A Phase I/II Trial of Plerixafor/G-CSF Combined with IV Bu/Flu Conditioning Regimen In AML/MDS Patients Undergoing Allogenic Stem Cell Transplantation

Abstract Abstract 2358 Allogeneic stem cell transplantation (alloSCT) is an effective treatment for pts with acute myeloid leukemia (AML) in first remission. However, only 10–20% of pts with relapsed disease achieve a durable remission. Microenvironment/leukemia interactions play a major role in chemoresistance of leukemic stem cells residing in the bone marrow niches. In pre-clinical in vivo leukemia models, inhibition of chemokine receptor CXCR4 results in mobilization of leukemic cells into circulation and sensitization to chemotherapy. We hypothesized that mobilization of leukemic stem cells by CXCR4 inhibition and G-CSF will result in improved anti-leukemia activity of a standard preparative regimen followed by alloSCT. In this Phase I/II study, G-CSF is administered at a standard dose beginning on day -9 daily for 6 days, and the CXCR4 inhibitor plerixafor (Mozobil®) from day -7 at one of the 4 dose levels 0 (control), 0.08, 0.16, or 0.24 mg/kg, 8 hours prior of each four daily doses of a standard preparative regimen (Fludarabine, 40mg/m2 and IV Busulfan, 130mg/m2, days -6 through -3). Twenty seven pts have been enrolled in the study to date with a median age of 48 yrs (range 25–65). Baseline characteristics include 13 pts (48%) with de novo AML, 6 (22%) with secondary AML, 5 with MDS and 3 with CML. Among the 24 AML/MDS pts, 14 (58%) had intermediate and 10 (42%) poor risk cytogenetics. Twelve pts (50%) had primary refractory AML, 5 were in 1st or 2nd relapse, 2 were untreated, 3 were in CR1 and 2 in CR2. The source of stem cells was sibling donor in 16 and unrelated donor in 11. After phase I plerixafor dose escalation in 16 pts, 11 pts received 0.24 mg/kg in Phase II. Common grade ≥ 3 adverse events which consisted primarily of neutropenic fever, infections, or rash were seen in 24/27 (89%) pts. There were no toxicities ascribed to the G-CSF/plerixafor component of the regimen. No evidence of significant delays in neutrophil (ANC >500/mm3, median 12.5d, range 10–19) or platelet recovery (plt >20k/mm3, median 12d, range 9–74d) were observed. Grade I-II GVHD was seen in 10/27 pts (37%), with no occurrences of Grade III-IV GVHD. Of the 19 pts with active disease at study entry, 18 achieved a CR. Treatment failure was due to persistent disease in 1 pt (4%), relapsed disease in 10 pts (37%) and early death due to complications from intracranial hemorrhage in 1 pt (4%). Median progression-free survival (PFS) for all pts was 26.6 wks (95%CI: 18.1–33.9 wks) and 15.7 wks (95% CI: 12.1–26.6 wks) in relapsed pts. Median follow-up for all study pts was 19.14 wks (range: 0.7–54.6 wks). Correlative studies analyzed from 16 pts enrolled in the Phase I portion of the trial demonstrate that G-CSF/plerixafor mobilizes CD34+ cells, with the mean fold increase of 5.9-fold at 0.08 mg/kg plerixafor; at 0.16 mg/kg, 13-fold; and at 0.24 mg/kg, 14.2-fold. Based on fitted longitudinal linear mixed models, G-CSF had a significant effect on cell mobilization over time (WBC and CD34+). In contrast, plerixafor at the doses of 0.16 and 0.24 mg/kg was significantly associated with increased cellular CXCR4 expression levels and with mobilization of CXCR4+ cells over time (p<0.02). To determine relative proportion of mobilization of leukemic and non-leukemic cells, we performed FISH analysis on peripheral blood samples from pts with informative cytogenetic abnormalities (n=12). Both, FISH+ and FISH- cell counts increased from day -8 to day -6 and remained relatively stable or decreased thereafter (between day -6 and day -3), with the initial increase much larger for the plerixafor dose level 0.16 mg/kg (mean fold increase FISH+, 24.3; FISH-, 10.3). Over time, the relative increase of FISH+ cells was significantly higher than that of FISH- cells, indicating preferential mobilization of cytogenetically abnormal leukemic over normal cells (p=0.005). In summary, G-CSF/plerixafor is safe in combination with the established IV busulfan/fludarabine preparative regimen for alloSCT in pts with advanced disease. Our data indicate preferential mobilization of clonal leukemic over normal cells. The objective of the ongoing Phase II study is to determine if the combination of G-CSF/plerixafor with busulfan/fludarabine improves PFS compared to historical controls receiving busulfan/fludarabine alone. We hypothesize that interventions disrupting stroma-leukemia interactions may enhance chemosensitivity and therefore the therapeutic efficacy in hematological malignancies. Disclosures: Konopleva: Genzyme: Research Funding. Off Label Use: Plerixafor for transplant in AML. Andreeff: Genzyme: Consultancy, Research Funding. Champlin: Genzyme: Consultancy, Research Funding.

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Leukemic Stem Cells of Monocytic AMLs Are Not-Resistant to BCL-2 Inhibition

Blood ◽

10.1182/blood-2021-149781 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 3469-3469

Author(s):

Simon Renders ◽

Aino-Maija Leppä ◽

Alexander Waclawiczek ◽

Maike Janssen ◽

Elisa Donato ◽

...

Keyword(s):

Stem Cells ◽

Flow Cytometry ◽

Research Funding ◽

Independent Study ◽

Leukemic Cells ◽

Leukemic Stem Cells ◽

Monocytic Differentiation ◽

Color Flow ◽

Travel Costs ◽

Nsg Mice

Abstract Treatment with Hypomethylating agents (HMA) such as 5-Azazytidine (AZA) in combination with the BCL-2 inhibitor Venetoclax (VEN) has recently become the standard of care for AML patients unsuitable for intensive induction chemotherapy and shows results superior to treatment with AZA alone (DiNardo et al., 2020, NEJM). However upfront resistance and relapse following initial response remain major obstacles. It has recently been proposed that monocytic differentiation predicts resistance to AZA/VEN treatment in AML (Pei et al., 2020 Cancer Discovery). This appears to be due to increased expression of other anti-apoptotic proteins such as MCL-1 in monocytic AMLs, which conveys resistance to AZA/VEN therapy, as survival of leukemic cells in these patients is no longer dependent on BCL-2. However, an independent study found no impaired outcome in patients with monocytic AMLs treated with HMA/VEN (Maiti et al., 2020, Blood, ASH abstract). Here, we show that monocytic AML cell lines and bulk cells of monocytic primary AML cells are indeed intrinsically resistant to AZA/VEN treatment. However, in a collective of 30 patients treated with HMA/VEN at Heidelberg University Medical Center between 2018 and 2020, monocytic differentiation assessed by flow cytometry was not an independent risk factor for refractory disease. We hypothesized that the conflicting data may be caused by intra-patient heterogeneity of AZA/VEN sensivitity and assessed killing efficiency in various immunophenotypic subpopulations of 12 primary AML patient samples in vitro. The CD64 +CD11b +, differentiated blast population made up >50% of leukemic cells in monocytic and <20% in primitive samples and showed high levels of resistance to AZA/VEN therapy in both primitive and monocytic leukemias but did not engraft when transplanted into NSG mice, arguing they do not contain leukemic stem cells (LSC). In contrast, we found immature CD64 -CD11b - GPR56 + LSC to be sensitive to AZA/VEN treatment irrespective whether they were derived from monocytic or primitive types of primary AMLs. As expected, LSCs from either monocytic or primitive AMLs initiated disease in NSG mice, highlighting that targeting LSCs is essential for the effect of AML therapy. Next, we investigated expression of BCL-2, MCL-1 and BCL-xL in the same primary patient samples and observed high MCL-1 expression in monocytic AML samples. However, MCL-1 expression was restricted to the CD64 +CD11b + population whereas in the LSC sub-populations robust expression of BCL-2 but low levels of MCL-1 and BCL-xL were detected, independent of whether monocytic or primitive AMLs were analyzed. To further validate the sensitivity of LSCs of monocytic AML to BCL-2-I, we established a platform combining BH-3 profiling with multi-color flow cytometry, allowing for single cell assessment of cellular dependencies on independent apoptotic pathways. We found that LSCs of both AML types show high VEN/BAD but low MS-1 induced apoptosis, functionally confirming the expression patterns of BCL-2 and MCL-1. As LSCs are rare in monocytic samples, investigation of samples in bulk are dominated by MCL-1 expressing and resistant non-LSCs, explaining the overall higher MCL-1 expression/survival of monocytic compared to immature AML cells. However, our data uncovers sensitivity of LSCs to AZA/VEN independent of overall monocytic or primitive sample classification and provide a mechanistic explanation for the clinical data of Maiti et al. and our Heidelberg AML collective, which found no increased resistance of monocytic AMLs to AZA/VEN treatment. Disclosures Unglaub: JazzPharma: Consultancy, Other: travel costs/ conference fee; Novartis: Consultancy, Other: travel costs/ conference fee. Schlenk: Abbvie: Honoraria; Agios: Honoraria; Astellas: Honoraria, Research Funding, Speakers Bureau; Celgene: Honoraria; Daiichi Sankyo: Honoraria, Research Funding; Hexal: Honoraria; Neovio Biotech: Honoraria; Novartis: Honoraria; Pfizer: Honoraria, Research Funding, Speakers Bureau; Roche: Honoraria, Research Funding; AstraZeneca: Research Funding; Boehringer Ingelheim: Research Funding. Müller-Tidow: Janssen: Consultancy, Research Funding; Bioline: Research Funding; Pfizer: Research Funding.

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Bmi-1 Overexpression Synergizes with p210-BCR-ABL to Induce Stem Cell and Progenitor Transformation.

Blood ◽

10.1182/blood.v114.22.3251.3251 ◽

2009 ◽

Vol 114 (22) ◽

pp. 3251-3251

Author(s):

Amitava Sengupta ◽

Jorden Arnett ◽

Susan Dunn ◽

Jose Cancelas

Keyword(s):

Stem Cells ◽

Stem Cell ◽

Research Funding ◽

Chronic Phase ◽

Fold Increase ◽

Myelogenous Leukemia ◽

Leukemic Stem Cells ◽

Blastic Transformation ◽

Bmi1 Expression ◽

Blastic Phase

Abstract Abstract 3251 Poster Board III-1 Chronic myelogenous leukemia (CML) is a hematopoietic stem cell (HSC) malignancy induced by p210-BCR-ABL and characterized by myeloproliferation followed by poor-prognosis acute blastic transformation. Persistence of BCR-ABL+ HSCs in patients under tyrosine kinase inhibitor therapy suggests that inhibition of ABL-kinase alone is not sufficient to completely eliminate the leukemic stem cells and progenitor (LSC/P) population and a group of patients continue developing accelerated/blastic phase despite therapy. Expression of p210-BCR-ABL is necessary and sufficient to develop CML but the molecular mechanisms necessary for its blastic transformation remain elusive. The polycomb group gene Bmi1 plays an essential role in regulating the proliferative capacity of both normal and leukemic stem cells (Lessard J, et al. Nature 2003). Recently, expression of Bmi1 has been found significantly elevated in CD34+ cells of patients with advanced phases compared with patients in chronic-phase CML (Mohty M et al. Blood 2007). We therefore intend to determine whether Bmi1 expression in CML stem cells and progenitors, isolated from Scl/p210-BCR-ABL-expressing mice, is sufficient to accelerate significantly the development of blastic phase. Since simultaneous co-expression of Bmi1 and BCR-ABL in normal HSC/P may not faithfully recapitulate the progression events in CML transformation, we have over-expressed Bmi1 in a model of stem cell-dependent chronic phase CML. This model is based on the tetracycline-dependent expression of p210-BCR-ABL driven by the Scl promoter (Scl-tTA x TRE-BCR-ABL, Koschmieder S et al. Blood 2005), where expression of BCR-ABL is restricted to the HSC/P compartment. Scl-driven expression of BCR-ABL is predominantly active in HSC (Lin-/Sca1+/c-kit+; LSK) and progenitors (Lin-/c-kit+; LK) and is transplantable into secondary recipients (Sengupta A et al., ASH 2008). Expression of Bmi1 into Scl/p210-BCR-ABL-expressing cells significantly increases the HSC/P proliferation (2.5 fold) and also increases the frequency of colony forming cells (CFU-Cs) after serial propagation in liquid culture, compared to Bmi1 (4.6-fold) or BCR-ABL-expressing CML cells alone (517-fold). Furthermore, Bmi1 expression into Scl/p210 leukemic progenitors increases the hypermigratory phenotype of leukemic progenitors (3-fold increase over 5.5-fold increase induced by BCR/ABL alone; P<0.005) in response to CXCL12. Although, Bmi1 overexpression in Scl/p210 cells does not decrease further the reduced adhesion to fibronectin of BCR/ABL-transformed CML HSC/P, leukemic progenitors co-expressing Bmi1 and SCL/p210 have significantly reduced homing in bone marrow compared to Bmi1-expressing HSC/P (7.7 fold, P≤0.005). Altogether, these data indicate that Bmi-1 synergistically enhances the transformation phenotype of p210-BCR-ABL-expressing HSC/P and emphasize the role of epigenetic changes inducing overexpression of self-renewal genes in the pathogenesis of CML. These data suggest that Bmi-1 may represent a novel therapeutic target for CML LSC/P. Disclosures: Cancelas: CERUS CO: Research Funding; CARIDIAN BCT: Research Funding; HEMERUS INC: Research Funding.

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Inhibiting the Mitochondrial Sulfhydryl Oxidase Alr Reduces Cox17 and Alters Mitochondrial Cristae Structure Leading to the Differentiation of AML and Stem Cells

Blood ◽

10.1182/blood.v130.suppl_1.881.881 ◽

2017 ◽

Vol 130 (Suppl_1) ◽

pp. 881-881

Author(s):

Danny V. Jeyaraju ◽

Veronique Voisin ◽

Changjiang Xu ◽

Samir H. Barghout ◽

Dilshad H. Khan ◽

...

Keyword(s):

Stem Cells ◽

Cord Blood ◽

Respiratory Chain ◽

Research Funding ◽

Annexin V ◽

Leukemic Cells ◽

Copper Chaperone ◽

Leukemic Stem Cells ◽

Sulfhydryl Oxidase

Abstract The vast majority of mitochondrial proteins are encoded in the nucleus, translated in the cytoplasm and then imported into the mitochondria. A subset of these imported proteins are folded into their mature and functional forms in the mitochondrial inter-membrane space (IMS) by the Mitochondrial IMS Assembly (MIA) pathway. We found that genes encoding substrates of the MIA pathway are over-expressed in leukemic stem cells compared to bulk AML cells. Therefore, we assessed the effects of inhibiting the MIA pathway in AML. We knocked down the mitochondrial sulfhydryl oxidase ALR, a key regulator of the MIA pathway. Knockdown of ALR with shRNA reduced the growth and viability of OCI-AML2, TEX and NB4 leukemia cells. In addition, knockdown of ALR reduced the engraftment of TEX cells into mouse marrow, demonstrating an effect on the leukemia initiating cells. The small molecule selective ALR inhibitor, MitoBloCK-6, mimicked the effects of ALR knockdown and killed AML cells with an IC50 of 5-10 μM. MitoBloCK-6 preferentially reduced the clonogenic growth of primary AML cells (n=4/5) over normal hematopoietic cells (n=4). However, only 3/10 bulk AML cells were sensitive to MitoBloCK-6 induced cell death by Annexin V/PI staining. Next, we evaluated the efficacy and toxicity of ALR inhibition in vivo . We injected primary AML cells or normal cord blood into the femurs of mice and then treated mice with MitoBloCK-6 (80 mg/kg i.p. 5 of 7 days x 2 weeks). MitoBloCK-6 strongly reduced the engraftment of primary AML samples but did not affect engraftment of cord blood. In secondary transplants, MitoBloCK-6 also targeted leukemic stem cells. No change in mouse body weight, serum chemistries, or organ histology was seen. As expression levels of ALR substrates are increased in AML stem cells, we assessed the effects of ALR inhibition on differentiation in AML. Genetic or chemical inhibition of ALR induced the differentiation of AML cells as evidenced by increased CD surface marker expression and increased non-specific esterase. In addition, ALR inhibition was preferentially cytotoxic towards undifferentiated cells and stem cells over differentiated bulk AML cells. Interrogation of the effects of ALR inhibition on its substrates identified the mitochondrial copper chaperone, Cox17 as the primary downstream target in leukemic cells. Inhibition of ALR selectively reduced levels of Cox17 protein and altered mitochondrial cristae structure. Validating the functional importance of these findings, knockdown of Cox17 phenocopied ALR inhibition and reduced AML proliferation, induced differentiation of AML cells, and altered mitochondrial cristae structure, without changing respiratory chain activity or oxygen consumption. Of note, cristae remodelling independent of respiratory chain function has been recently implicated in cellular differentiation and in yeast, Cox17 regulates the cristae organizing machinery. Thus, we have identified novel mechanisms by which mitochondrial pathways regulate the fate and differentiation of AML cells and stem cells Moreover, inhibition of ALR may be a novel therapeutic strategy to promote the differentiation of AML cells and stem cells. Disclosures Schimmer: Takeda Pharmaceuticals: Research Funding; Medivir: Research Funding; Novartis Pharmaceuticals: Honoraria.

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AMD3100 Mobilizes Acute Promyelocytic Leukemia Cells from the Bone Marrow into the Peripheral Blood and Sensitizes Leukemia Cells to Chemotherapy.

Blood ◽

10.1182/blood.v106.11.246.246 ◽

2005 ◽

Vol 106 (11) ◽

pp. 246-246 ◽

Cited By ~ 3

Author(s):

Bruno Nervi ◽

Matthew Holt ◽

Michael P. Rettig ◽

Gary Bridger ◽

Timothy J. Ley ◽

...

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Mouse Model ◽

Acute Promyelocytic Leukemia ◽

Reporter Gene ◽

Peripheral Blood ◽

Fold Increase ◽

Promyelocytic Leukemia ◽

Leukemic Cells ◽

Leukemia Cells

Abstract CXCR4/SDF-1 axis regulates the trafficking of normal stem cells to and from the bone marrow (BM) microenvironment. SDF-1 is a chemokine widely expressed by many tissues especially BM stromal cells and osteoblasts. AMD3100 (AMD) is a novel bicyclam molecule that is a competitive inhibitor of SDF-1/CXCR4 binding and has been used to enhance stem cell mobilization when combined with G-CSF in mouse, dog and man. We are interested in evaluating whether leukemic cells “mobilize” similar to normal stem cells after treatment with AMD, and if so, whether this mobilization increases the efficacy of chemotherapy. Therefore, we utilized a mouse model of human acute promyelocytic leukemia (APL) in which the PML-RARα transgene was knocked into a single allele of the murine cathepsin G locus. To more efficiently track the leukemic cells, we transduced banked APL tumors with a dual function reporter gene that encodes a fusion protein comprised of click beetle red (CBR) luciferase, a bioluminescence imaging (BLI) optical reporter gene, and EGFP for ex vivo cell sorting (CBR/EGFP). We generated large numbers of CBR/EGFP+ APL cells by isolating EGFP+ cells using a MoFlo cell sorter, and passaging them in secondary syngeneic recipients. Importantly, the secondary recipients developed a rapidly fatal acute leukemia after intravenously (iv) or intraperitoneal injection, which displayed an APL phenotype (CD34/GR1 co-expression) and exhibited luciferase activity. Upon iv injection into syngeneic recipients, the CBR/EGFP+ APL cells rapidly migrated to the BM microenvironment, as evidenced by the significantly increased BLI signal in the femurs, spine, ribs, and skull of recipients at 4 days after injection. Over the next 2–3 days the CBR/EGFP+ cells migrated to the spleen followed rapidly by widespread dissemination and death due to leukostasis by 14–16 days. To our knowledge, this represents the only mouse leukemia model in which leukemia cells home preferentially to the BM microenvironment in a manner that is similar to what is seen in human AML. Therefore, we used this model to study the effect of AMD on the “mobilization” of APL cells into the peripheral blood (PB) and on their sensitivity to chemotherapeutic agents that are known to affect the proliferation of these cells. Surprisingly, injection of AMD (5 mg/kg) immediately at the time of APL infusion had no impact on the engraftment (short term or long term) of either normal BM stem cells or the leukemic cells. However, we observed rapid mobilization of the leukemic cells when AMD was administered 11 days after APL injection. In fact, 40% of mice that received a single dose of AMD on day +11 after APL injection died 2 to 4 hours after AMD injection as a result of the rapid and massive mobilization of blasts. Overall, we found that AMD treatment on day +11 induced a 3-fold increase in total WBC counts with a 10-fold increase in the leukemic blasts into PB. Interestingly, the administration of AMD concomitant with cytarabine (AraC) (200 mg/kg) on day +11 significantly prolonged the overall survival of mice, compared with mice treated only with AraC. In summary, we developed a mouse model to study the APL cell trafficking, and we have shown leukemia cell mobilize from the BM into PB after AMD administration. We propose that CXCR4/SDF-1 is a key regulator for leukemia migration and homing to the BM. In these preliminary results, we observed that AMD sensitizes APL cells to AraC.

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

Blood ◽

10.1182/blood.v112.11.1637.1637 ◽

2008 ◽

Vol 112 (11) ◽

pp. 1637-1637

Author(s):

Huiyu Li ◽

Yi-Mei Du ◽

Linlin Guo ◽

Tiannan Guo ◽

Shenghua Jie ◽

...

Keyword(s):

Stem Cells ◽

Cell Migration ◽

Leukemic Cell ◽

Cxcr4 Expression ◽

Leukemic Cells ◽

K Channel ◽

Leukemic Stem Cells ◽

Lower Chamber ◽

Herg Current ◽

K Current

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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A Phase I Study of the HDAC Inhibitor LBH589 in Combination with Imatinib for Patients with CML in Cytogenetic Remission with Residual Disease Detectable by Q-PCR.

Blood ◽

10.1182/blood.v114.22.2194.2194 ◽

2009 ◽

Vol 114 (22) ◽

pp. 2194-2194 ◽

Cited By ~ 3

Author(s):

Ravi Bhatia ◽

David S. Snyder ◽

Allen Lin ◽

Jennifer Arceo ◽

Linda Seymour ◽

...

Keyword(s):

Stem Cells ◽

Phase I ◽

Research Funding ◽

Residual Disease ◽

Leukemia Stem Cells ◽

Gi Symptoms ◽

Level 1 ◽

Grade 3 ◽

Level 2 ◽

Dose Limiting Toxicity

Abstract Abstract 2194 Poster Board II-171 Imatinib mesylate (IM) is effective in inducing remission and improving survival in CML patients. However IM-treated patients continue to harbor residual leukemia stem cells (Blood 101:4701, 2003). Most patients relapse if treatment is discontinued, and it is generally recommended that treatment with IM be continued indefinitely. The inability of IM to cure CML, the potential for side effects and the financial burden of life-long treatment provide an impetus to develop approaches to eliminate residual leukemia stem cells. We have shown in preclinical studies that treatment with the HDAC inhibitor LBH589 (LBH) combined with IM effectively eliminates CML stem cells resistant to IM alone. The safety and MTD of LBH589 in combination with Imatinib has not been previously evaluated. We have initiated a phase I, open label clinical trial to determine the safety and tolerability of LBH589 given in combination with IM in CML patients, and to determine the MTD and dose-limiting toxicity (DLT). CML patients in chronic phase (CP) treated with IM 400mg/d for >1 year with major or complete cytogenetic response and residual disease on Q-PCR are eligible. LBH589 is administered in combination with IM 400mg PO daily in 28 day cycles, with successive cohorts of patients receiving escalating doses of LBH 3 times a week (level 1: 10mg; level 2: 15mg; level 3: 20mg). Treatment is scheduled for 6 cycles of 28 days each. Five patients have been enrolled thus far (Table). No dose limiting toxicity (DLT), defined as Grade 3 hematological or non-hematological toxicity in the first 28 days, was observed in 3 patients enrolled at dose level 1. DLT (Grade 3 thrombocytopenia) was observed in 1 of the 2 patients enrolled at dose level 2. Other toxicities included thrombocytopenia (Grade 3 [n=2]; Grade 1-2 [n=4]), hypophosphatemia (Grade 3 [n=1]; Grade 2 [n=2]), fatigue (Grade 1 [n=3]), hypocalcemia (Grade 1 [n=2] and Grade 1-2 GI symptoms (diarrhea [n=2]; nausea [n=3]; anorexia [n=2]; vomiting [n=3]; constipation [n=1]). Of note, significant QTc prolongation was not observed on intensive EKG monitoring. IM did not require to be held for any of these toxicities. Two patients have completed 6 cycles of treatment, with one opting to receive an additional 3 cycles, 2 are currently receiving treatment, and one withdrew after 1.5 cycles because of fatigue and GI symptoms. Bone marrow aspirates for assessment of BCR-ABL status were performed at the end of cycles 3 and 6 of treatment. Q-PCR analyses showed reduction in BCR-ABL levels in patient #2 (LBH 10mg) after 3 months, which was not sustained at 6 months. Patient #4 (LBH 15mg), followed for 5 months so far, had undetectable BCR-ABL after 3 months of treatment. These results suggest that LBH589 can be safely administered in combination with IM. Reduction in BCR-ABL levels was seen in two patients but the durability is unclear as yet. We are continuing accrual of patients to define the MTD and safety of this combination. Disclosures: Bhatia: Novartis: Consultancy, Research Funding. Snyder:Novartis: Consultancy, Honoraria, Speakers Bureau. Deininger:Novartis: Consultancy. Radich:Novartis: Consultancy, Honoraria, Research Funding.

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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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Nilotinib and Dasatinib Produce Synergistic Growth-Inhibitory Effects In Imatinib-Resistant CML Cells, Including Subclones Bearing the Multi-Resistant BCR/ABL Mutant T315I

Blood ◽

10.1182/blood.v116.21.2280.2280 ◽

2010 ◽

Vol 116 (21) ◽

pp. 2280-2280

Author(s):

Karoline V. Gleixner ◽

Harald Herrmann ◽

Irina Sadovnik ◽

Karina Schuch ◽

Winfried F Pickl ◽

...

Keyword(s):

Stem Cells ◽

Research Funding ◽

Synergistic Effects ◽

Chronic Phase ◽

Leukemic Cells ◽

Inhibitory Effects ◽

Blast Phase ◽

Drug Concentrations ◽

Growth Inhibitory ◽

Imatinib Resistant

Abstract Abstract 2280 In most patients with chronic myeloid leukemia (CML), complete cytogenetic remission can be achieved with the BCR/ABL tyrosine kinase inhibitor (TKI) imatinib. However, not all patients are long-term responders. A major cause of acquired resistance against imatinib is the development of BCR/ABL mutations in subclones. In most of these patients, a second generation TKI is prescribed. However, the T315I mutant of BCR/ABL introduces resistance against most TKI, including nilotinib and dasatinib. One approach to overcome drug resistance in BCR/ABL T315I+ CML cells may be to apply drug combinations. Recent data suggest that the mechanisms through which dasatinib and nilotinib act on BCR/ABL differ from each other and that both drugs act on multiple additional targets in CML cells. Here, we show that dasatinib and nilotinib cooperate with each other in producing growth inhibition in imatinib-sensitive and imatinib-resistant CML cells, including subclones bearing BCR/ABL T315I. The drug combination was tested on leukemic cells obtained from 9 patients with chronic phase (CP) CML and 3 with blast phase (PB) of CML. Samples were assessed from 4 patients at the time of diagnosis, and against cells from 8 patients (CP, n=5; BP, n=3) who had developed resistance against one or more BCR/ABL TKI. In all 3 patients in PB, the T315I mutant was detectable. As expected, nilotinib and dasatinib failed to inhibit proliferation of cells harbouring BCR/ABL T315I when applied as single agents. However, the combination xnilotinib+dasatinibx produced synergistic effects in most samples, including primary CML cells and Ba/F3 cells harbouring BCR/ABL T315I. Interestingly, in all 3 patients with BP (BCR/ABL T315I+), strong cooperative or even synergistic growth-inhibitory effects were observed in primary CML cells, resulting in substantial anti-leukemic effects seen at reasonable (pharmacologic) drug concentrations (< 1 μ M) (figure). Based on these results, we treated one patient with TKI-resistant CML in hematologic relapse in whom 2 BCR/ABL mutant-bearing subclones, one clinically resistant against nilotinib (F359V) and one clinically resistant against dasatinib (F317L) had been detected, with a combination of nilotinb (800 mg p.o. daily) and dasatinib (50 mg/day p.o., days 1–5 every third week). A transient hematologic response was obtained in this patient, and except for mild bone pain, no side effects were recorded. Moreover, we were able to show that during treatment with xnilotinib+dasatinibx, the number of CD34+/CD38-/CD33+ CML stem cells decreased from clearly measurable levels (0.005%) to nearly undetectable levels (0.0002%). Finally, ex vivo analyses of leukemic blood cells confirmed, that the combination xnilotinib+dasatinibx produced strong cooperative growth-inhibitory effects in both disease-components, i.e. the F359V-bearing subclone and the F317L-bearing subclone. In summary, our data show that the combination of dasatinib and nilotinib can override acquired TKI resistance in CML, and can suppress growth of various imatinib-resistant subclones including cells that bear BCR/ABL T315I or other BCR/ABL mutants. Whether this combination can suppress imatinib-resistant subclones in CML for prolonged time periods or even can eradicate neoplastic stem cells remains in CML patients to be determined. Synergistic effects of nilotinib and dasatinib on primary leukemic cells obtained from a patient with a BCR/ABL T315I+ blast phase of CML Disclosures: Valent: Novartis: Research Funding; Bristol-Myers Squibb: Research Funding.

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ABT-737 Targets Leukemic Stem Cells In Mouse Models of Mutant NRASD12/hBCL-2- Mediated Acute Myeloid Leukemia Progression with Increased Survival

Blood ◽

10.1182/blood.v116.21.3308.3308 ◽

2010 ◽

Vol 116 (21) ◽

pp. 3308-3308

Author(s):

Rose Ann Padua ◽

Stephanie Beurlet ◽

Patricia Krief ◽

Nader Omidvar ◽

Carole Le Pogam ◽

...

Keyword(s):

Stem Cells ◽

Disease Progression ◽

Board Of Directors ◽

Research Funding ◽

Leukemic Stem Cells ◽

Spleen Cells ◽

Employment Equity ◽

Advisory Committees ◽

Equity Ownership ◽

Bh3 Mimetic

Abstract Abstract 3308 Background: Animal models enable us to understand disease progression and provide us with reagents to test various therapeutic strategies. We have previously developed a mouse model of myelodysplasia/acute myelogenous leukemia (MDS/AML) progression using mutant NRASD12 and overexpression of human hBCL-2 (Omidvar et al Cancer Res 67:11657-67, 2007). Expanded leukemic stem cells (LSC) were identified as Lin-/Sca1+/KIT+ (LSK) populations, with increased myeloid colony growth and were transplantable. Increased hBCL-2 and RAS-GTP complex were observed in both MDS/AML diseases. The MDS-like disease had increased apoptosis, whilst the AML-like mice had liver apoptosis patterns similar to wild type. The single NRASD12 line also had increased apoptosis. In this present study using a BCL-2 homology domain 3 (BH3) mimetic ABT-737 (Abbott), we have evaluated the effects of targeting BCL-2 in our preclinical models. Methods & Results: Treatment with the inhibitor shows a reduction of LSK cells, reduced progenitor numbers in colony assays and clearance of the liver infiltrations in both MDS and AML models. Gene expression profiling of the MDS mice shows regulation of 399 genes upon treatment including 58 genes expressed by the single mutant RAS mice and not expressed in the untreated AML mice. 78 genes were shared between single NRASD12 and diseased mice and not the treated mice. These studies potentially identify the contribution of NRASD12 genes to disease progression. By confocal microscopy we observed that in the MDS mice the majority of the RAS and BCL-2 co-localized to the plasma membrane, where active pro-apoptotic RAS is normally located, whereas in the AML disease RAS and BCL-2 co-localized in the mitochondria, where BCL-2 is normally found (Omidvar et al 2007). After treatment with the inhibitor the AML co-localization of RAS and BCL-2 shifted to the plasma membrane where single NRASD12 is normally localized. Furthermore, increased RAS-GTP levels was detected in both Sca1+ and Mac1+ enriched spleen cells and interestingly an increase in BCL-2 expression was observed in peripheral blood and in spleen cells after treatment; this increase in BCL-2 was associated with a decrease in the phosphorylation of serine 70 and an increase in phosphorylation of threonine 56 of BCL-2. ABT-737 treatment led to increased phosphorylated ERK resembling RAS and reduced MEK and AKT phosphorylation, changes detected by western blots and the nanoimmunoassay (NIA, NanoPro, Cell Biosciences) that might account for the increased apoptosis, measured by TUNEL and In vivo imaging by single-photon emission computed tomography (SPECT) using Tc-99m-labelled AnnexinV (SPECT). In contrast, although treated MDS mice had increased apoptosis they did not have an increase in overall expression of BCL-2 or in RAS-GTP levels. Treatment of both MDS and AML models with this inhibitor significantly extended lifespan from diagnosis with mean survival of 28 days untreated vs 80 days treated (p=0.0003) and mean survival from birth of 39 untreated vs 85 days treated (p<0.0001) respectively Conclusions: Genomics, proteomics and imaging have been employed in the MDS/AML models to characterize disease progression and follow response to treatment to the BH3 mimetic ABT-737 in order to gain molecular insights in the evaluation of the efficacy. ABT-737 appears to target LSCs, induce apoptosis, regulating RAS and BCL-2 signalling pathways, which translated into significantly increased survival. Disclosures: Padua: Vivavacs SAS: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees. Auboeuf:GenoSplice technology: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees. de la Grange:GenoSplice technology: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties. Fenaux:Celgene: Honoraria, Research Funding; Novartis: Honoraria, Research Funding; Janssen Cilag: Honoraria, Research Funding; ROCHE: Honoraria, Research Funding; AMGEN: Honoraria, Research Funding; GSK: Honoraria, Research Funding; Merck: Honoraria, Research Funding; Cephalon: Honoraria, Research Funding. Tu:Cell Biosciences Inc;: Employment. Yang:Cell Biosciences Inc;: Employment. Weissman:Amgen, Systemix, Stem cells Inc, Cellerant: Consultancy, Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees. Felsher:Cell Bioscience:. Chomienne:Vivavacs SAS: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees.

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