib. MCL-1 accumulated in CD34(+) AML cells upon bortezomib treatment and inhibition of MCL-1 by shRNA, or Obatoclax, significantly improved the sensitivity of CD34(+) AML cells to bortezomib. These results demonstrate that combining bortezomib with specific NF-κB or MCL-1 inhibitors might potentially target the leukemic stem cells.

CD82 Regulates STAT5/IL-10 and Supports Survival of Acute Myelogenous Leukemia Cells.

Abstract 2981We recently reported that adhesion molecule CD82 is aberrantly expressed in CD34+/CD38− leukemia stem cells (LSCs) (ASH 2010; Abstract 2168.). Here, we report the results of a functional analysis of CD82 in CD34+/CD38− acute myelogenous leukemia (AML) cells. Short hairpin (sh)RNA-mediated downregulation of CD82 resulted in dephosphorylation of STAT5 and a decrease in the level of IL-10. In contrast, forced expression of CD82 in CD34+/CD38+ AML cells by transduction with CD82-expressing lentiviral particles resulted in an increase in the levels of p-STAT5 and IL-10. Chromatin immunoprecipitation (ChIP) assay results indicated that STAT5A binds to the promoter region of the IL-10 gene, while reporter gene assay results indicated stimulation of IL-10 expression at the transcriptional level. These results suggest that CD82 positively regulates the STAT5/IL-10 signaling pathway. Notably, exposure of CD34+/CD38− AML cells to IL-10 stimulated clonogenic growth of these cells. Moreover, shRNA-mediated downregulation of CD82 expression in CD34+/CD38− AML cells impaired AML reconstitution in immunodeficient mice in parallel with downregulation of STAT5. Taken together, our data suggest that the CD82/STAT5/IL-10 signaling pathway is involved in the survival of CD34+/CD38− AML cells and may thus be a promising therapeutic target for eradication of AML LSCs. Disclosures:No relevant conflicts of interest to declare.

Anti-Leukemic Activity of the CRM1 Inhibitor KPT-330 in Advanced CML and Ph+ ALL

AbstractAbstract 35As tyrosine kinase inhibitors (TKIs) do not induce long-term response in blast crisis chronic myeloid leukemia (CML-BC) or Philadelphia chromosome-positive acute lymphoblastic leukemia (Ph+ ALL), and are unable to kill quiescent Ph+ hematopoietic stem cells, alternative therapies targeting dysregulated pathways in BCR-ABL1+ acute leukemias are needed. CRM1, a karyopherin aberrantly overexpressed in several cancers, controls the nuclear export of proteins (e.g. ABL1, SET, p53, p21, FOXO and RB) that regulate normal and malignant hematopoietic cell survival, self-renewal and proliferation. Here we show that enhanced CRM1 expression also occurs in Ph+ leukemias and the clinical stage small molecule inhibitor of CRM1, KPT-330, has a detrimental effect on malignant but not normal hematopoietic progenitors.Specifically, a substantial upregulation (≥ 65% increase) in the expression levels of CRM1 was detected by immunoblot in CD34+ progenitors from bone marrow (BM) of leukemic primary samples (n=3), when compared to CD34+ progenitors from healthy (n=3) donors. Interestingly, CRM1 protein levels in CML CD34+ cells were markedly but not totally reduced by treatment with the ABL1 kinase inhibitor Imatinib (1μM, 72h), suggesting that BCR-ABL1-driven pathways are not the only factor contributing to CRM1 upregulation. Additionally, treatment of primary CML cells with KPT-330 (1μM, 72h) resulted in a 75% reduction in BCR-ABL1 kinase activity, consistent with an interrelationship between BCR-ABL1 and CRM1 activities. Finally, the ability of BCR-ABL1 to induce CRM1 expression was demonstrated upon ectopic BCR-ABL1 expression in myeloid 32Dcl3 precursor cells in which CRM1 protein levels became ∼10-fold higher.To determine the therapeutic relevance of CRM1 inhibition, cell survival was assessed in MACS-purified CD34+ progenitors. Treatment with KPT-330 resulted in ≥80% induction of apoptosis in Ph+ (n=5) CML (2μM, 72hr) and B-ALL (500nM, 72h) CD34+ cells. Conversely, KPT-330 exerted only a minimal effect on the survival of CD34+ progenitors from healthy donor BM (15–30% Annexin V positive, KPT-330 0.5–2μM, 72h). Consistent with the existence of BCR-ABL1-independent signaling leading to increased CRM1 expression, marked apoptosis was also observed in KPT-330-treated CD34+ progenitors isolated from a Ph-negative B-ALL patient. Thus, CRM1 inhibitor-based therapies might also benefit BCR-ABL1-negative leukemias. To formally determine the effects of KPT-330 on leukemic cell survival, methylcellulose-based clonogenic assays were performed. KPT-330 treatment (1μM) resulted in almost total suppression (97% reduction) of the clonogenic potential of leukemic but not normal (30% reduction) CD34+ progenitors.Because of evidence that CRM1 directly interacts with the protein phosphatase 2A (PP2A) inhibitor SET, and that CRM1 inhibition alters trafficking of hnRNP A1, a direct regulator of the SET-PP2A interplay in Ph+ leukemias, it is conceivable that the anti-leukemic activity of KPT-330 is, at least in part, mediated by PP2A activation. Indeed, KPT-330 treatment (1μM, 48hr) of BCR-ABL1+ cell lines resulted in full restoration of PP2A activity, comparable to levels observed in BCR-ABL1-negative cells. Accordingly, KPT-330 treatment (1μM, 12h) of 32Dcl3BCR-ABL1 cells caused a nuclear accumulation (4-fold increase) and cytoplasmic decrease (95% lower) of SET protein levels, as indicated by both confocal microscopy and immunoblots of subcellular fractions. Moreover, KPT-330-treated cells showed altered hnRNP A1 cellular distribution and overall downregulation. However, unexpectedly, hnRNP A1 protein levels were proportionally higher in the cytoplasm and lower in the nucleus, suggesting that the deleterious effect of CRM1 inhibition on hnRNP A1 might not depend on direct inhibition of hnRNP-A1 nuclear export.Because KPT-330 displays favorable ADME properties and has been shown to alleviate leukemic burden using in vivo models of different cancers, we are currently testing the anti-leukemic activity of this compound in a mouse model of CML-BC. We do expect a profound inhibition of leukemogenesis, and all treated mice are currently alive with no signs of toxicity. Thus, CRM1 inhibition by KPT-330 represents a potential new therapeutic avenue which could easily be exploited in malignancies like CML-BC and Ph+ ALL that show a dismal outcome to current available therapies. Disclosures:Walker:Karyopharm Therapeutics Inc: Research Funding. Landesman:Karyopharm Therapeutics Inc: Employment. Shacham:Karyopharm Therapeutics: Employment. Kauffman:Karyopharm Therapeutics Inc: Employment. Perrotti:Karyopharm Therapeutics Inc: Research Funding.

Role of Integrin Linked Kinase in Expansion and Chemotaxis of CD34+ Cord Blood Cells.

Abstract 3616 Poster Board III-552 Despite being an excellent alternative to bone marrow or mobilized peripheral blood as a source of hematopoietic stem progenitor cells (HSPCs), the limiting factor to wider use of cord blood (CB) in transplantation is the 10-fold lower cell dose in a typical CB unit compared to harvested bone marrow or peripheral blood stem cells. Successful ex-vivo expansion of HSPCs as well as increasing transplantation efficiency by adopting protocols that enhance homing and engraftment of transplanted HSPC provides hope of making the applicability of a single unit of CB wider in the adult population. Interaction and adhesion of HSPCs with extracellular matrix (ECM) is an important event that regulates cell differentiation, proliferation, survival/ apoptosis as well as migration of HSPCs. Based on evidence present in the literature it appears that in addition to cooperative action between adhesion and growth factors, integrin mediated cell adhesion also provides distinct growth regulatory cues. Although it is known that in hematopoietic system, β1 integrin plays an important role in the interaction of HSPCs with integrin ligands, the underlying molecular mechanisms regulating βl integrin activity in hematopoietic cells remains largely unknown. Based on studies in other systems- Integrin linked kinase (ILK) appears to be an important molecule. ILK not only modulates β1 integrin activity, but also functions as an adapter protein, physically coupling downstream signals from both integrins and growth factors. ILK is preferentially expressed in murine stem / early progenitor cells (based on I. Lemiscka's Stem cell database) and we have found that ILK is expressed in both primitive (CD38-/loCD34+) and committed progenitor (CD38+ CD34+) cells from CB by western blot. Moreover, using a co-immunoprecipitation approach, we found that in freshly isolated CD34+CB cells ILK interacts with β1 integrin. To investigate the role of ILK in adhesion-dependent and -independent growth of CB CD34+ cells both in the presence and absence of growth factors we expressed constitutive active ILK (CAILK) or dominant negative ILK (DNILK) in CD34+CB cells. In addition, we have also evaluated the effect of modulating ILK activity on chemotaxis of CD34+ CB cells towards stromal derived factor-1 (SDF-1)/CXCL12. We found that expression of CAILK enhances expansion of total CD34+ cells as well as colony forming cells stimulated ex-vivo by growth factors (stem cell factor- SCF; Flt-3 ligand and thrombopoietin-TPO), compared to cells transfected with vector alone. In contrast, expression of DNILK inhibited expansion of CD34+CB cells; this effect was more pronounced when cells were cultured in the absence of fibronectin, the ECM. Expression of CAILK increases cell-cycling since a greater proportion of cells were in ‘S’-phase compared to cells expressing DNILK or vector alone, both when the cells were expanded in the presence or absence of fibronectin for 20h. Expression of CAILK also leads to improved survival of CD34+ cells in the absence of serum and growth factors. Interestingly, β-catenin could be detected in CD34+ cells expressing CAILK, but not in cells transfected with DNILK or vector alone. Furthermore, in response to growth factor stimulation, Akt is phosphorylated in cells expressing either CAILK or DNILK; however, the amount of Akt phosphorylation was lower in cells expressing DNILK. Expression of CAILK improved modestly but not significantly the chemotaxis of CD34+ cells towards SDF-1 compared to cells expressing vector alone. However, DNILK significantly impaired chemotaxis of CD34+CB cells towards SDF-1. This impairment of chemotaxis is associated with defective actin polymerization in response to SDF-1, both at the ‘leading-edge’ and ‘tail’ of a polarized DNILK expressing CD34+ cell. Our findings implicate a role for ILK in both growth factor stimulated ex-vivo expansion of HSPCs as well as SDF-1 mediated chemotaxis. This may have potential implications in the therapeutic use of CB cells. Disclosures: No relevant conflicts of interest to declare.

Notch ligands potentiate IL‐7‐driven proliferation and survival of human thymocyte precursors

Notch and IL-7 are both well-characterized factors involved in T-cell development. In contrast to the mouse model, their precise requirements in the differentiation and/or proliferation of various stages of human thymic development have not been fully explored. Here, we demonstrate that IL-7 alone is sufficient to induce the differentiation of ex vivo purified CD34(+) triple negative (TN) surface (s) CD3(-) CD4(-)CD8(-) (CD3(-)CD4(-)CD8(-)), CD4 immature single positive (ISP) (sCD3(-)CD4(+)CD8(-)) and double positive (DP) (sCD3(-)CD4(+)CD8(+)) human thymic precursors to mature DP expressing sCD3 (sCD3(+)CD4(+)CD8(+)). We show that activation of Notch signaling by its ligands Delta-1 or Delta-4 potentiates IL-7-driven proliferation and survival of CD34(+) TN and to a lesser extent of CD4(+) ISP precursors. This effect of Notch is related to a sustained induction of IL-7 receptor alpha chain expression on thymocytes through a decreased methylation of its gene promoter. Thus, we show here that proliferation and differentiation of T-cell precursors are differentially modulated by IL-7 depending on the presence or absence of external signals. These results may have important implications for the clinical use of this cytokine as a strategy aimed at improving immune restoration.

The Effect of Cryopreservation on Clonogenic Capacity and In Vitro Expansion Potential of Umbilical Cord Blood Progenitor Cells

Background Umbilical cord blood progenitor cells have been demonstrated to possess significant advantages over bone marrow in terms of proliferative capacity and immunologic reactivity. But the low number of hematopoeitic stem cells (HSC) is the most important limitation of its use. The ex vivo expansion of cord blood progenitor cells is the current strategy to overcome this problem. Futhermore, among the factors that enable successful cord blood transplantation is the ability to store and subsequently recover a sufficient number of viable cells. Since it would be costly to expand umbilical cord blood (UCB) progenitor cells, it is important to determine the feasibility and reproducibility of progenitor cell expansion after cryopreservation. We evaluated whether cryopreservation procedures might impair the clonogenic capacity and in vitro expansion of UCB. Materials and methods We evaluated the cell viability, clonogenic capacity, CD34 +38 − content and in vitro expansion potential of progenitor cells from UCB ( n = 10) separated mononuclear cells (MNC), before and after 1 month of cryopreservation by programmed rate freezing. Results Although cell viability decreased after cryopreservation ( P < .05), there was no significant difference in CD34 + or CD34 +38 − absolute count, colonogenic capacity and in vitro expansion potential of cord blood progenitor cells ( P > .05). Conclusions Since the survival of CD34 + cells was greater than other elements, CD34 + cells seem more tolerant to cryopreservation than the other nucleated populations. Moreover in vitro expansion of UCB progenitor cells may be obtained following cryopreservation. Our results suggest that cryopreservation procedures do not impair the clonogenic capacity and in vitro expansion potential of cord blood stem/progenitor cells.

Tacrolimus: In Vitro Effects on Myelopoiesis, Apoptosis, and CD11b Expression

Tacrolimus is a common component of multi-drug immunosuppressive regimens that are used for the prevention of rejection in transplant recipients. Tacrolimus therapy has been associated with anemia after transplantation, and recent clinical evidence in children suggests its association with the development of neutropenia for which an alternative etiology is not apparent. Mechanisms of suspected tacrolimus-related neutropenia have not been previously elucidated. We hypothesized that this variety of neutropenia might be due to a negative effect of tacrolimus on neutrophil production and/or survival. We designed in vitro studies to determine the dose-dependent effect of tacrolimus on myeloid cell production and/or apoptosis. CD34+ cells and neutrophils isolated from umbilical cord blood of term gestations were cultured with tacrolimus (0-1,000 ng/ml). To evaluate apoptosis, cells cultured for 24 hours were stained with annexin V-fluorescein isothiocyanate (V-FITC) and 7-amino-actinomycin D (7-AAD) and analyzed by flow cytometry. For clonal analysis, CD34+ cells cultured in cytokine-enhanced semi-solid media were scored for their myeloid/erythroid mix colony forming units (CFU-Mix) and myeloid (CFU-GM) progenitor cell contents. Tacrolimus induced a dose-dependent enhancement of clonogenesis and survival of CD34+ cells at clinically relevant doses. Conversely, tacrolimus had no effect on the survival of mature neutrophils or on the upregulation of CD11b in response to chemotactic stimulation. In contrast to our initial hypothesis, we observed that tacrolimus at clinically relevant concentrations enhanced clonogenesis of neutrophil progenitors and promoted their survival. Our in vitro studies suggest that tacrolimus alone is unlikely to be a significant factor in the neutropenia observed during immunosuppressive therapy.

The viability of cryopreserved PBPC depends on the DMSO concentration and the concentration of nucleated cells in the graft

DMSO is widely used as a cryoprotectant for PBPC. It is desirable to reduce the amount of DMSO without jeopardizing the quality of the stem cell product. The present study was undertaken to investigate whether recovery and survival of CD34+ cells would be significantly altered when PBPC used for autologous transplantations were cryopreserved with four different DMSO concentrations. Apheresis samples of PBPC from 20 consecutive patients were mixed in parallel with 2%, 4%, 5% and 10% DMSO, frozen with identical cell concentrations at a controlled rate, and stored in liquid nitrogen for 6-8 weeks. PBPC samples from 11 consecutive patients were also cryopreserved with two different cell concentrations (150 and 300 x 10(6) nucleated cells/mL) to investigate the effect of increasing the cell concentrations while decreasing the DMSO concentration. The flow cytometric absolute count method, based on ISHAGE guidelines, was used to measure the absolute count of total and viable CD34+ cells in the post-thaw samples. PBPC cryopreserved at 150 x 10(6) cells/mL with 2% DMSO yielded significantly inferior CD34+ cell recovery (P < 0.001) and survival (P < 0.001) compared with cryopreservation with 4% and 5% DMSO. This was also observed when comparing higher cell concentrations. However, a reduced cell survival (P = 0.02) was observed when the nucleated cell concentration was increased from 150 to 300 x 10(6) cells/mL in samples cryopreserved with 5% DMSO. We conclude that 5% DMSO may be the optimal dose for cryopreserving PBPC as long as the cells have not been concentrated at much more than 200 x 10(6) nucleated cells/mL.

CD34‐positive acute promyelocytic leukemia is associated with leukocytosis, microgranular/hypogranular morphology, expression of CD2 and bcr3 isoform

Acute promyelocytic leukemia (APL) has a favorable prognosis. Current therapy includes chemotherapy used in combination with all-trans-retinoic acid (ATRA). Although the differentiating effects of ATRA on promyelocytes have been well established, in vitro studies have shown that less-differentiated APL blasts (CD34(+)) demonstrate a variable responsiveness to ATRA. To assess the clinical relevance of this finding, we analyzed a cohort of 38 patients with t(15;17) and/or PML-RARalpha APL to determine the incidence and laboratory features of CD34(+) APL. Thirty-two percent (12/38) of cases were CD34(+). There was a difference in WBC at presentation between CD34(+) and CD34(-) cases (34.6 +/- 9.2, mean +/- standard error vs. 5.4 +/- 2.0, P = 0.009). Patients with CD34(+) APL demonstrated a micro/hypogranular phenotype (75%) (P = 0.001), co-expression of CD2(+) (83%) (P = 0.001), and the bcr3 isoform (100%) (P = 0.017). In contrast, CD34(-) cases demonstrated hypergranular morphology (65%), CD2(+) (15%), and the bcr1 isoform (50%). A high presenting WBC count (\G10 x 10(9)/L) was associated with an inferior overall survival (Log rank = 0.0047). Patients with CD34(+) APL demonstrated an incidence of early mortality of 50%. Despite a marked correlation between CD34 positivity and increased WBC count, overall survival of CD34(+) and CD34(-) cases did not differ significantly in our small cohort. Immunophenotypic analysis for CD34 expression should be included in future large APL trials to determine if detection of CD34(+) blasts represents an independent adverse prognostic factor.

Effect of nitric oxide production and oxygen tension on progenitor preservation in ex vivo culture

Many cell types are capable of expressing inducible nitric oxide synthase (iNOS) in response to cytokines or endotoxin. We detected iNOS mRNA by reverse transcriptase polymerase chain reaction in CD34 + human bone marrow cells after 48-hour incubation with interferon-gamma (IFN-γ)/tumor necrosis factor alpha (TNF-α) and IFN-γ/ lipopolysaccharide. Based on this finding, we examined the effect of nitric oxide (NO) on hematopoiesis and particularly on proliferation and survival of CD34 + marrow cells in in vitro culture. When CD34 + cells were cultured in the presence of an NO donor, S-nitroso-N-acetyl-D,L-penicillamine (SNAP), a dose-dependent inhibition of cell proliferation was observed. Addition of the selective iNOS inhibitor, L-N 6-(1-iminoethyl)-lysine hydrochloride (L-NIL) at a dose of 500 μM increased the cell counts by 23% (range 0–89%). The expansion of total CD34 + cell number was 4-fold with a hematopoietic cytokine cocktail compared to 5.2-fold with the addition of L-NIL to this combination. At days 7 and 14 of culture, SNAP induced apoptosis in CD34 + human bone marrow cells detected by an in situ terminal deoxynucleotidyl transferase assay. The apoptosis was partially inhibited with the addition of L-NIL. SNAP also inhibited cell cycling, as evidenced by a 56% decrease in the number of cells in S phase after 5 days of culture in the presence of SNAP. To investigate if NO production was dependent on oxygen tension, J774A mouse macrophage cells were induced with lipopolysaccharide/IFN-γ, and nitrite production measured by the Griess reaction. Nitrite production was persistently less in 5% compared to 21% oxygen. CD34 + marrow cells from normal donors were also grown under variable oxygen tension, and the cell proliferation in 5% oxygen was significantly greater at both 7 and 14 days of culture. CFU-GM colony growth also was increased in the low oxygen setting. The concentration of varous cytokines was measured in CD34 + progenitor culture supernatants, including interleukin (IL)-1α, IL-1β and TNF-α. SNAP increased IL-1α production by CD34 + cells as well as from light-density bone marrow cells, whereas the effect on IL-1β and TNF-α production was not significant. Manipulation of oxygen tension and the inhibition of production of reactive oxygen and nitrogen intermediates may have potential to optimize cell expansion and progenitor preservation in ex vivo culture systems.

Effect of stromal cell coculture on progenitor cell expansion and myeloid effector function in vitro.

Stimulation of CD34(+)-enriched marrow or light density marrow with various growth factor combinations can generate granulocyte progenitors and mature neutrophils in vitro. In this work, we have examined the influence of irradiated marrow stromal layers on growth factor-induced myeloid and early multipotential progenitor expansion from enriched marrow CD34+ progenitors. We have also explored whether the addition of early-acting growth factors known to enhance myelopoiesis in long-term culture, such as fibroblast growth factor (b-FGF), insulin growth factor (IGF-1), c-kit ligand or stem cell factor (SCF), and flk-2flt-3 ligand (FL), can lengthen survival of CD34+ progenitors in these cultures. Stromal cell coculture resulted in greater numbers of total cells and CFU-GM at day 7 and day 14, but with the addition of multiple growth factors, these effects of stromal cell coculture were diminished. At day 14, generally < 1% of the expanded cells over stromal coculture conditions were CD34+, with up to 90% demonstrating CD15 positivity. Culture of CD34+ cells in the presence of early-acting growth factors did not cause significant expansion of CD34+ cells over a 14-day life span, even in the presence of marrow stromal cells. These data suggest that although stromal cell coculture for a period up to 14 days can enhance expansion of total cell numbers and CFU-GMs, stromal cell presence does not lead to expansion of CD34+ cells in these cultures and may diminish the number of clonogenic cells present when growth factors with differentiating capacity are present. Mature neutrophils harvested from such cultures are capable of chemotaxis, actin polymerization, and migration, suggesting a replete functional status.

Nerve growth factor is involved in the supportive effect by bone marrow- -derived stromal cells of the factor-dependent human cell line UT-7 [published erratum appears in Blood 1996 Oct 1;88(7):2818

We previously demonstrated that murine MS-5 and SI/SI4 cell lines induce the proliferation of human factor-dependent UT-7 cells in the absence of normally required human cytokines and also stimulate the differentiation of CD34+/CD38-LTC-ICs. We report in this study that the effect of MS-5 cells on UT-7 cells can be completely explained by the synergistic action of nerve growth factor (NGF) and stem cell factor (SCF) produced by these murine stromal cells. Purified murine NGF was able to support short-term clone formation and long-term growth of UT-7 cells in suspension cultures as efficiently as rhu-granulocyte-macrophage colony-stimulating factor. NGF action was mediated through the TrkA receptor, in which messenger RNA (mRNA) was easily detected in UT-7 cells by Northern blot. MS-5 cells strongly expressed NGF mRNA in Northern blot, and direct implication of MS-5-derived NGF in the induction of UT-7 cells proliferation was demonstrated in inhibition assays with an anti-NGF monoclonal antibody (MoAb) that neutralized by 84% +/- 4.1% (n = 5) UT-7 clone formation. However, NGF did not act alone, and several arguments demonstrated the synergistic action of MS-5-derived SCF: (1) an anti-c-kit partially inhibited UT-7 cells clone formation in coculture assays, (2) SCF and NGF synergized in an H3-TdR incorporation assay, and (3) the stimulatory effect of 10x-concentrated MS-5 supernatant was completely inhibited by an anti-c-kit but not by an anti-NGF, and levels of soluble NGF (1.2 ng/mL) detected by enzyme-linked immunosorbent assay in 10x supernatant of MS-5 cells cultures were below the biologically active concentrations. In contrast, although MS-5 cells also promoted the differentiation of very primitive CD34+/CD38- human stem cells both in colony assays and long-term cultures, we could not incriminate MS-5-derived NGF in the observed effect: an anti-NGF MoAb did not inhibit the synergistic effect of MS-5 cells in colony assays or long-term cultures nor did soluble muNGF duplicate MS-5 effect and survival of CD34+/CD38- clonogenic progenitor cells promoted by MS-5 was unaffected by an anti-NGF and was not induced by soluble NGF alone or combined with SCF. In contrast, NGF in synergy with SCF supported the short-term maintenance of high numbers of CD34+/CD38+ mature erythroid progenitors probably through an indirect mechanism implying macrophages. These results suggest that NGF, in which the primary target cells are outside the hematopoietic system, is present in the marrow environment and might act at some steps of hematopoietic stem cell development. These results also underline that the response of cell lines and normal stem cells to stromal cells is mediated by different pathways.