Combined Pharmacologic Inhibition of Bcl-Xl/Bcl-2 and mTORC1/2 Survival Signals Trigger Apoptosis in BCR-ABL1+in Vitro Models of Blast Crisis Chronic Myelogenous Leukemia (CML-BC), and Primary CD34+/CD38− Stem and CD34+ progenitor Cells From CML-BC Patients

Abstract Abstract 2738 Tyrosine kinase inhibitors (TKIs) have become frontline therapy for CML; however, alternative therapies are required, as TKIs do not induce long-term response in CML patients undergoing blastic transformation and are ineffective against Philadelphia-positive (Ph+) quiescent stem cells, which show innate resistance to BCR-ABL1 kinase inhibitors. Therapeutic targets of interest are survival factors conferring resistance to TKI-induced apoptosis and/or those increasing proliferation of leukemic progenitors. We previously reported (Harb JG et al., ASH 2007) that genetic inactivation of Bcl-x did not inhibit BCR-ABL1 leukemogenesis in an inducible mouse model of CML. Thus, we hypothesize that BCR/ABL mediated post-translational modification and inactivation of pro-apoptotic BAD negates the requirement for the anti-apoptotic function of Bcl-xL in stem/progenitor cells from SCLtTA-BCR/ABL1/Bcl-x−/− mice. Following this rationale, we tested if simultaneous pharmacologic BAD activation and Bcl-xL inhibition may be an efficient way of killing CML stem/progenitor cells. To test this, loss of Bcl-xL function with increased levels of active BAD was achieved by expressing Bcl-x shRNA in 32D-BCR/ABL mouse myeloid precursors that were then treated with LY294002 (LY), which suppresses the inhibitory effects of PI-3K/Akt activation on BAD. Flow cytometric analysis of Annexin V+ cells revealed that levels of apoptosis were three times higher in BCR-ABL1+ cells expressing the Bcl-x shRNA when compared with vector-transduced BCR-ABL1+ cells. As expected, increased levels of dephosphorylated (active) BAD at the mitochondrial membrane were found in LY-treated BCR-ABL+ cells. Interestingly, co-treatment of Bcl-x shRNA-expressing BCR-ABL1+ cells with LY and the Bcl-xL/Bcl-2 antagonist ABT-263 (ABT) did not further promote apoptosis, suggesting that decreased survival of BCR-ABL1+ cells was dependent on downregulation of Bcl-xL and not Bcl-2. To determine efficacy of combined pharmacologic Bcl-xL inhibition and BAD activation, 32D-BCR/ABL and K562 cells were treated with compounds expected to activate BAD upon inhibition of PI-3K/Akt/mTOR-generated signals, used alone or in combination with ABT. Individually, at suboptimal doses, LY, Rapamycin (RAP), mTORC1/2 inhibitor PP242, and ABT were tolerated with apoptosis levels lower than 20%. Notably, when combined with ABT, all three efficiently induced apoptosis (∼90% Annexin V+) of BCR-ABL1+ cells. As with LY, increased levels of active BAD were found at the mitochondrial membrane of RAP- and PP242-treated BCR-ABL1+ cells. We found that PP242 downregulated p-Akt (92%), Mcl-1 (67%) and Bcl-xL (51%) more efficiently than RAP or LY. It has been shown that PP242 impairs the clonogenic potential of TKI-resistant mononuclear BM CML-BC cells; however, its effects when used alone or in combination with ABT on survival of normal and leukemic hematopoietic stem (HSCs) and progenitor cells is still unknown. Thus, HSC-enriched (CD34+/CD38-) and progenitor (CD34+) CML-BC cell fractions were isolated from bone marrow and peripheral blood and used in colony forming (CFC) assays with ABT, PP242 or ABT/PP242. ABT alone did not suppress colony formation of Ph+ CD34+/CD38− cells, while PP242 reduced it by nearly 50%. Conversely, ABT/PP242 combination decreased Ph+ stem and progenitor colony formation by ∼80%. Furthermore, the self-renewal of Ph+ CD34+/CD38− cells was markedly impaired by ABT/PP242 as demonstrated by the 80% decrease in replating efficiency. To assess if non-leukemic stem cells would tolerate ABT/PP242, colony assays were performed with LSK from wild type mice treated with ABT, PP242, RAP and ABT/PP242. We did not find a significant effect of ABT or PP242 on clonogenic potential when given as single agents. More importantly, combined treatment decreased CFC output by only 35% while RAP, which has an acceptable toxicity profile as it has been used in clinical trials for patients unresponsive to TKIs, decreased LSK colony forming potential by 50%. In summary, our data showing that combined treatment with the mTORC1/2 inhibitor/BAD activator PP242 and the BCl-xL/Bcl-2 antagonist ABT-263 markedly induces apoptosis of BCR-ABL+ cell lines, in HSCs and in progenitors from CML-BC patients. This approach warrants further pre-clinical investigation aimed at inclusion in clinical protocols for treating blast crisis CML. Disclosures: No relevant conflicts of interest to declare.

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Cooperative Targeting of Bcl-2 Family Proteins By ABT-199 (GDC-0199) and Tyrosine Kinase Inhibitors to Eradicate Blast Crisis CML and CML Stem/Progenitor Cells

Blood ◽

10.1182/blood.v124.21.512.512 ◽

2014 ◽

Vol 124 (21) ◽

pp. 512-512 ◽

Cited By ~ 2

Author(s):

Bing Z Carter ◽

Po Yee Mak ◽

Hong Mu ◽

Hongsheng Zhou ◽

Duncan H Mak ◽

...

Keyword(s):

Stem Cells ◽

Tyrosine Kinase ◽

Progenitor Cells ◽

Tyrosine Kinase Inhibitors ◽

Kinase Inhibitors ◽

Blast Crisis ◽

Single Agent ◽

Specific Inhibitor ◽

Chronic Phase

Abstract Bcr-Abl tyrosine kinase supports CML cell survival in part by regulating antiapoptotic Bcl-2 proteins such as Bcl-xL and Mcl-1. Tyrosine kinase inhibition, the front-line therapy for patients with chronic phase CML, is less effective in blast crisis (BC) patients and inactive against quiescent CML stem/progenitor cells. We reported that ABT-737, a dual Bcl-2/Bcl-xL inhibitor, induces apoptosis in BC CML cells including CD34+quiescent CML cells. ABT-199, a potent Bcl-2 specific inhibitor, has entered clinical trials for various hematological malignancies. We hypothesized that cooperative targeting of antiapoptotic Bcl-2 proteins using a combination of ABT-199 and tyrosine kinase inhibitors (TKIs) would exert enhanced activity against BC CML and CML stem/progenitor cells. Cells from patients (n=4) with TKI-resistant BC CML were treated with ABT-199, TKIs, and combinations. Although exerting low activity by itself, ABT-199 in combination with TKIs synergistically induced apoptosis (CI<0.1) in bulk and CD34+38- cells from these patients regardless of their previous clinical responses to TKIs. The combinations had minimal activity against normal CD34+cells (n=3). Mechanistic studies demonstrated that nilotinib inhibited the expression of Bcl-xL and Mcl-1 mRNA and protein, even in cells from TKI (including nilotinib) resistant patients. Individual inhibition of Bcl-xL or Mcl-1, and even more so inhibition of both, by siRNAs increased the sensitivity of cells to ABT-199, suggesting that cooperative inhibition of Bcl-2 by ABT-199 and Bcl-xL/Mcl-1 by TKIs contributes to the synergy. To evaluate the effect of these combinations on TKI-insensitive quiescent stem/progenitor CML cells, BC CML patient cells were stained with the cell division-tracking dye carboxyfluorescein succinimidyl ester (CFSE) and then co-cultured with human bone marrow (BM)-derived mesenchymal stromal cells (MSCs). Once proliferating and quiescent cells were distinguishable by flow cytometry, cells were treated with ABT-199, TKIs, and their combinations for 48 hours with or without MSC co-culture. Apoptosis was measured in proliferating and quiescent progenitor cells, defined as the percentage of annexin V positivity in CD34+CFSEdim and CD34+CFSEbright cells, respectively. ABT-199 as a single agent decreased viability of CML cells cultured alone or co-cultured with MSCs in both proliferating (IC50=191±103nM and 194±64nM, respectively) and quiescent (IC50=221±75nM and 205±123nM, respectively) CD34+ CML cells. Combinations of ABT-199 with TKIs, including imatinib, nilotinib, dasatinib, or ponatinib, synergistically induced death (CI<0.2) and decreased the number of viable cells in proliferating as well as quiescent CD34+progenitor cell populations (n=6). All 6 patients were resistant to TKIs, and 4 had mutations in the BCR-ABL gene, including three with the T315I mutation. To further test the ability of ABT-199 and TKI combinations to eradicate CML stem cells, we used an inducible transgenic CML mouse model in which the BCR-ABL gene is expressed under control of a tet-regulated enhancer of the murine stem cell leukemia (Scl) gene, allowing targeted BCR-ABL expression in stem/progenitor cells. Once BM cells from transgenic Scl-tTa-BCR-ABL/GFP mice were engrafted in wild type recipient mice, the mice were treated with ABT-199, nilotinib, or both. At the end of a 3-week treatment period, each single agent alone, and even more so with the combinations, significantly decreased blood total GFP+ WBC (12.9±1.4, 5.2±0.3, 6.1±0.4, and 1.6±0.3 x106/ml in controls, ABT-199, nilotinib, and combination, respectively) and neutrophils (1.43±0.03, 0.49±0.06, 0.32±0.03, and 0.25±0.05 x106/ml in the respective groups). ABT-199 (P=0.02), and more so with the combination (P<0.01) but not nilotinib alone (P=0.29), significantly decreased BM GFP+ LSK cells (12.0±1.2, 6.8±0.6, 9.5±1.6, and 2.2±0.2 x103 cells in the respective groups). The in vivo experiments are ongoing. Conclusions: ABT-199 and TKIs cooperatively target antiapoptotic Bcl-2 family proteins. This combination is highly effective in killing bulk and CD34+38- CML cells and quiescent CD34+ CML stem/progenitor cells from BC CML patients in vitro and in suppressing leukemia and leukemia stem cells in vivo. This strategy has the potential to eradicate BC CML cells and CML stem/progenitor cells, neither of which are effectively targeted by TKIs alone. Disclosures Carter: AbbVie, Inc.: Research Funding. Leverson:AbbVie, Inc.: Employment. Konopleva:AbbVie, Inc: clinic trial Other.

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Activation of PP2A by FTY720 Inhibits Survival and Self-Renewal of the Ph(+) Chronic Myelogenous Leukemia (CML) CD34+/CD38− Stem Cell through the Simultaneous Suppression of BCR/ABL and BCR/ABL– independent Signals

Blood ◽

10.1182/blood.v112.11.189.189 ◽

2008 ◽

Vol 112 (11) ◽

pp. 189-189 ◽

Cited By ~ 7

Author(s):

Paolo Neviani ◽

Ramasamy Santhanam ◽

Yihui Ma ◽

Guido Marcucci ◽

John C. Byrd ◽

...

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Stem Cell ◽

Progenitor Cells ◽

Blast Crisis ◽

Healthy Individuals ◽

Self Renewal ◽

Pp2a Activity ◽

Cd34 Cells ◽

Clonogenic Potential

Abstract CML is a clonal disorder of the pluripotent hematopoietic stem cell characterized by the sustained kinase activity of the BCR/ABL oncoprotein. We reported that the BCR/ABL-dependent and SET-mediated inhibition of protein phosphatase PP2A tumor suppressor activity is essential for the leukemogenic potential of CD34+ CML bone marrow progenitors, as molecular and pharmacologic restoration of PP2A inhibits the activity of BCR/ABL and that of several important regulators of cell survival/proliferation, thus resulting in marked apoptosis, impaired clonogenic potential and in vivo leukemogenesis of imatinib/dasatinib-sensitive and -resistant Ph(+), but not normal, CD34+ blasts and/or BCR/ABL+ mouse marrow progenitors. Here we show that SET-dependent suppression of PP2A activity is a common feature of Ph(+) progenitors (CMP and GMP) and imatinib/ dasatinib-insensitive CD34+/CD38- BCR/ABL+ (n=3) stem cells but not of the equivalent cell fractions from healthy individuals (n=3). To determine the biological importance and therapeutic implications of impaired PP2A activity in Ph(+) stem cells, we evaluated by clonogenic, CFC/replating, LTC-IC and CFSE-mediated cell division-tracking assays, the effects of FTY720 (2.5 mM), a PP2A activator currently in phase III trials for MS patients, and lentiviral-mediated ectopic PP2Ac expression on survival and self-renewal of BCR/ ABL+ stem/progenitor cells isolated from bone marrow of CML blast crisis patients (ntot=8; Ph1≥90%) and/or SCL-tTA-BCR/ABL transgenic animals (ntot=10). FTY720 treatment (2.5–5mM) severely suppressed the clonogenic potential of CD34+/CD38− and CD34+/CD38+/CD45RA−/+ CML stem/progenitor cells. Accordingly, self-renewal and long-term repopulating potential of CML leukemic stem cells was markedly impaired by pharmacologic PP2A reactivation. In fact, the CFC output of LTC-IC cultures (6 weeks) deriving from FTY720-treated (2.5 mM; 72h) Ph(+) CD34+ cells was more than 95% inhibited if compared to that of LTC-IC cultures from untreated CML cells. By contrast, imatinib (5 mM) and dasatinib (200 nM) treatment led to a 3.5 and 5-fold increase in CFC output, respectively. Consistent with the ability of FTY720 to impair self-renewal of CML stem cells, a 50–90% reduction of the CFSEMAX/quiescent cell population was observed in CFSE-stained CD34+ CML cells treated for 6–9 days with FTY720. Notably, FTY720 did not exert any significant effect on CFSE-stained CD34+ cells from healthy individuals whereas, as expected, imatinib (5 mM) and dasatinib (200 nM) treatment led to a 22% and 27% increase in CFSEMAX CML cells, respectively. Interestingly, only FTY720 triggered apoptosis of CFSEMAX CML cells (41% Annexin V+ cells) although BCR/ABL activity (phospho-ABL intracellular flow-cytometry staining) in CFSEMAX cells was efficiently inhibited by FTY720, Imatinib and dasatinib, suggesting that BCR/ABL-independent PP2A-regulated signals control the survival and self-renewal of CML stem cells. Indeed, lentiviral-driven PP2Ac-overexpression as well as treatment with FTY720, but not imatinib, significantly decreased (40–90% reduction) CFC/serial replating efficiency, colony size and percentage of CFSEMAX fraction (66–96% reduction) of Lin−/ Sca+/Kit+ (LSK) cells isolated from bone marrow and spleen of leukemic SCL-tTA-BCR/ ABL mice. Mechanistically, the detrimental effect of PP2A activation on survival and self-renewal of CML stem cells might depend on the ability of PP2A to inactivate b-catenin that, reportedly, is a PP2A target essential for the self-renewal of the CML blast crisis GMP progenitors. In fact, immunoblotting, direct immunofluorescence and LET/TCF luciferase assays showed that ectopic PP2Ac expression and/or FTY720, but not imatinib, treatment leads to inactivation/degradation of nuclear b-catenin in BCR/ABL+ primary mouse LSK and/or 32D-BCR/ABL cells. Altogether our data not only highlight the importance of PP2A inactivation for survival and self-renewal of CML stem cells but also suggest the existence of BCR/ABL-independent, PP2A-sensitive and b-catenin-mediated signals that may account for resistance of CML quiescent stem cells to tyrosine kinase inhibitor monotherapy. Thus, FTY720 treatment has the potential to eradicate CML by efficiently targeting both stem and progenitor Ph(+) cells regardless of their degree of sensitivity to imatinib and dasatinib.

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Effective and Selective Elimination of CML Stem Cells Using Novel Ethacrynic Acid Derivatives

Blood ◽

10.1182/blood.v124.21.4508.4508 ◽

2014 ◽

Vol 124 (21) ◽

pp. 4508-4508

Author(s):

Su Chu ◽

YinWei Ho ◽

Guisen Zhao ◽

Tessa L. Holyoake ◽

Samuel Waxman ◽

...

Keyword(s):

Stem Cells ◽

Protein Expression ◽

Progenitor Cells ◽

Ethacrynic Acid ◽

Conflicts Of Interest ◽

Blast Crisis ◽

Chronic Phase ◽

Cd34 Cells ◽

Induced Apoptosis

Abstract Tyrosine kinase inhibitors (TKI) are highly effective in the treatment of CML but do not eliminate primitive, quiescent leukemia stem cells (LSC), which persist as a potential source of leukemia relapse. Additional strategies to enhance eradication of LSC are required to increase the possibility of treatment free remissions for CML patients. Glutathione S-transferase P1-1 (GSTP1-1) is a Phase II detoxifying enzyme which is overexpressed in several cancers and causes drug resistance. The diuretic Ethacrynic acid (EA) is a GSTP1-1 activity inhibitor and also induces celldeath in malignant cells at high concentrations. We have synthesized two EAoxadiazole analogs, 6U and 6S, which demonstrate enhancedpro-apoptotic effects in CML K562 cells expressing high levels of GSTP1-1. Previously we found that 6U and 6S induced apoptosis through downregulation of anti-apoptotic protein MCL-1 in addition to their GSTP1-1 activity inhibition. We extended these observations to primary CML stem/progenitor cells. We observed increased expression of GSTP1-1 mRNA and protein, associated with increased expression of MCL-1, BCL2 and BCL-xL, in chronic phase (CP) and blast crisis (BC) CML compared to normal CD34+ cells. Treatment of CP CML CD34+ cells with 6U or 6S (1 to 6µM) for 24 to 48 hours resulted in a significant dose-dependent induction of apoptosis, inhibition of cell growth, and reduction in colony forming cell (CFC) numbers, with 6U demonstrating greater efficacy than 6S. Treatment with 6U did not induce significant apoptosis of normal (NL) CD34+ cells at doses below 4µM. 6U induced significantly less apoptosis in NL compared with CML CD34+ cells (2µM, p<0.05). We further tested the activity of 6U against purified CML and normal CD34+CD38- stem/primitive progenitors and CD34+CD38+ committed progenitors with or without the BCR-ABL TKI Dasatinib (DAS). 6U treatment induced apoptosis of CML, but not normal, CD34+CD38- and CD34+CD38+ cells (Table). Combination of 6U with DAS (50nM) selectively enhanced apoptosis of CML compared to normal cells, including quiescent, slowly dividing CML LSC that are resistant to TKI-induced apoptosis (p≤0.01). Treatment with 6U alone or with DAS, significantly increased G1, and decreased S/G2/M phase of CML, but not in normal CD34+ cells, and reduced CFC growth from CML CD34+CD38+ cells (Table). CML, but not normal CD34+ cells, treated with 6U, with or without DAS, prior to transplant, showed significantly reduced engraftment in NSG mice, indicating selective inhibition of in vivo repopulating CML LSC (Table). Treatment with 6U was also effective in inducing apoptosis and inhibiting CFC growth in BC CML progenitor cells (Table). 6U treatment resulted in down-regulation of GSTPI1-1 and MCL-1 protein expression in CP and BC CML, but not in normal CD34+ cells. Interestingly 6U treatment also reduced BCR-ABL protein expression in CP and BC CML CD34+ cells. We conclude that CML CP and BC LSC express high levels of GSTP1-1 and anti-apoptotic proteins, which can be targeted by the novel EA derivative 6U through a new mechanism. Since 6U has significantly lesser effects on normal stem cells, it may offer a promising and innovative approach to selectively target CP and BC CML LSC in combination with TKI inhibitors. Abstract 4508. Table CML CP Normal CML BC Ctrl 6U DAS DAS+ 6U Ctrl 6U DAS DAS+ 6U Ctrl 6U DAS DAS+ 6U Apoptosis (normal, CP CML: CD34+CD38-; CML BC CD34+) 3.4± 0.9 15.9±6.7 9.4± 2.6 47.4±13.6 ** 3.3± 0.9 5.1± 1.0 1.6± 0.2 7.0± 1.2 * 3.4± 0.7 30±12.7 10.6±1.8 43.3±14.1 ** CFU-GM (normal, CP CML: CD34+CD38+; CML BC CD34+) 71.3± 7.8 7± 3.2 ** 21± 7.3 ** 5 ± 2.3 ** 121±19.3 102.7±6.2 134.3±15.9 103±5.1 288.5±89.4 26.5±11.3 *** 82.7±33.1 ** 8 ± 3.6 *** NSG engraftment (CD34+) 1.8± 0.3 0.4± 0.1 *** 0.8± 0.3 ** 0.4± 0.04 *** 68.2± 4.9 61± 2.2 68.1± 2.9 64.2± 3.9 Data shown are mean ± SEM of 3-6 samples. Significance, compared to controls. *p≤0.05,**p≤0.01, ***p≤0.001 Disclosures No relevant conflicts of interest to declare.

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Identification of the TAK1-NF-κB Axis As Critical Regulator of AML Stem and Progenitor Cell Survival.

Blood ◽

10.1182/blood.v120.21.2982.2982 ◽

2012 ◽

Vol 120 (21) ◽

pp. 2982-2982

Author(s):

Matthieu C.J. Bosman ◽

Jan J. Schuringa ◽

Wim J. Quax ◽

Edo Vellenga

Keyword(s):

Stem Cells ◽

Cell Death ◽

Cell Survival ◽

Cell Lines ◽

Combined Treatment ◽

Annexin V ◽

Fold Increase ◽

Dependent Manner ◽

Leukemic Stem Cells ◽

Induced Apoptosis

Abstract Abstract 2982 A small population of leukemic stem cells is resistant to chemotherapy and is responsible for the leukemic out-growth and relapse in acute myeloid leukemia (AML) patients. Evasion of apoptosis might be one of the essential mechanisms involved in this process. In order to gain more insight into the differences in the apoptotic programming between normal and leukemic (stem) cells, we recently performed gene array analysis by comparing CD34+ AML cells versus CD34+ normal bone marrow (NBM) cells. Gene ontology (GO) analysis of the differentially expressed genes between AML and NBM cells revealed differences in GO terms metabolic processes and apoptosis. In order to characterize differences in apoptotic programming in more detail 429 apoptotic related genes were selected and cluster analysis showed that CD34+ AML and CD34+ NBM cells could be separated into two distinct groups. In particular TGF-β activated kinase 1 (TAK1)/MAP3K7 was one of the apoptosis-related genes that was significantly higher expressed in CD34+ AML cells compared to CD34+ NBM cells (p = 1.8e−7). This increased expression of TAK1 could be confirmed by Q-PCR, showing an increase of on average 5.8 fold in TAK1 expression in the studied CD34+ AML cells. In mice it has been demonstrated that TAK1 is required for the survival of hematopoietic cells which is largely dependent on TNFR1 and TNFR2. In accordance with these data, we showed that TAK1 is also necessary in human hematopoiesis. Colony-forming cell (CFC) assays showed that inhibition of TAK1 in human cord blood CD34+cells, either by shRNAs targeting TAK1 or the TAK1 inhibitor 5z–7-oxozeaenol, resulted in a 2 fold reduction in CFU-GM and BFU-E frequencies compared to control cells. The efficacy was strongly further enhanced by the addition of TNFα, which resulted in a 9.4 fold decrease in CFC colonies upon TAK1 inhibition. Subsequently, we questioned whether TAK1 inhibition would affect CD34+ AML cell survival. Treatment of the AML cell lines MOLM13, OCI-M3 and HL60 with the TAK1 inhibitor 5z–7-oxozeaenol alone only induced modest effects, but in combination with TNFα for 24 hrs a strong induction of apoptosis was observed (IC50 respectively = 23nM, 215nM and 60 nM). Comparable results were observed in HL60 cells transduced with shRNAs targeting TAK1 whereby a downmodulation of TAK1 resulted in a 5.4 fold increase in Annexin V+ cells upon TNFα addition. In accordance with previous data, Western blot analysis showed that TAK1 inhibition reduced the levels of p-IκBα, p-p38, p-ERK and p-C-JUN. To test which of these pathways would be important for cell survival, AML cell lines were treated with either the p38 inhibitor SB203580, MEK/ERK inhibitor U0126, JNK inhibitor SP600125 and the NF-κB inhibitor BMS-345541, alone or in combination with TNFα. Addition of the NF-κB inhibitor BMS-345541 induced apoptosis in OCI-M3 and MOLM13 which was significantly increased in combination with TNFα (2.4 fold, p = 0.02). In contrast, inhibition of p38, MEK/ERK and JNK, either alone or in combination with TNFα, did not induce cell death in the AML cell lines. These data suggest that cell death induced by TAK1 inhibition is mainly due to inhibition of the NF-κB pathway. To determine the effects of TAK1 inhibition on primary AML cells, long-term expansion of the leukemic stem cell enriched CD34+ AML cell fraction was evaluated in MS5 stromal co-cultures in the absence or presence of TAK1 inhibitor and/or TNFα. Combined treatment for a period of 2 weeks completely abrogated the out-growth of CD34+ AML cells, indicating that both leukemic progenitors as well as leukemic stem cells were targeted. In contrast, addition of the single agents did not efficiently reduce cell growth. Similarly, downmodulation of TAK1 using shRNAs strongly sensitized primary CD34+ AML cells for TNFα-induced apoptosis, showing a 6 fold increase in Annexin V+ cells compared to control cells. Results on the in vivo efficacy of TAK1 inhibition on primary AML cells are in progress. In conclusion, our results show that TAK1 is frequently overexpressed in CD34+ AML cells, and that inhibition of TAK1 in combination with TNFα is highly efficient in inducing apoptosis of leukemic stem/progenitor cells in a NF-κB-dependent manner. Disclosures: No relevant conflicts of interest to declare.

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Poly-L-arginine: Enhancing Cytotoxicity and Cellular Uptake of Doxorubicin and Necrotic Cell Death

Anti-Cancer Agents in Medicinal Chemistry ◽

10.2174/1871520618666180412114750 ◽

2019 ◽

Vol 18 (10) ◽

pp. 1448-1456 ◽

Cited By ~ 3

Author(s):

Bahareh Movafegh ◽

Razieh Jalal ◽

Zobeideh Mohammadi ◽

Seyyede A. Aldaghi

Keyword(s):

Cell Death ◽

Cellular Uptake ◽

Combined Treatment ◽

Annexin V ◽

Cell Penetrating Peptides ◽

Short Exposure ◽

Dependent Manner ◽

Du145 Cells ◽

Induced Apoptosis ◽

Resistance To Doxorubicin

Objective: Cell resistance to doxorubicin and its toxicity to healthy tissue reduce its efficiency. The use of cell-penetrating peptides as drug delivery system along with doxorubicin is a strategy to reduce its side effects. In this study, the influence of poly-L-arginine on doxorubicin cytotoxicity, its cellular uptake and doxorubicin-induced apoptosis on human prostate cancer DU145 cells are assessed. Methods: The cytotoxicity of doxorubicin and poly-L-arginine, alone and in combination, in DU145 cells was evaluated at different exposure times using MTT assay. The influence of poly-L-arginine on doxorubicin delivery into cells was evaluated by fluorescence microscopy and ultraviolet spectroscopy. DAPI and ethidium bromide- acridine orange stainings, flow cytometry using annexin V/propidium iodide, western blot analysis with anti-p21 antibody and caspase-3 activity were used to examine the influence of poly-L-arginine on doxorubicininduced cell death. Results: Poly-L-arginine had no cytotoxicity at low concentrations and short exposure times. Poly-L-arginine increased the cytotoxic effect of doxorubicin in DU145 cells in a time-dependent manner. But no significant reduction was found in HFF cell viability. Poly-L-arginine seems to facilitate doxorubicin uptake and increase its intracellular concentration. 24h combined treatment of cells with doxorubicin (0.5 µM) and poly-L-arginine (1 µg ml-1) caused a small increase in doxorubicin-induced apoptosis and significantly elevated necrosis in DU145 cells as compared to each agent alone. Conclusion: Our results indicate that poly-L-arginine at lowest and highest concentrations act as proliferationinducing and antiproliferative agents, respectively. Between these concentrations, poly-L-arginine increases the cellular uptake of doxorubicin and its cytotoxicity through induction of necrosis.

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Quantitative and cell-cycle differences in progenitor cells mobilized by recombinant human interleukin-7 and recombinant human granulocyte colony-stimulating factor

Blood ◽

10.1182/blood.v88.11.4139.4139 ◽

1996 ◽

Vol 88 (11) ◽

pp. 4139-4148 ◽

Cited By ~ 1

Author(s):

KJ Grzegorzewski ◽

KL Komschlies ◽

JL Franco ◽

FW Ruscetti ◽

JR Keller ◽

...

Keyword(s):

Stem Cells ◽

Progenitor Cells ◽

Combined Treatment ◽

Fold Increase ◽

Granulocyte Colony Stimulating Factor ◽

Colony Stimulating Factor ◽

Interleukin 7 ◽

Stimulating Factor

Abstract Administration of recombinant human interleukin-7 (rhIL-7) to mice increases the exportation of myeloid progenitors (colony-forming unit [CFU]-c and CFU-granulocyte erythroid megakaryocyte macrophage [CFU-GEMM]) from the bone marrow (BM) to peripheral organs, including blood, and also increases the number of primitive progenitor and stem cells in the peripheral blood (PB). We now report that combined treatment of mice with rhIL-7 and recombinant human granulocyte-colony stimulating factor (rhG-CSF) stimulates a twofold to 10-fold increase in the total number of PB CFU-c, and a twofold to fivefold increase in the total number of PB CFU-spleen at day 8 (CFU-S8) over the increase stimulated by rhIL-7 or rhG-CSF alone. In addition, the quality of mobilized cells with trilineage, long-term marrow-repopulating activity is maintained or increased in mice treated with rhIL-7 and rhG-CSF compared with rhIL-7 or rhG-CSF alone. These differences in mobilizing efficiency suggest qualitative differences in the mechanisms by which rhIL-7 and rhG-CSF mobilize progenitor cells, in fact, the functional status of progenitor cells mobilized by rhIL-7 differs from that of cells mobilized by rhG-CSF in that the incidence of actively cycling (S-phase) progenitors obtained from the PB is about 20-fold higher for rhIL-7-treated mice than for mice treated with rhG-CSF. These results suggest the use of rhIL-7-mobilized progenitor/stem cells for gene-modification and tracking studies, and highlight different functions and rates of repopulation after reconstitution with PB leukocytes obtained from mice treated with rhIL-7 versus rhG-CSF.

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NF-kB Family Proteins Participate in Multiple Steps of Hematopoiesis through Elimination of Reactive Oxygen Species (ROS).

Blood ◽

10.1182/blood.v104.11.1694.1694 ◽

2004 ◽

Vol 104 (11) ◽

pp. 1694-1694

Author(s):

Soichi Nakata ◽

Itaru Matsumura ◽

Hirokazu Tanaka ◽

Yusuke Satoh ◽

Takumi Era ◽

...

Keyword(s):

Stem Cells ◽

Hematopoietic Stem Cells ◽

Progenitor Cells ◽

Es Cells ◽

Hematopoietic Cells ◽

Hematopoietic Stem ◽

Growth And Survival ◽

Ros Accumulation ◽

Dependent Growth ◽

Induced Apoptosis

Abstract NF-kB family proteins have been reported to induce the expression of over 150 target genes, thereby crucially regulating immune responses, stress responses, and inflammation. These proteins also play important roles in cell growth and survival in various cell types. However, the precise roles of NF-kB in hematopoiesis and their mechanisms remain undetermined. To examine the roles for NF-kB family proteins in the growth and survival of hematopoietic cells, we expressed dominant negative NF-kB (IkBSR) in a murine IL-3-dependent cell line Ba/F3 using a Lac-inducible system, in which IkBSR was inducibly expressed by the IPTG treatment; this clone was designated Ba/F3/IkBSR. Furthermore, we introduced EPO receptor (R), TPOR, and G-CSFR/gp130 consisting of the extracelluar domain of G-CSFR and cytoplasmic domain of gp130 into this clone. At first, we confirmed that these clones could survive and proliferate under the cultures with IL-3, EPO, TPO, G-CSF, respectively. Although IPTG-induced IkBSR slightly suppressed IL-3- and EPO-dependent growth at low concentrations, it did not affect TPO- or gp130L-dependent growth, suggesting that NF-kB might not be so important for cytokine-dependent growth of hematopoietic cells. In contrast, IkBSR prominently enhanced factor-deprived apoptosis, which was accompanied by the ROS accumulation. To access the roles of ROS in IkBSR-enhanced apoptosis, we overexpressed ROS scavenger enzymes MnSOD and thioredoxin X (TRX) in Ba/F3/IkBSR, respectively. As a result, MnSOD and TRX significantly canceled IkB-SR-enhanced apoptosis, suggesting that ROS would be responsible for this apoptosis. We next analyzed the effects of IkBSR on the growth and survival of normal hematopoietic cells. When IkBSR was introduced into murine Lin−Sca-1+ hematopoietic stem/progenitor cells with the retrovirus system, it induced apoptosis even in the presence of appropriate cytokines. This apoptosis was also accompanied by the ROS accumulation due to the downregulated expression of anti-oxidants such as glutathione, MnSOD, glutathione peroxidase, and TRX. In addition, the expression of antiapoptotic BCl-2 family members, Bcl-XL, Bcl-2, and A1 was found to be repressed by IkBSR. However, since antioxidants such as MCI (3-methyl-1-phenyl-2-pyrazolin-5-one), N-acetylecysteine and TRX cancelled this apoptosis, ROS were supposed to be more important for IkBSR-induced apoptosis in normal hematopoietic stem/progenitor cells. To further analyze the roles for NF-kB proteins in the development of hematopoietic cells, we expressed IkBSR in an inducible fashion at various stages of hematopoiesis using the OP9 system, in which hematopoietic cells are induced to develop from ES cells. When IkBSR was expressed at the stage of hemangioblasts, IkBSR induced apoptosis and inhibited the development of hematopoietic stem cells, which was also cancelled by MCI. Furthermore, when IkBSR was expressed after the development of hematopoietic stem cells, it also inhibited terminal differentiation towards granulocytes, erythrocytes, and megakaryocytes through ROS-mediated apoptosis; IkBSR inhibited granulopoiesis before the development of myeloblasts, erythropoiesis after the development of proerythroblasts, and megakaryopoiesis during polyploidization of megakaryocytes. These results indicate that NF-kB family proteins play essential roles to prevent apoptosis at multiple steps of hematopoiesis by eliminating ROS.

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Inhibition of Bcl-2/Bcl-XL Promotes Apoptosis in Blast Crisis CML Including Quiescent Primitive Progenitor Cells Regardless of Cellular Responses to Tyrosine Kinase Inhibitors.

Blood ◽

10.1182/blood.v114.22.646.646 ◽

2009 ◽

Vol 114 (22) ◽

pp. 646-646

Author(s):

Duncan H. Mak ◽

Wendy D. Schober ◽

Marina Konopleva ◽

Jorge Cortes ◽

Hagop M. Kantarjian ◽

...

Keyword(s):

Tyrosine Kinase ◽

Tyrosine Kinase Inhibitor ◽

Progenitor Cells ◽

Kinase Inhibitors ◽

Kinase Inhibitor ◽

Blast Crisis ◽

Antiapoptotic Proteins ◽

Cell Compartments ◽

Cd34 Cells

Abstract Abstract 646 The advent of imatinib, a Bcr-Abl tyrosine kinase inhibitor revolutionized the treatment for patients with CML. Development of resistance, limited activity in blast crisis CML, and more importantly, insensitivity of quiescent primitive CD34+ CML progenitor cells are evolving problems facing this therapy. Antiapoptotic Bcl-2 proteins were known to be highly expressed in Bcr-Abl expressing cells and inhibition of Bcl-2/Bcl-XL by the selective inhibitor ABT-737 was reported to augment the killing of tyrosine kinase inhibitors in CML cells. However, its effect on quiescent primitive CD34+ CML progenitor cells is unknown. To investigate the effect of activating the apoptotic machinery in quiescent primitive CD34+CML progenitor cells, which are resistant to current therapies, we first compared the expression of antiapoptotic proteins in proliferating and quiescent primitive CD34+CML progenitor cells. Cells obtained from patients with blast crisis CML were stained with the fluorescent 5-(and 6-) carboxy-fluorescein diacetate succinimidyl ester, a cell proliferation tracking dye, and cultured in vitro for 4-6 days. Cells were then stained with CD34 antibody and FACS sorted into proliferating and quiescent CD34+/PI- CML progenitor cells. RNA levels of antiapoptotic proteins in these two cell populations (n=8) were determined by real-time RT-PCR: quiescent and proliferating primitive CD34+ CML progenitor cells expressed similar levels of Bcl-2, Bcl-XL, Mcl-1, and XIAP implying that like total blast cells, quiescent primitive CD34+CML progenitor cells may also be sensitive to agents targeting these proteins. We next treated 5 samples obtained from patients with blast crisis CML with ABT-737 and measured apoptosis in total CD34+ cells, proliferating CD34+ cells, and quiescent CD34+ cells. All 5 patients were resistant to or relapsed from imatinib and nilotinib and/or dasatinib treatments and they were insensitive to imatinib in vitro as expected. However, cells from 4 patients were sensitive to ABT-737, in bulk blasts and in both proliferating and quiescent CD34+ CML cell compartments: % specific apoptosis with 100 nM of ABT-737=40.8±7.7, 38.4±8.5, 40.0±5.1, respectively at 24 hours. Interestingly, when ABT-737 was combined with imatinib, cell death was greatly enhanced in cells from all 5 patients in all cell compartments (combination index=0.059±0.032, 0.041±0.025, 0.111±0.042, respectively). Furthermore, we showed previously, that triptolide, an antitumor agent from a Chinese herb, induces apoptosis in both proliferating and quiescent primitive CD34+CML progenitor cells by decreasing Mcl-1 which is a resistant factor for ABT-737, XIAP, and Bcr-Abl protein levels (Mak D. et al., MCT in press). When ABT-737 was combined with triptolide, a significant increase of cell death was found in total CD34+ and proliferating as well as quiescent primitive CD34+CML cells with combination index at EC50=0.57, 0.55, and 0.56, respectively in cells from the 5 patients suggesting a high degree of synergism. In summary, Bcl-2, Bcl-XL, Mcl-1, and XIAP are equally expressed in proliferating and quiescent primitive CML cells and targeting Bcl-2/Bcl-XL promotes death of blast crisis CML cells, tyrosine kinase inhibitor resistant CML cells, and quiescent primitive CD34+ CML progenitor cells. Researches suggest that the combination of apoptosis inducing agents and tyrosine kinase inhibitor is a novel strategy to overcome tyrosine kinase resistance, eradicate quiescent primitive CML progenitor cells, and improve current therapy for patients with CML. Disclosures: No relevant conflicts of interest to declare.

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Targeting of CLEC12A In Acute Myeloid Leukemia by Antibody-Drug-Conjugates and Bispecific CLL-1×CD3 BiTE Antibody

Blood ◽

10.1182/blood.v116.21.2890.2890 ◽

2010 ◽

Vol 116 (21) ◽

pp. 2890-2890

Author(s):

Paul Noordhuis ◽

Monique Terwijn ◽

Arjo P Rutten ◽

Linda Smit ◽

Gert J. Ossenkoppele ◽

...

Keyword(s):

Stem Cells ◽

T Cells ◽

Colony Formation ◽

Myeloid Leukemia ◽

Splice Variants ◽

Annexin V ◽

Rt Pcr ◽

Self Renewal ◽

Bite Antibody

Abstract Abstract 2890 Response rates of ±80% in Acute Myeloid Leukemia (AML) are observed after conventional therapy but ±30% of patients experience a relapse. In the elderly the outcome is even worse. A small population of therapy resistant leukemia cells, minimal residual disease (MRD), are thought to be responsible for relapse of AML. The leukemic stem cells (LSC) herein have self renewal potential and reside in the CD34+CD38- stem cell compartment and side population (SP) compartment and can be identified via aberrant marker expression and scatter properties. Several markers are identified that show differential expression on AML (stem) cells versus normal hematopoietic stem cells (HSC). Previously we showed that CLEC12A (CLL-1, MICL, KLRL1, DCAL-2) is expressed on blasts of 90% of AML patients with varying expression. Importantly, CLEC12A is expressed on LSC but not on normal HSC (van Rhenen, Blood 110(7), 2007). This unique expression pattern paves the way to develop therapies that potentially eliminate CLEC12A-positive LSC and preserves CLEC12A-negative HSC. Drug-conjugated antibodies (ADCs) targeting CLEC12A and Bispecific T cell Engager (BiTE) scFv-antibodies targeting T-cells to CLEC12A positive cells could be instrumental to achieve this goal. We evaluated the response of AML cells to ADCs conjugated via cleavable and non-cleavable linkers to the maytansine derivates DM1 and DM4 and to the BiTE antibody CLL-1×CD3. ADC activity was assessed by colony formation capacity after 24 hours exposure to 0.1–100 nM ADC in 29 freshly obtained AML samples. The response to the BiTE antibody was tested by flow cytometry in 9 AML samples via induction of apoptosis (Annexin V/7AAD) after 24 hours exposure. To determine the effect of ADC on self-renewal in normal bone marrow (NBM), colony formation capacity was asseses during long term liquid culture after 24 hours exposure to 1–100 nM ADC. Furthermore internalisation of CLEC12A in AML progenitor and stem cells was tested. Several splice variants of CLEC12A are reported (CLL-1, MICLα, MICLβ, MICLγ) that have different intra-cellular signalling motifs or lack the transmembrane motif or the extra-cellular c-type lectin-like domain. Since these variants could not all be distinguished or detected by extra-cellular antibody binding, we evaluated these splice variants by Q-RT-PCR. After 24 hours exposure, a median IC50 value of >100 nM was observed for the unconjugated antibody CR2357. The median IC50 values for ADCs with non-cleavable linkers were 10 nM for CR2357-SMCC-DM1 (4,3 DM1/mAb), 2 nM for CR2357-PEG4-MAL-DM1 (5.9 DM1/mAb) and 0.8 nM for CR2357-PEG4-MAL-DM1 (10 DM1/mAb). For CR2357-SPDB-DM4 (4 DM4/mAb) which has a cleavable linker the median IC50 was 4 nM. The median IC50 of ADCs with non-cleavable linkers were significantly correlated to each other (r=0.730-0.784, p<0.01). CR2357-PEG4-MAL-DM1 (10 DM1/mAb) was significantly correlated to CLEC12A membrane expression (r=0.649, p<0.05). Prelimanary data of colony formation capacity during long term liquid culture of NBM showed that at >5 weeks after exposure, this was reduced to 15–50% for CR2357 and CR2357-PEG4-MAL-DM1 (10 DM1/mAb) relative to the untreated control. Exposure of AML cells to the CLL-1×CD3 BiTE antibody with donor T-cells (E:T=10:1 and 1:1) showed a dose dependent activation of T-cells as measured by increased CD25 and CD69 expression on CD4+ and CD8+ T-cells. Importantly, besides T-cell activation, Annexin V/7AAD staining of AML cells showed a specific decrease of CLEC12A-positive viable cells while in CLEC12A-negative cells viability remained constant. Internalisation of CR2357 antibody in CD34+/CD38+ progenitor cells and in CD34+/CD38- LSC was clearly demonstrated. Q-RT-PCR of CLEC12A splice variant expression showed highest expression for MICLα > MICLβ ∼F MICLγ > CLL-1 indicating that MICLα is the main variant expressed on the cellular membrane. Downstream signalling will therefore mainly be mediated by SHP-1/2 phosphatases. Although expression levels in AML, NBM, and sorted sub-populations varied, the ratio between the splice variants remained almost similar suggesting that the individual splice variants play a similar role in the different cell populations. In conclusion: these result show that targeting of CLEC12A-positive AML cells by ADCs and BiTE antibodies results in specific cell kill and might be a promising approach for the eradication of LSC that survive conventional therapy. Disclosures: No relevant conflicts of interest to declare.

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5-Azacytidine and Decitabine Induce Demethylation and Re-Expression of FAS (CD95) and Promote Apoptosis in Neoplastic Cells in Acute Myeloid Leukemia (AML),

Blood ◽

10.1182/blood.v118.21.3463.3463 ◽

2011 ◽

Vol 118 (21) ◽

pp. 3463-3463

Author(s):

Heidrun Karlic ◽

Rene Reitermaier ◽

Viviane Ghanim ◽

Harald Herrmann ◽

Roman Thaler ◽

...

Keyword(s):

Stem Cells ◽

Flow Cytometry ◽

Acute Myeloid Leukemia ◽

Progenitor Cells ◽

Myeloid Leukemia ◽

Neoplastic Cells ◽

Drug Induced ◽

Secondary Aml ◽

Induced Apoptosis ◽

Acute Myeloid

Abstract Abstract 3463 Epigenetic and apoptosis-regulating mechanisms have been implicated as critical factors contributing to the progression from myelodysplastic syndromes (MDS) to secondary acute myeloid leukemia (AML). However, the exact molecular mechanisms and genes involved in disease evolution have not been identified yet. We screened for epigenetically regulated pro-apoptotic effector molecules in neoplastic cells in patients with MDS (n=50) and AML (n=30). Among a series of potential regulators, we identified FAS (CD95) as an epigenetically regulated critical death regulator in neoplastic cells. As assessed by qPCR, bone marrow cells obtained from patients with low risk MDS were found to display high levels of FAS, whereas FAS mRNA levels were lower or undetectable in patients with advanced MDS (with excess of blasts) or secondary AML. Moreover, we were able to show by multicolor flow cytometry that CD34+/CD38+ progenitor cells and CD34+/CD38- stem cells in MDS and AML display measurable FAS (CD95) on their surface, with slightly higher levels detectable in progenitor cells in low risk MDS compared to high risk MDS and secondary AML. Methylation-specific PCR and qPCR revealed that the FAS-promoter is hypermethylated in primary AML cells as well as in various AML cell lines including KG1 and HL60 thus repressing mRNA-synthesis. In addition, we found that exposure to 5-Azacytidine or Decitabine leads to demethylation of CpG-rich regions closest to the transcription starting sites, and thus to re-expression of FAS in AML cells. In vitro-targeting of AML cells by demethylating drugs was also found to revert epigenetic inactivation of other tumor suppressor genes such as CDKN2B (P15), CDKN2A (P16), ESR1 (estrogen-receptor alpha), with subsequent normalization of mRNA expression levels. Next, we asked whether CD95 acts as a critical death regulator involved in drug-induced apoptosis in neoplastic cells. In these experiments, both demethylating agents, 5-Azacytidine or Decitabine, were found to induce dose-dependent apoptosis and growth inhibition in primary AML cells, primary MDS cells, and in all AML cell lines examined. Drug-induced apoptosis in AML cells was accompanied by activation of caspase 8 and caspase 3 as well as increased expression of proapoptotic FAS/CD95 as determined by qPCR, Western blotting, and flow cytometry. Moreover, both drugs were found to induce expression of the FAS-ligand and DAPK1 in neoplastic cells. We then applied a siRNA against FAS. The siRNA-induced knock-down of FAS was found to block drug-induced FAS expression and FAS-induced apoptosis in KG1 cells and HL60 cells. In conclusion, our data show that FAS is hypermethylated in neoplastic cells in patients with advanced MDS and AML, that demethylating agents lead to re-expression of FAS, and that drug-induced FAS expression mediates apoptosis in leukemic cells. As FAS is also expressed on neoplastic stem cells, these observations may have clinical implications and may explain beneficial effects seen with 5-Azacytidine or Decitabine in patients with advanced MDS. Disclosures: Valent: Novartis: Consultancy, Honoraria, Research Funding.

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