HERG K+ Channel Is Essential for Leukemic Cell Migration Induced by SDF-1.

Abstract Background: Recent studies suggest that HERG K+ channel is an important regulator of non excitable cell proliferation and migration, and has been found in tumor cells including acute myeloid leukemia(AML), where HERG K+ channel is generally considered to be absent from their healthy counterparts. Bone marrow stromal cells constitutively secrete the stromal cell-derived factor-1 (SDF-1) which is a homeostatic chemokine that signals through CXCR4, SDF-1/CXCR4 axis and plays an important role in hematopoiesis development and leukemic cells migration. In this study, we investigated whether SDF-1-induced leukemic cell migration associated with HERG K+ channel. Methods: primary CD34+/CD38− leukemic stem cells (LSCs) were isolated by cell sorting using a FACS Vantage. Transwell was used to assess the effect of E-4031, a specific HERG K+ channel inhibitor, on leukemic cell migration, the lower chamber was filled with serum-free RPMI-1640 with 100ng/ml SDF-1. Flow cytometry was used to analyze the CXCR4 expression as well as phenotypical analysis of leukemia samples. HERG K+ channels were expressed in Xenopus oocyte by microinjection and the resulting currents were measured using the standard two microelectrode voltage clamp techniques. Results: numbers of HL-60 cells with and without E-4031 treatment migrated towards SDF-1 in the lower chamber were 1.58±0.98 ×104 and 3.47±0.81 ×104 respectively, indicating E-4031 significantly blocked the cell migration induced by SDF-1. The similar results were also observed in primary leukemic cells (n=7) and leukemic stem cells(n=3). From a holding potential of −80 mV varying potentials from −70 mV to +50 mV in 10 mV increments (2s) were applied to elicit activating currents. Each pulse was followed by a constant return pulse to −50 mV (2s) to evoke outward tail currents. 100 ng/ml SDF-1 increased HERG K+ current expressed in oocytes, for example, at +50 mV, HERG current increased about 30% (n=5). The HERG K+ current increase by SDF-1 might contribute to the mechanism of SDF-1 induced leukemic cell migration. There were no significant changes of CXCR4 expression on both HL-60 cells and primary leukemic cells regardless of untreated and treated with E-4031 for 24 hours (p>0.05), suggesting that the leukemic cell migration induced by SDF-1 were specifically associated with HERG K+ channel, not by regulating CXCR4 expression. Conclusion: the data showed that HERG K+ channel was essential for leukemic cell migration induced by SDF-1. SDF-1 enhanced herg current suggested that SDF-1 promotes leukemic cell migration. Blocking HERG K+ channel with specific inhibitor could decrease leukemic cell and leukemic stem cell migration caused by SDF-1. Prospectively, HERG K+ channel may be a potential therapeutic target with specific inhibitors in leukemia treatment.

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Competition In Engraftment of Normal Hematopoietic Stem Cells and Leukemic Stem Cells

Blood ◽

10.1182/blood.v116.21.4836.4836 ◽

2010 ◽

Vol 116 (21) ◽

pp. 4836-4836

Author(s):

Gyeongsin Park ◽

Michael Heuser ◽

Tobias Berg ◽

R. Keith Humphries

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Hematopoietic Stem Cells ◽

Bone Marrow Cells ◽

Conflicts Of Interest ◽

Leukemic Cell ◽

Fixed Number ◽

Leukemic Cells ◽

Leukemic Stem Cells ◽

Hematopoietic Stem

Abstract Abstract 4836 Engraftment is a process including homing to bone marrow, implantation and proliferation. Implantation implies interactions with specialized microenvironments, niches, in which hematopoietic stem cells (HSCs) live and are regulated. Studies have demonstrated the possibility that leukemic stem cells (LSCs) interact with niches in a similar manner to HSCs. We investigated whether HSCs and LSCs compete with each other in their engraftment. We employed a mouse transplantation assay with unmanipulatated bone marrow cells (BMCs) as a source of normal HSCs and LSCs generated by transduction of BMCs with Meningioma 1 (MN1), a potent oncogene causing myeloid leukemia in mice. In irradiated recipients (750 cGy), cotransplantation of leukemic cells (1×105) with various numbers of BMCs (1×105, 1×106 and 1×107) demonstrated that the engraftment level of leukemic cells is influenced by BMCs in a dose dependant manner (5.2%, 41.3% and 82.2% at 2-weeks; 52.3%, 69.5% and 86.9% at 4weeks; mice died before the 5 weeks bleeding, 94.9% and 97.5% at 5weeks, respectively). Cotransplantation of various numbers of leukemic cells (1×104, 1×105 and 1×106) with a fixed number of BMCs (1×106) demonstrated a similar pattern of leukemic engraftment (7.0%, 59.5% and 87.1% at 2weeks; 62.0%, 85.7% at 4 weeks, and mice died before the four week bleeding, respectively). To further elucidate the competition between HSCs and LSCs, we transplanted the cells at different time intervals. Transplantation of normal BMCs (1×106) 2 days prior to transplantation of LSCs (1×105) resulted in much reduced levels of leukemic engraftment compared to that seen in mice simultaneously transplanted (3.5% vs 59.5% at 2 weeks; 73.1% vs 85.76% at 4weeks). This competitive suppression of leukemic engraftment was further enhanced by transplanting larger numbers of normal BMCs (2×107) as little as 12 hours prior LSC transplantation (5×105) compared to simultaneous injection (0% vs 7.26% at 2weeks, 0.9% vs 35.3% at 3 weeks, and 6.0% vs 60.6% at 4 weeks). When BMCs (1×105) or leukemic cells (1×105) were transplanted at equal doses of 1×105 together with normal helper cells (1×106) the leukemic cells expanded 280-fold compared to only 7.3 fold for normal BMCs at 2 weeks (total cell count from two femurs and two tibias per 1×105 transplanted cells). Thus the competitive suppression of leukemic cell growth seen upon sequential transplantation of normal BMCs is not readily explained by enhanced kinetics of normal BMC growth but rather by competition at the level of initial engraftment. In conclusion, our data demonstrate that there is a competition between normal and leukemic cells during the engraftment process, suggesting niche competition of HSCs and LSCs. Disclosures: No relevant conflicts of interest to declare.

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The Target Receptor Siglec-3 (CD33) Is Expressed on AML Stem Cells in a Majority of All Patients with AML.

Blood ◽

10.1182/blood.v106.11.4324.4324 ◽

2005 ◽

Vol 106 (11) ◽

pp. 4324-4324

Author(s):

Alexander W. Hauswirth ◽

Stefan FLorian ◽

Maria-Theresa Krauth ◽

Gerit-Holger Schernthaner ◽

Edgar Selzer ◽

...

Keyword(s):

Stem Cells ◽

Acute Myeloid Leukemia ◽

Myeloid Leukemia ◽

Staining Technique ◽

Scid Mice ◽

Leukemic Cells ◽

Cytostatic Drug ◽

Leukemic Stem Cells ◽

Targeting Therapy ◽

Acute Myeloid

Abstract The cell surface antigen Siglec-3 = CD33 is becoming increasingly important as target of therapy in acute myeloid leukemia (AML). In particular, a conjugate consisting of the humanized CD33 antibody P67.6 (gemtuzumab) and the cytostatic drug calicheamicin has been developed for clinical use and was found to work as an effective antileukemic agent (Mylotarg®) in patients with CD33+ AML. In normal myelopoiesis, expression of CD33 is restricted to advanced stages of differentiation, whereas primitive stem cells do not express CD33 (Siglec-3). In line with this notion, CD33-targeting therapy is a non-myeloablative approach. In the present study, we asked whether leukemic stem cells in patients with AML express CD33. For this purpose, a multicolor-staining technique was applied in eleven patients with AML. Leukemic stem cells were defined as CD34+/CD38−/CD123+ cells. In all patients in whom the majority of myeloblasts expressed CD33 (=CD33+ AML, n=8), the AML progenitor cells reacted with the CD33 antibody P67.6. Repopulation experiments utilizing NOD/SCID mice confirmed that the AML stem cells in these patients reside within the CD33+ subpopulation of leukemic cells. Moreover, AML stem cells (CD34+/CD38−/CD123+ cells) highly purified (>98% purity) from patients with (CD33+) AML by cell sorting, were found to express CD33 mRNA in RT-PCR analyses. To demonstrate that AML stem cells can also reside within the CD33-negative fraction of the AML clone, we purified CD33-negative cells in a patient with AML in whom a majority of leukemic stem cells were found to lack CD33. In this particular patient, the CD33-negative cells were found to repopulate NOD/SCID mice with leukemias in the same way as the entire leukemic clone did. The CD33 antigen was neither detectable on CD34+/CD38− cells in the normal bone marrow nor on leukemic stem cells in patients with CD33-negative AML. In summary, our data show that leukemic stem cells in patients with CD33+ AML frequently express the target receptor CD33. This observation is in favor of novel treatment concepts employing CD33-targeting antibodies (Mylotarg®) in acute myeloid leukemia.

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The Transcription Factor ELF4 Promotes Survival of Myeloid Leukemic Stem Cells.

Blood ◽

10.1182/blood.v116.21.1209.1209 ◽

2010 ◽

Vol 116 (21) ◽

pp. 1209-1209

Author(s):

Chun Shik Park ◽

Koramit Suppipat ◽

H. Daniel Lacorazza

Keyword(s):

Stem Cells ◽

Transcription Factor ◽

Kinase Inhibitor ◽

Conflicts Of Interest ◽

Blast Crisis ◽

Philadelphia Chromosome ◽

Lymphocytic Leukemia ◽

Leukemic Cells ◽

Leukemic Stem Cells ◽

Hematopoietic Stem

Abstract Abstract 1209 Chronic myeloid leukemia (CML) is a myeloproliferative disease that originate in hematopoietic stem cells (HSCs) as a result of the t(9;22) translocation, giving rise to the Ph (Philadelphia chromosome) and BCR-ABL oncoprotein. Although treatment of CML patients with tyrosine kinase inhibitor can efficiently eliminate most leukemic cells, chemoresistant leukemic stem cells (LSCs) can survive and drive recurrence of CML in these patients. A number of genes have been described to promote or inhibit proliferation of LSCs. Some of them have similar roles in normal HSCs. The transcription factor ELF4 promotes cell cycle entry of quiescent HSCs during homeostasis (Lacorazza et al., 2006). Thus, to investigate the function of ELF4 in CML initiation and maintenance, we developed a BCR-ABL-induced CML-like disease using retroviral transfer of BCR-ABL in Elf4-null bone marrow (BM) cells. We first investigated whether ELF4 is required for the induction of CML. Recipient mice of BCR-ABL-transduced WT BM cells developed CML and died with a latency 16–23 days, whereas recipient mice of BCR-ABL-transduced Elf4-/- BM cells showed longer latency of 45–47 days (n=20; p<0.0005). Progression of leukemia was monitored in peripheral blood, BM and spleen by flow cytometry. In mice transplanted with BCR-ABL-transduced Elf4-null BM cells, Gr-1+ leukemic cells expanded the first two weeks after BM transplantation followed by a decline at expense of a secondary expansion of B220+ cells. In contrast, Gr-1+ leukemic cells continuously expanded in mice receiving BCR-ABL-transduced WT BM cells. These results suggest that loss of ELF4 causes a profound abrogation in BCR-ABL-induced CML, while allowing progression of B-cell acute lymphocytic leukemia. Since loss of Elf4 led to impaired maintenance of myeloid leukemic cells, we postulated that ELF4 may affect survival of LSCs. Thus, we analyzed the frequency of Lin-c-Kit+Sca-1+ (LSK) cells that are BCR-ABL positive in BM and spleen. We found that BCR-ABL+ LSK cells were significantly reduced in recipients of BCR-ABL-transduced Elf4-/- BM cells. These studies indicate that ELF4 is essential to maintain the LSC pool in CML acting as a molecular switch between myeloid and lymphoid blast crisis. Disclosures: No relevant conflicts of interest to declare.

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Role of hERG1 K+ Channels as a Positive Regulator In SDF-1alpha-Mediated Proliferation of Leukemia Cells and Leukemia Stem/Progenitor Cells

Blood ◽

10.1182/blood.v116.21.4786.4786 ◽

2010 ◽

Vol 116 (21) ◽

pp. 4786-4786

Author(s):

Fang Zheng ◽

Huiyu Li ◽

Fang Liu ◽

Wen Du ◽

Shiang Huang

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Cell Proliferation ◽

Progenitor Cells ◽

Signaling Pathway ◽

Cell Apoptosis ◽

Leukemic Cell ◽

Bone Marrow Microenvironment ◽

Leukemic Cells ◽

K Channels

Abstract Abstract 4786 Background: Mounting evidence that leukemia stem cells (LSCs) occupy and receive important signals from specialized areas (“niches”) that alter the stromal microenvironment and disrupt normal hematopoiesis. The innovative therapeutic strategies focus on targeting of microenvironmental interactions in leukemia. Therefore, it is important to fully elaborate the mechanisms of microenvironment- mediated leukemogenesis. Stromal-cell derived factor-1alpha (SDF-1à) is the main cytokine produced by bone marrow stromal cells. The SDF-1à/CXCR4 axis specifically mediates homing and migration of leukemic blasts. While our previous work has shown that SDF-1à significantly increases hERG1 K+ tail current and a specific hERG1 K+ channels inhibitor significantly blocks SDF-1à- induced migration of leukemic cells. In fact, recent studies suggested that the human ether à-go-go-related gene (HERG) K+ channels are constitutively expressed in AML stem/progenitor cells, and regulate cell proliferation as well as clinical prognosis. Here we investigate the hypothesis that a new leukemic blast–stromal interaction is mediate by hERG1 K+ channels and SDF-1à. Methods: Proliferation assay, apoptosis and cell cycle analysis were used to analyze effects of E-4031(a specific hERG1 K+ channels inhibitor) in the presence of SDF-1à on leukemia cell lines HL-60. RT–PCR and western blot analysis were used to determine changes in herg1 expression and Wnt/β-catenin signaling pathway in response to SDF-1à in the presence and absence of E-4031. Primary leukemias obtained from the bone marrow of de novo AML patients (n=6) at diagnosis. Mononuclear cells were isolated from the samples using Ficoll-Paque density gradient separation, and cultured with SDF-1à in the presence and absence of E-4031. AML colony-forming cell (CFC) assays and flow cytometry were performed to assess the effects of E-4031 in the presence of SDF-1à on LSCs. Results: SDF-1a enhanced cell proliferation in a dose-dependent manner. The maximal increase by 1.6 times was obtained for 100ng/ml. While this effect was impaired by E-4031, which significantly impaired cell proliferation induced by SDF-1a with a concentration of 100ng/mL by (40.3±8.4)%. In addition, E-4031 inhibited SDF-1a-stimulated leukemic cell proliferation by inducing G0/G1 arrest. Cell apoptosis analysis revealed that either E-4031 or SDF-1a has direct effect on HL-60 cell apoptosis. Unexpected, there was no significant synergistic effect upon apoptosis. After exposures to 100ng/ml SDF-1à, hERG1 mRNA and protein levels increased significantly, by approximately 1.5-fold above control levels. Moreover, SDF-1a increased the expression of Wnt/β-catenin target genes, including β-catenin, cyclin-D1, and c-myc. Interestingly, this manner was abolished by E-4031. The presence of progenitor cells was evaluated by plating suspension cells cultured with SDF-1a in CFC assays. E-4031 decreased numbers of CFC in suspension to 77.3%. Upon expansion with SDF-1a, E-4031 resulted in a significant reduction in the number of progenitors to 31.8%. The effects on LSCs were determined on phenotypically described stem cells from AML. Treatment with 1μ M E-4031 for 48 hours inhibited the proliferation of LCSs compared with untreated controls, a mean viability of 11.8% for CD34+CD38- and 10.4% for CD34+CD38+. In contrast, a significant decrease in the viability of stem cells after E-4031 in the present of SDF-1a treatment, with only 9.6% for CD34+CD38- and 9.5% for CD34+CD38+. Conclusions: Initial studies provided evidence that the hERG1 K+ channels and SDF-1 emerged as mediators of stromal/leukemic cell interactions, which largely contribute to the proliferation mediated by the microenvironment. Likewise, other components of bone marrow microenvironment, such as Wnt/β-catenin signaling pathway, may modulate hERG1 K+ channels in leukemic cells. Taken together, these results provided rationale for studies of new molecular events involved in bone marrow microenvironment and leukemogenesis. Disclosures: No relevant conflicts of interest to declare.

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Whole Genome Sequencing Of Chronic Myeloid Leukemia (CML)-Derived Induced Pluripotent Stem Cells (iPSC) Reveals Faithful Genocopying Of Highly Mutated Primary Leukemic Cells

Blood ◽

10.1182/blood.v122.21.514.514 ◽

2013 ◽

Vol 122 (21) ◽

pp. 514-514

Author(s):

Ivan Sloma ◽

Maria Teresa Mitjavila-Garcia ◽

Olivier Feraud ◽

Noufissa Oudrhiri ◽

Lucie Tosca ◽

...

Keyword(s):

Stem Cells ◽

Chronic Myeloid Leukemia ◽

Pluripotent Stem Cells ◽

Myeloid Leukemia ◽

Leukemic Cell ◽

Jak2 V617f ◽

Leukemic Cells ◽

Whole Genome ◽

Induced Pluripotent ◽

Short Indels

Abstract Genetic instability is a hallmark of chronic myeloid leukemia (CML). Recently, several major abnormalities in DNA repair mechanisms have been identified in primitive CML cells that likely explain the additional mutations these cells develop leading to their selective growth under tyrosine kinase inhibitor (TKI) therapies. It seems likely that such mechanisms also underlie disease progression in CML. However, an understanding of the specific somatic mutations involved and investigations of their resulting effects on the biological behavior of primary sources of primitive chronic phase (CP) CML cells is extremely challenging. As an alternative approach, we have now explored the possibility of applying whole genome sequencing (WGS) to induced pluripotent stem cells (iPSCs) derived from primitive CML cells to determine if such iPSCs, genocopy the mutations present in the diagnostic sample from which they were generated and whether primitive hematopoietic cells derived from these iPSCs might be useful for future drug screening experiments. To this end, we chosen a CML patient whose CP clonogenic cells contained both the Ph1 chromosome and the JAK2 V617F mutation and whose disease progressed into an accelerated phase (AP) during TKI therapy. iPSC were generated from leukemic cells obtained at the time of AP using Oct4, Sox2, Klf4 and c-Myc gene transfer. The presence of both BCR-ABL and JAK2 V617F was confirmed in 24/24 iPSC colonies. A control iPSC line negative for both genes was similarly established from the patient’s CD34+CD31+ endothelial progenitors purified from peripheral blood. We then performed WGS on DNA prepared from the leukemic cells obtained at diagnosis of CP (CML 006), the AP cell-derived iPSCs (PB34), and the control non-leukemic iPSCs (PB13), using a HighSeq Illumina platform. WGS revealed 845,175 somatic SNVs and 68,817 somatic short Indels in the CP leukemic cells at diagnosis that were not present in the non-leukemic iPSCs (PB13). 49,225 of these SNVs and 11,665 of the short Indels were novel (absent in the dbSNP database), and 419 were found in the COSMIC database. We identified 274 novel SNVs (3 missense, 161 nonsense, 108 synonymous and 2 splice site mutations) and 46 short Indels (19 insertions and 27 deletions). Most of the novel coding SNVs and Indels were heterozygous and an estimation of the variant allele frequency indicated these were present in virtually all leukemic cells. In addition to the JAK2 V617F mutation that was present at diagnosis, we found a novel frame shift mutation in exon 12 of ASXL1 gene (p.S871YfsX5) leading to protein truncation, a genetic event that has also been associated with myeloproliferative neoplasms (MPNs) and AML. We also identified several novel SNVs predicted by SIFT, Provean and PolyPhen-2 algorithms to be deleterious for protein structure. These novel mutations were found in genes relevant for the pathophysiology of MPNs, including the catenin (CTNNA1 R204C, and AIDA K235T), RAS (RREB1 P789T), autophagy (ULK1 R553C) cellular antioxidant defense (GSR S293C), RNA nuclear transport (NUP160 start loss) pathways. Individual sequencing confirmed the presence of these mutations in PB34 and their absence in PB13 (non-leukemic iPSC). We next compared the sequence data from the AP leukemic cell-derived iPSCs (PB34) with the diagnostic data (CML006). This analysis showed only 799 additional somatic SNVs and 96 new short Indels compared with those already evident in the cells present at diagnosis. Only 4 (3 non synonymous and 1 synonymous) SNVs and no Indels were found in exons. These mutations could have appeared during the application of the reprogramming process to the AP leukemic cell-derived iPSCs; none was an obvious contributor to MPN pathophysiology. Finally, we showed that day16 embryoid bodies derived from the PB34 iPSCs contained expected numbers of CD34+ cells (18±11%, n=6) and BCR-ABL-expressing hematopoietic colony-forming cells (CFCs, 143±64 / 105 cells, n= 6). These CFCs showed a slight inhibitory response to imatinib (54±15% colonies obtained in 1 µM IM, n=4) whereas a combination of IM and Pimozide (a STAT5 phosphorylation inhibitor), reduced survival another ∼10-fold. In conclusion, we have provided proof-of-principle results illustrating the potential of iPSC technology in combination with WGS to dissect the clonal evolution of disease progression in CML and develop patient specific drug screens that could build on this data. Disclosures: Turhan: BMS, Novartis: Honoraria, Research Funding.

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Targeting Cell-Bound MUC1 on Myelomonocytic and Monocytic Leukemias and Leukemic Stem Cells: Therapeutic Implications

Blood ◽

10.1182/blood.v126.23.2899.2899 ◽

2015 ◽

Vol 126 (23) ◽

pp. 2899-2899

Author(s):

Thierry Guillaume ◽

Virginie Dehame ◽

Patrice Chevallier ◽

Pierre Peterlin ◽

Marc Grégoire ◽

...

Keyword(s):

Stem Cells ◽

Flow Cytometry ◽

Cell Surface ◽

Board Of Directors ◽

Leukemic Cell ◽

Muc1 Expression ◽

Leukemic Stem Cells ◽

Advisory Committees ◽

Alpha Chain ◽

Myelomonocytic Leukemia

Abstract Monocytic neoplasms comprise a heterogeneous group of hematologic malignancies including chronic myelomonocytic leukemia (CMML), juvenile myelomonocytic leukemia (JMML), acute myelomonocytic and monocytic leukemia (AML-M4 and AML-M5), and monocytic sarcoma. Monocytic or granulomonocytic hyperplasia is a finding frequently-if not invariably-shared by these different entities, as is a poor therapeutic outcome in the absence of hematopoietic stem cell transplantation. Cell surface molecules aberrantly expressed or overexpressed by leukemic cells represent potential disease-specific therapeutic targets. MUC1, a polymorphic type I high molecular weight glycoprotein represents such a molecule. MUC1 consists of an extracellular domain containing 20 to 125 tandem repeats of a 20 amino acid-long sequence, followed by a transmembrane domain and a short cytoplasmic tail leading to intracellular signaling. Cleavage of MUC1 yields two unequal chains: a large extracellular alpha subunit containing the tandem repeat array bound in a strong non-covalent interaction to a smaller beta subunit containing the transmembrane and cytoplasmic domains. Essentially all anti-MUC1 antibodies reported to date target the highly immunogenic tandem repeat of the MUC1 alpha chain. Because the alpha chain binds the cell-bound domains of MUC1 only intermittently in an 'on-and-off' manner, agents directed against the alpha chain will not effectively target MUC1+ cells. In contrast, the MUC1 SEA domain represents a stable structure fixed to the cell surface at all times. We therefore generated mAbs that specifically recognize the cell-bound MUC1 SEA domain. One of them, a partially humanized murine mAb termed DMB-5F3 was used to examine the expression of MUC1 on AML cells by flow cytometry. A series of twenty-two AML samples (blood-derived n=12; bone marrow-derived n=10; AML0=2, AML1=2, AML2=10, AML4=1, AML5=5, AML6=2) collected either at the time of diagnosis or at relapse were analysed for MUC1 expression by flow cytometry. A murine mammary tumor cell line stably transfected with human MUC1 DNA served as control. Blasts cells from 5 AML samples highly expressed MUC1, and significantly, all were of monocytic or myelomonocytic lineage (AML4=1, AML5=4). Leukemic stem cells (CD34pos or CD34neg linneg) from the MUC1+ AMLs were examined and likewise found to express MUC1. In addition, AML cell lines MV411, MOLM14, and SHI-1 derived from monocytic leukemic lineage clearly expressed cell surface MUC1, while non- monocytic leukemic cell lines U937, K562, and HL60 had little or no expression. Normal monocytes and monocytes derived from patients with activated monocytosis were also found to express MUC1. Based on these findings we examined MUC1 expression in a series of myelomonocytic leukemia (CMML and JMML). In fifteen CMML samples examined (type 1 n=11, type 2 n=4) (blood n=7, BM n=7) 92%-100% (median 99.7%) of CD14+CD56+ CMML cells bound mAb DMB-5F3 to cell-surface MUC1. CD14+CD16+CD56+ blast cells from 2 pts with JMML were also found to express MUC1 (between 64% and 71 % positive). Based on these findings we conclude that expression of MUC1 is restricted to monocytic and myelomonocytic leukemias and that MUC1 represents an effective target for leukemic immunotherapy. Significantly, anti-MUC1 mAb also targets monocytic leukemic stem cells, reinforcing its therapeutic potential. The fact that the anti-MUC1 antibody DMB-5F3 can enter cells and thereby ferry Ab-bound toxin opens the way for us to demonstrate leukemic cell killing with anti-MUC1 mAb-immunotoxin conjugates. Disclosures Moreau: Bristol-Myers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees; Janssen-Cilag: Honoraria, Membership on an entity's Board of Directors or advisory committees; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees; Millennium: Honoraria, Membership on an entity's Board of Directors or advisory committees.

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Inhibition of CML Stem Cells with an Alkaloid That Reduces β-Catenin

Blood ◽

10.1182/blood.v128.22.1882.1882 ◽

2016 ◽

Vol 128 (22) ◽

pp. 1882-1882

Author(s):

Shawnya J. Michaels ◽

Ngoc DeSouza ◽

Yi Shan ◽

Shaoguang Li ◽

Saira Bates

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Bone Marrow Transplantation ◽

Marrow Transplantation ◽

Bone Marrow Cells ◽

Leukemic Cells ◽

Leukemic Stem Cells ◽

Solvent Control ◽

Equity Ownership ◽

Marrow Cells

Abstract Although the majority of CML patients initially respond positively to BCR-ABL tyrosine kinase inhibitors (TKIs), they fail to eradicate the leukemia stem cells (LSCs) from which the disease arises. Only a minority of patients is able to discontinue TKI therapy, presumably due to the survival of LSCs. Therefore, the development of new therapeutics which ablate CML stem cells through a non-TKI, BCR-ABL independent pathway is needed. The Wnt/β-catenin pathway has been identified as an LSC survival pathway which provides proliferative signals and controls the stability of BCR-ABL1 through the increased expression of β-catenin. While previous research has demonstrated that Wnt/β-catenin is necessary for the survival and self-renewal of all CML cells and LSCs, it is not essential for maintenance of normal hematopoietic stem cells (HSCs). Tetrandrine (ES-3000, TET) is a natural product alkaloid used clinically in China as an analgesic and an anti-inflammatory. Its known mechanism of action is the inhibition of voltage-gated calcium channels and calcium activated big potassium (BK) channels which are commonly overexpressed in malignancies. However, TET has recently been demonstrated to inhibit the Wnt/β-catenin pathway resulting in a reduced expression of β-catenin, putatively through the inhibition of CaMKII-γ activation. This study investigated the efficacy of TET in models of CML stem cells. To demonstrate that TET can reduce β-catenin in leukemic cells, an in vitro assay with leukemic K562 cells was performed. Cells were exposed to TET for 24 hours at concentrations between 0-40 μM. Cell lysates were assayed by Western blot for β-catenin and actin. The results demonstrated that TET reduces β-catenin expression in a dose dependent manner. The effectiveness of TET was tested on CML stem cells using an in vivo mouse CML model. After priming donor C57BL/6 (B6) mice with intravenous injections of 5-fluorouracil for four days, bone marrow cells were harvested from femurs and tibia, then transfected twice with retrovirus containing MSCV-BCR-ABL-IRES-GFP. Recipient mice were lethally irradiated by two doses of 550 cGy before bone marrow transplantation by intravenous injection with 5x105 cells/mouse. Blood from recipient mice was tested for disease induction one week after transduction by FACS analysis for GFP. All mice tested positive. Treatments started on day 8 after bone marrow transplantation. Mice were randomized into four groups and treated orally with vehicle [3x/day, 2x], imatinib [100 mg/kg, 2x/day], TET [150 mg/kg, 1x/day] or imatinib + TET [3x/day: 2x with imatinib, 1x with TET]. The results of this study demonstrated that TET given orally once a day is superior to imatinib given twice a day in inhibiting the development of both circulating leukemic cells and leukemic stem cells while the combination of TET with imatinib further improves efficacy (See Figure). To determine whether TET has efficacy on human CML stem-like cells, a colony forming cell (CFC) assay with bone marrow cells from a de-novo CML patient was performed. The bone marrow cells were treated with 10 µM (IC50) TET for 14 days. After treatment, primary and secondary colonies were grown and analyzed by qPCR to determine BCR-ABL or ABL only cells. Replating efficiency of TET treated cells was 54% compared to 67% of solvent controls. In primary colonies, 95% of colonies from solvent control cultures were BCR-ABL positive compared to 70% of colonies treated with ES-3000. In secondary colonies (representing stem-ness), the TET treatment group was negative for BCR-ABL colonies while 79% of solvent control colonies still tested positive for BCR-ABL, indicating efficacy of TET in CML stem-like cells (See Table). We conclude that TET reduces leukemic stem cells in both a murine model of CML and a CFC assay which demonstrates its potential for development as an adjuvant therapy for CML patients demonstrating a lack of optimal response to TKIs, alone. Figure Figure. Table Table. Disclosures Michaels: Escend Pharmaceuticals, Inc.: Equity Ownership. Bates:Escend Pharmaceuticals, Inc.: Equity Ownership.

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