Combined Therapy With BRD4 Antagonist and FLT3 Inhibitor Exerts Synergistic Activity Against Cultured and Primary AML Blast Progenitors Expressing FLT-ITD

Abstract Mutations in FLT3 are detected in approximately 30% of AML and are associated with poor overall survival. Although first (PKC412, sorafenib and CEP701) and second generation (AC220) FLT3 tyrosine kinase inhibitors (TKI) induce remissions, resistance-causing gatekeeper (F691I/L), activation loop (AL) mutations (D835V/Y/F) or compound FLT3-ITD/F691I AL mutations are known to impair the in vitro and in vivo activity of the FLT3-TKIs. The BET (bromodomain and extraterminal) protein family members including BRD4 bind to acetylated lysines on the histone proteins, help assemble transcriptional regulators at the target gene promoters and enhancers, and regulate the expression of important oncogenes, e.g., MYC and BCL-2. BRD4 antagonists JQ1 and I-BET151 disrupt the binding of the bromodomain of BRD4 to acetylated lysines on histone proteins, thereby inhibiting expressions of c-MYC and BCL-2 and inducing apoptosis of AML cells. Based on this, we evaluated the in vitro and in vivo activity of JQ1 and FLT3 antagonists AC220 and ponatinib against cultured mouse lymphoid (Ba/F3/FLT3-ITD), as well as human cultured (MOLM13 and MV4-11) and primary AML blast progenitor cells (BPCs) expressing FLT3-ITD. JQ1, but not its inactive enantiomer R-JQ1, potently induced apoptosis of not only Ba/F3/FLT3-ITD but also of Ba/F3/FLT3-ITD expressing the highly FLT3 TKI-resistant mutations F691L and D835V (IC50 values for JQ1 were 697, 1588 and 909 nM, in the three cell lines, respectively). This was associated with attenuation of c-MYC, but the induction of BIM levels. Both JQ1 and I-BET151 dose-dependently induced apoptosis of MOLM13 and MV4-11 cells, as well as of primary AML BPCs expressing FLT3-ITD. Concomitantly, JQ1 treatment attenuated c-MYC, BCL2 and CDK6, while inducing p21, p27, BIM and cleaved PARP levels. JQ1 and I-BET151 did not induce apoptosis of CD34+ normal bone marrow progenitor cells. Following engraftment of NOD/SCID mice with MOLM13 cell xenograft, treatment with JQ1 (50 mg/kg, formulated in 10% 2-hydroxypropyl-β-cyclodextrin, daily x 5 days per week x 3 weeks), versus treatment with vehicle control, significantly improved survival of the mice (p< 0.05), without causing any toxicity. This was associated with the in vivo attenuation of c-MYC and BCL-2 levels in the harvested AML cells from the mice. Co-treatment with JQ1 or I-BET151 and FLT3 antagonist AC220 or ponatinib synergistically induced apoptosis of MOLM13 and MV4-11 cells. This was associated with greater reduction in the levels of MYC, BCL2 and CDK6, but more induction of BIM, p27 and cleaved PARP levels. Knockdown of BRD4 by treatment with specific shRNA phenocopied the effects of JQ1 and sensitized MOLM13 cells to ponatinib and AC220. As compared to each agent alone, treatment with JQ1 and ponatinib or AC220 also induced more apoptosis of primary AML BPCs expressing FLT3-ITD, associated again with greater reduction of the levels of MYC, BCL2 and CDK6, but more induction of BIM, p27 and cleaved PARP levels. We also determined the effects of JQ1 against MOLM13/TKIR cells that were generated under the continuous selection pressure of FLT3 TKI, and exhibited > 10 fold resistance to ponatinib but > 50 fold resistance to AC220. Importantly, as compared to the parental MOLM13, the MOLM13/TKIR cells were markedly more sensitive to JQ1-induced apoptosis (p< 0.001). Additionally, co-treatment with JQ1 and ponatinib but not AC220 synergistically induced apoptosis of MOLM13/TKIR cells. Supporting our previous findings (Blood. 2005;105:1768) that FLT3-ITD is a heat shock protein (hsp) 90 client-oncoprotein, the non-geldanamycin hsp90 inhibitor AUY922 was equally effective in inducing apoptosis of MOLM13 versus MOLM13/TKIR cells. Collectively, these findings demonstrate that BRD4 antagonist exhibits potent activity against cultured and primary AML cells expressing FLT-3-ITD, as well as against cellular models of FLT3 with gate-keeper and activation loop mutations. These findings also highlight the novel and synergistic activity of the combination of BRD4 antagonist and AC220 or ponatinib against AML BPCs expressing FLT3-ITD, and support the rationale for testing ponatinib and BRD4 antagonist against TKI-refractory AML expressing FLT3-ITD. Disclosures: No relevant conflicts of interest to declare.

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Anti-AML activity of a novel beta-catenin antagonist BC2059.

Journal of Clinical Oncology ◽

10.1200/jco.2012.30.15_suppl.10605 ◽

2012 ◽

Vol 30 (15_suppl) ◽

pp. 10605-10605

Author(s):

Kapil N. Bhalla ◽

Warren Fiskus ◽

Sunil Sharma ◽

Stephen Horrigan ◽

Uma Mudunuru ◽

...

Keyword(s):

Transcription Factor ◽

Cyclin D1 ◽

Progenitor Cells ◽

Survivin Expression ◽

Beta Catenin ◽

Tail Vein ◽

Nsg Mice ◽

Induced Apoptosis

10605 Background: The canonical WNT-β-catenin pathway is essential for self-renewal, growth and survivalof AML stem and progenitor cells. Deregulated WNT signaling inhibits degradation of β-catenin, causing increased nuclear translocation and interaction of β-catenin with the TCF/LEF transcription factor, which up regulates cyclin D1, Myc and survivin expression in AML progenitor cells. BC2059 (β-Cat Pharmaceuticals) is a potent, small molecule, anthraquinone oxime-analog, which inhibits WNT-β catenin pathway by promoting the degradation and attenuation of β-catenin levels. Methods: We determined the in vitro anti-AML activity of BC2059 (BC) (250 to 1000 nM) against cultured and primary human AML blast progenitors, as well as evaluated the in vivo anti-AML efficacy of BC in NOD-SCID and NOD-SCID-IL2γ receptor deficient (NSG) mice. Results: BC induced cell cycle G1 phase accumulation and apoptosis (40%) of the cultured OCI-AML3, HL-60 and HEL92.1.7 (HEL) AML cells. BC dose-dependently also induced apoptosis of primary AML versus normal progenitors. Treatment with BC resulted in proteasomal degradation and decline in the nuclear levels of β-catenin, which led to decreased activity of the LEF1/TCF4 transcription factor highlighted by reduced TOP-FLASH luciferase activity in the AML cells. This was associated with reduced protein levels of cyclin D1, MYC and survivin. Co-treatment with BC and the histone deacetylase inhibitor panobinostat (PS) (10 to 20 nM) synergistically induced apoptosis of cultured and primary AML blasts. Following tail vein infusion and establishment of AML by OCI-AML3 or HEL cells in NOD-SCID mice, treatment with BC (5, 10 or 15 mg/kg b.i.w, IV) for three weeks demonstrated improved survival, as compared to the control mice (p <0. 001). Survival was further improved upon co-treatment with BC and PS (5 mg/kg IP, MWF). BC treatment (5 or 10 mg/kg IV) also dramatically improved survival of NSG mice with established human AML following tail-vein injection of primary AML blasts expressing FLT3 ITD. Mice did not experience any toxicity or weight loss. Conclusions: These findings highlight the notable pre-clinical in vitro and in vivo activity and warrant further development and in vivo testing of BC against human AML.

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Neuroprotective and Neurorestorative Effects of Holothuria scabra Extract in the MPTP/MPP+-Induced Mouse and Cellular Models of Parkinson’s Disease

Frontiers in Neuroscience ◽

10.3389/fnins.2020.575459 ◽

2020 ◽

Vol 14 ◽

Author(s):

Kunwadee Noonong ◽

Prasert Sobhon ◽

Morakot Sroyraya ◽

Kulathida Chaithirayanon

Keyword(s):

Dopaminergic Neurons ◽

Substantia Nigra Pars Compacta ◽

Motor Deficits ◽

Holothuria Scabra ◽

Cellular Models ◽

Anti Oxidant ◽

Pre Treatment ◽

Induced Apoptosis

Extracts from Holothuria scabra (HS) have been shown to possess anti-inflammation, anti-oxidant and anti-cancer activities. More recently, it was shown to have neuroprotective potential in Caenorhabditis elegans PD model. Here, we assessed whether HS has neuroprotective and neurorestorative effects on dopaminergic neurons in both mouse and cellular models of PD. We found that both pre-treatment and post-treatment with HS improved motor deficits in PD mouse model induced with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) as determined by grid walk test. This was likely mediated by HS protective and restorative effects on maintaining the numbers of dopaminergic neurons and fibers in both substantia nigra pars compacta (SNpc) and striatum. In a cellular model of PD, HS significantly attenuated 1-methyl-4-phenylpyridinium (MPP+)-induced apoptosis of DAergic-like neurons differentiated from SH-SY5Y cells by enhancing the expression of Bcl-2, suppressing the expression of cleaved Caspase 3 and preventing depolarization of mitochondrial membrane. In addition, HS could stimulate the expression of tyrosine hydroxylase (TH) and suppressed the formation of α-synuclein protein. Taken together, our in vivo and in vitro findings suggested that HS is an attractive candidate for the neuroprotection rather than neurorestoration in PD.

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Leptin Receptor As a Marker for a Subset of Long-Term Functional Hematopoietic Stem Cells

Blood ◽

10.1182/blood-2020-139595 ◽

2020 ◽

Vol 136 (Supplement 1) ◽

pp. 31-32

Author(s):

Thao Trinh ◽

James Ropa ◽

Arafat Aljoufi ◽

Scott Cooper ◽

Edward F. Srour ◽

...

Keyword(s):

Progenitor Cells ◽

Leptin Receptor ◽

Conflicts Of Interest ◽

Gene Set Enrichment Analysis ◽

Type I ◽

Hematopoietic Stem ◽

Therapeutic Implications

The hematopoietic system is maintained by the hematopoetic stem and progenitor cells (HSCs/HPCs), a group of rare cells that reside in a hypoxic bone marrow (BM) microenvironment. Leptin (Lep) is well-known for its neuroendocrine and immunological functions, and its receptor (Lepr) has been studied extensively in the BM niche cells. Yet, its biological implications in HSC/HPC biology remained largely unknown. In this study, we hypothesized that Lepr-expressing HSCs/HPCs are functionally and transcriptomically distinct from their negative counterparts. To test our hypothesis, we utilized both in vitro and in vivo approaches. We first employed Fluorescence-activated cell sorting (FACS) analysis to confirm expression of Lepr on HSCs/HPCs in adult mouse BM. We then isolated equal numbers of Lepr+Lineage-Sca1+cKit+ (LSK cells - a heterogenous population of long-term, short-term HSCs and multipotent HPCs) and Lepr-LSK cells from C57BL/6 (CD45.2+) mouse BM to perform colony-forming unit (CFU) assay and competitive transplantation assay, which also included using competitor cells from BoyJ (CD45.1+) unseparated BM and lethally-irradiated F1 (CD45.1+CD45.2+) as hosts. To determine whether Lepr can further hierarchize HSCs into two distinct populations, we repeated the competitive transplants using freshly isolated C57BL/6 Lepr+HSCs or Lepr-HSCs cells instead. At the end of primary transplants, whole BM were analyzed for donor chimerisms in the peripheral blood (PB) and BM as well as transplanted in a non-competitive fashion into lethally-irradiated secondary recipients. To gain mechanistic insights, we assessed homing potential as homing plays a role in increased engraftment. We also performed bulk RNA-seq using freshly sorted BM Lepr+HSCs or Lepr-HSCs to elucidate potential molecular pathways that are responsible for the differences in their functional capacity. By phenotypic studies, our FACS analyses showed that Lepr+ cells represented a smaller population within the hematopoietic compartment in the BM. However, HSCs contained a higher percentage of Lepr+ cells than other HPC populations. By functional assessments, Lepr+LSK cells were more highly enriched for colony-forming progenitor cells in CFU assay as compared to Lepr-LSK cells. Interestingly, Lepr+LSK cells exhibited more robust engraftment capability in primary transplants and substantial self-renewal capacity in secondary transplants throughout different time points in both PB and BM. In addition, Lepr+HSCs showed significantly higher donor chimerisms in PB month 1, 2, 4 and BM month 4 with similar lineage output compared to Lepr-HSCs. Higher engraftment could be due to increased homing of HSCs to the BM; however, Lepr+HSCs and Lepr-HSCs showed similar homing capacity as well as levels of surface CXCR4 expression. Molecularly, Fast Preranked Gene Set Enrichment Analysis (FGSEA) showed that Lepr+HSCs were enriched for Type-I Interferon and Interferon-gamma response pathways with Normalized Enrichment Scores of 2 or higher. Lepr+HSC transcriptomic study also revealed that these cells as compared to Lepr-HSCs expressed significantly higher levels of genes involved in megakaryopoiesis and proinflammatory immune responses including the NF-κB subunits (Rel and Relb). Interestingly, both IFN-γ and NF-κB signalings have been demonstrated to be critical for the emergence of HSCs from the hemogentic endothelium during embryonic development. In summary, although Lepr+LSK cells occupied a minor fraction compared to their negative counterparts in the BM, they possessed higher colony-forming capacity and were more highly enriched for long-term functional HSCs. In line with this, Lepr+HSCs engrafted significantly higher and self-renewed more extensively than Lepr-HSCs, suggesting that Lepr not only can be used as a marker for functional HSCs but also further differentiate HSCs into two functionally distinguishable populations. Intriguingly, Lepr+HSCs were characterized with a proinflammatory transcriptomic profile that was previously suggested to be critical for the development of HSCs in the embryo. All together, our work demonstrated that Lepr+HSCs represent a subset of highly engrafting adult BM HSCs with an embryonic-like transcriptomic signature. This can have potential therapeutic implications in the field of hematopoietic transplantation as Lepr is highly conserved between mice and human. Disclosures No relevant conflicts of interest to declare.

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Combined Targeting of BCR-ABL and JAK2 with ABL and JAK2 Inhibitors Is Effective Against CML Patients' Leukemic Stem/Progenitor Cells.

Blood ◽

10.1182/blood.v116.21.3404.3404 ◽

2010 ◽

Vol 116 (21) ◽

pp. 3404-3404

Author(s):

Donna DeGeer ◽

Paolo Gallipoli ◽

Min Chen ◽

Ivan Sloma ◽

Heather Jorgensen ◽

...

Keyword(s):

Progenitor Cells ◽

Kinase Inhibitor ◽

Conflicts Of Interest ◽

Integration Site ◽

Single Agent ◽

K562 Cells ◽

High Rate ◽

Jak2 Inhibitors

Abstract Abstract 3404 Imatinib mesylate (IM) is a tyrosine kinase inhibitor (TKI) that induces clinical responses in most chronic myeloid leukemia (CML) patients. Nevertheless, early relapses and later emergence of IM-resistant disease pose serious concerns for many. The inadequacies of IM therapy are due, at least in part, to the unique properties of CML stem/progenitor cells that make them generally less responsive to IM and, indeed, other TKIs, and also confer on them a genetic instability that leads to a high rate of formation of BCR-ABL mutants. Improved treatment approaches to prevent the development of resistant subclones by targeting other key molecular elements active in CML stem/progenitor cells are thus clearly needed. One candidate is a complex that forms in CML stem/progenitor cells between the oncoproteins encoded by AHI-1 (Abelson helper integration site 1), BCR-ABL and the JAK2 kinase. This complex contributes to the transforming activity of BCR-ABL both in vitro and in vivo and also plays a role in the IM response/resistance of primary CML stem/progenitor cells. We now describe the results of experiments designed to test the ability of ABL and JAK2 inhibitors to block the activity of this protein complex in CML cells. K562 cells engineered to stably overexpress AHI-1 showed a significantly reduced sensitivity to both IM (at 1 and 5 μM) and TG101209, a JAK2 inhibitor, (at 0.5 and 1 μM), as determined by assays for cell viability, apopotosis, and colony-forming activity. K562 cells engineered to suppression AHI-1 showed an opposite effect, with a heightened sensitivity to IM at concentrations as low as 1 μM. In addition, IM together with TG101209 was more effective at killing AHI-1-overexpressing K562 cells, IM-resistant K562 cells and IM-resistant T315I-mutant cells than either treatment alone. Western blot and co-IP experiments demonstrated a significant reduction of p-BCR-ABL, p-JAK2 and p-STAT5 in cells treated with IM plus TG101209 compared to cells treated with IM or TG101209 alone. Importantly, treatment with 5 μM IM, 150 nM dasatinib (DA) or 5 μM nilotinib (NL) in combination with 100 nM TG101209 caused a significantly greater reduction in the viability of primary CD34+CD38− and CD34+CD38+ CML cells when these responses were compared to any of the TKIs or TG101209 alone (~2-4 fold, n=3). Apoptotic cells at 72 hours were also significantly increased for all drug combinations compared to single agent treatments (40%-52% for the combinations vs 15%-18% for the single agents). CFSE tracking analysis of cell division in these cells further demonstrated additive anti-proliferative activity from the TKI plus TG101219 combinations, although some rare undivided cells were not eliminated. Nevertheless, exposure of CD34+ CML cells from IM-nonresponders (n=4) to TG101209 plus IM or DA did cause a greater inhibition (81% and 85%) of patients' colony-forming cells as compared to the same cells treated with the combination of IM plus DA only, or IM or DA only (60%, 41% and 50% inhibition, p<0.05). Long-term culture-initiating cell assays were undertaken to compare the effect of these combination treatments versus the effects of TKIs or TG101209 alone on very primitive CML cells. The results again showed a more significant reduction of these cells treated with the combination (n=3). Intracellular staining revealed a greater reduction in the levels of p-CrKL and p-STAT5 in CD34+ CML cells treated for 24 hours with the combination of TKIs plus TG101219 as compared to single TKI-treated cells (~44% vs 65% for p-CrKL and 36% vs 57% for p-STAT5, n=3). Strikingly, the combination treatment produced an even greater inhibition of both p-CrKL and p-STAT5 after 72 hours while p-CrKL was almost fully reactivated with TKIs alone (~29% vs 89% for p-CrKL and 23% vs 50% for p-STAT5). These results point to the possibility of achieving improved therapeutic outcomes in CML patients by simultaneously targeting both BCR-ABL and JAK2 activities in the critical TKI-insensitive CML stem/progenitor reservoir. Disclosures: No relevant conflicts of interest to declare.

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IFNs Upregulate Sca-1 and Block Proliferation in Murine Hematopoietic Stem and Progenitor Cells,

Blood ◽

10.1182/blood.v118.21.3394.3394 ◽

2011 ◽

Vol 118 (21) ◽

pp. 3394-3394

Author(s):

Kaitlyn Shank ◽

Yusup Shin ◽

Carson Wills ◽

Nicole Cunningham ◽

Alevtina Domashenko ◽

...

Keyword(s):

Cell Cycle ◽

Progenitor Cells ◽

Caspase 3 ◽

Inflammatory Responses ◽

Conflicts Of Interest ◽

Brdu Incorporation ◽

Apparent Paradox ◽

Hematopoietic Stem

Abstract Abstract 3394 Hematopoietic stem cells (HSC) replenish the cellular components of the blood throughout life by a homeostatic process in which the majority of HSCs remain quiescent while a small percentage enter the cell cycle to either self-review or differentiate. During inflammatory responses to infections, Interferons (IFNa, IFNg) perturb HSC homeostasis, presumably in response to the demand for increased numbers of inflammatory cells. Previous studies have highlighted an apparent paradox, i.e. IFNs suppress the proliferation of normally cycling murine hematopoietic progenitor cells (HPCs), yet increase the fraction of normally quiescent Sca+ HSCs that proliferate. To investigate the mechanisms underlying this paradox, we dissected the dynamics of cell surface phenotypes, cell cycle kinetics, pro- and anti-apoptotic pathways within the HSC and HPC compartments in response to pIpC and IFNs both in vivo and in vitro. Forty-eight hours after pIpC injection, bone marrow (BM) cellularity declined by 60%, the proportion of Sca- kit+ HPCs fell from 0.45% to 0.05%, while the proportion of BM cells with the Sca+ kit+ HSC phenotype increased from 0.17 to 0.26%. To determine whether the increase in Sca+kit+ cells was due to proliferation of HSCs or upregulation of Sca-1 on HPCs, we cultured purified CD150+ Sca-Kit+ HPCs and CD150+Sca+kit+ HSCs in vitro with IFNa, IFNg, or PBS. Sca expression was induced on previously Sca- HPCs, and the level of Sca expression on HSCs was also increased. This induction was detectable as early as 6 hours after treatment and accompanied by an increase in Sca mRNA. BrdU incorporation into both HPC and HSC populations decreased from pre-treatment baselines, further indicating that the increase in cells with the HSC phenotype was not due to HSC proliferation, but rather the appearance of cycling HPCs within the HSC staining gate following IFN-induced upregulation of Sca. Staining with FITC-DEVD-FMK identified active cleaved capase-3 in pIpC- or IFN-treated cells, suggesting that the reduced cellularity following IFN reflected a cellular stress that killed Lin+ precursors cells and some HPCs, but spared HSCs. In contrast to lin+kit- precursors, all kit + HPCs and HSCs expressed bcl-2, suggesting that expression of anti-apoptotic proteins may prevent IFN-induced stress from resulting in HSC/HPC apoptosis despite the initial triggering of caspase-3 cleavage. In summary, acute treatment with IFNs has anti-proliferative effects on all hematopoietic cells, including precursors, HPCs and HSCs, with the apparent increase in HSC proliferation the result of HPCs masquerading as Sca+HSCs after exposure to IFN. Unlike precursors, HSCs and some HPCs survive treatment to IFNs despite activation of cleaved caspase-3, possibly due to their expression of bcl-2, and likely related anti-apoptotic regulators. The previously observed increase in HSC proliferation days and weeks following IFN treatment is most likely due to the homeostatic response of HSCs to the depopulation of the precursor and HPCs caused by acute IFN exposure. Disclosures: No relevant conflicts of interest to declare.

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Lycorine Modulates the Expression of p21 Via a p53-Independent Pathway in HL-60 Cells

Blood ◽

10.1182/blood.v118.21.4297.4297 ◽

2011 ◽

Vol 118 (21) ◽

pp. 4297-4297

Author(s):

Jing Liu ◽

Shu-Ling Wang ◽

Lin Fang ◽

Mao Ye ◽

Zhi-Wei Sun ◽

...

Keyword(s):

Molecular Mechanisms ◽

Kinase Inhibitor ◽

Conflicts Of Interest ◽

Fold Increase ◽

Alternative Pathways ◽

Cyclin Dependent Kinase Inhibitor ◽

M Phase ◽

Induced Apoptosis

Abstract Abstract 4297 Leukemia is one of the most life-threatening cancers today, and acute promyelogenous leukemia is a common type of leukemia. We have previously shown that lycorine, a natural alkaloid extract from Amaryllidaceae, exhibited anti-leukemia effects in vitro and in vivo. Lycorine treatment of HL-60 cell arrested cell cycle at G2/M phase and induced apoptosis. In the present study, we sought to explore the molecular mechanisms for the anti-leukemia action of lycorine. Gene chip analysis revealed that lycorine treatment of HL-60 cells induced more than 9 fold increase of p21, a cyclin-dependent kinase inhibitor, whose expression is mainly regulated by p53. Since HL-60 cells are p53 null, the above findings suggest that lycorine activates p21 expression through p53-independent pathway. To further explore the alternative pathways for the activation of p21 induced by lycorine, we examined the effect of lycorine on the expression of Rb, pRb, E2F, c-Myc and HDACs which have shown to regulate p21 expression. We show that expression of pRb (ser780) and c-Myc was down-regulated, Rb and E2F were up-regulated, while the expression of HDAC1 and HDAC3 was not changed. Together these findings suggest that lycorine exerts its anti-leukemia effect by activating p21 expression via pRb/E2F and c-Myc pathways. Disclosures: No relevant conflicts of interest to declare.

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Combination of the mTOR inhibitor rapamycin and CC-5013 has synergistic activity in multiple myeloma

Blood ◽

10.1182/blood-2004-06-2281 ◽

2004 ◽

Vol 104 (13) ◽

pp. 4188-4193 ◽

Cited By ~ 137

Author(s):

Noopur Raje ◽

Shaji Kumar ◽

Teru Hideshima ◽

Kenji Ishitsuka ◽

Dharminder Chauhan ◽

...

Keyword(s):

Multiple Myeloma ◽

Mtor Inhibitor ◽

Mtor Inhibitors ◽

Mitogen Activated Protein Kinase ◽

Synergistic Activity ◽

Growth And Survival ◽

Mitogen Activated Protein ◽

Induced Apoptosis

Abstract Previous studies have demonstrated the in vitro and in vivo activity of CC-5013 (Revlimid), an immunomodulatory analog (IMiD) of thalidomide, in multiple myeloma (MM). In the present study, we have examined the anti-MM activity of rapamycin (Rapamune), a specific mTOR inhibitor, combined with CC-5013. Based on the Chou-Talalay method, combination indices of less than 1 were obtained for all dose ranges of CC-5013 when combined with rapamycin, suggesting strong synergism. Importantly, this combination was able to overcome drug resistance when tested against MM cell lines resistant to conventional chemotherapy. Moreover, the combination, but not rapamycin alone, was able to overcome the growth advantage conferred on MM cells by interleukin-6 (IL-6), insulin-like growth factor-1 (IGF-1), or adherence to bone marrow stromal cells (BMSCs). Combining rapamycin and CC-5013 induced apoptosis of MM cells. Differential signaling cascades, including the mitogen-activated protein kinase (MAPK) and the phosphatidylinositol 3′-kinase/Akt kinase (PI3K/Akt) pathways, were targeted by these drugs individually and in combination, suggesting the molecular mechanism by which they interfere with MM growth and survival. These studies, therefore, provide the framework for clinical evaluation of mTOR inhibitors combined with IMiDs to improve patient outcome in MM.

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Combined Targeting of β-Catenin and FLT-3 Is Synergistically Lethal Against Cultured and Primary AML Blast Progenitor Cells Expressing FLT3 Internal Tandem Duplication (ITD)

Blood ◽

10.1182/blood.v124.21.618.618 ◽

2014 ◽

Vol 124 (21) ◽

pp. 618-618

Author(s):

Saikat Saha ◽

Warren Fiskus ◽

Sunil Sharma ◽

Bhavin Shah ◽

Anna T Rogojina ◽

...

Keyword(s):

Cyclin D1 ◽

Progenitor Cells ◽

Nuclear Localization ◽

Target Genes ◽

Conflicts Of Interest ◽

Adaptor Protein ◽

Beta Catenin ◽

Induced Apoptosis ◽

Nuclear Levels

Abstract β-catenin acts as a co-activator for the T-cell factor (TCF) 4/lymphoid enhancer factor (LEF) 1 bipartite transcription factor at the promoters of the WNT-β-catenin target genes, including cyclin D1, c-Myc and survivin. The canonical WNT-β-catenin pathway is documented to be essential for self-renewal, growth and survival of the AML stem and blast progenitor cells (BPCs), which has also been correlated with a poor prognosis in AML. In AML stem/BPCs expressing mutant FLT3-ITD, increased PI3K/AKT activity causes phosphorylation and inactivation of GSK3β, thereby preventing degradation, promoting stabilization and nuclear localization of β-catenin. Additionally, FLT3 can also directly mediate the tyrosine phosphorylation of β-catenin, thereby stabilizing and promoting the nuclear localization and binding of β-catenin to TCF4. TBL1 (transducin beta-like) is an adaptor protein, which binds to nuclear β-catenin and promotes its co-factor activity with TCF4/LEF1 in mediating transcription of the target genes, including c-Myc, cyclin D1 and survivin. Therefore, we hypothesized that targeted disruption of TBL1-β-catenin binding or depletion of TBL1 would abrogate the pro-growth and oncogenic signaling of β-catenin in AML BPCs, especially those expressing FLT3-ITD. Here, we demonstrate that treatment with 20 to 100 nM of BC2059 (β-Cat Pharmaceuticals), a small molecule, anthraquinone oxime-analog, disrupts the binding of β-catenin to TBL1 (by anti-TBL1 pull down and immunofluorescence analyses) and promotes proteasomal degradation of β-catenin, thereby attenuating the nuclear levels of β-catenin in the cultured (OCI-AML3, MOLM13 and MV4-11), as well as in primary (p) AML BPCs. Concomitantly, BC2059 treatment inhibited the mRNA and protein expression of c-Myc, cyclin D1 and survivin, while de-repressing p21 and Axin2. BC2059 also dose dependently inhibited growth and induced apoptosis of cultured and CD34+ pAML BPCs expressing FLT3-ITD (40 to 60%), but not of normal CD34+ bone marrow progenitor cells (p < 0.01). Transient knockdown of TBL1 or beta catenin (60 to 70%) by lentivirus-transduced shRNA caused loss of viability in MOLM13 cells, which was significantly enhanced by treatment with BC2059 (p < 0.01). BC2059 also induced apoptosis of MOLM13-TKIR cells that were isolated in vitro to exhibit resistance to FLT3 antagonists (approximately 50-fold). Notably, BC2059 treatment (10 mg/kg, t.i.w., by IV injection) also exerted potent in vivo anti-AML activity and significantly improved the survival of immune depleted mice engrafted with cultured and patient-derived pAML BPCs (p < 0.001). Since compared to the control OCI-AML3 cells, BC2059 demonstrated significantly greater lethality against the OCI-AML3 cells ectopically overexpressing FLT3-ITD (approximately 8-fold), we hypothesized that co-treatment with a FLT3 antagonist would further reduce the nuclear levels of β-catenin and enhance the lethal activity of FLT3-antagonist against AML BPCs expressing FLT3-ITD. Indeed, co-treatment with BC2059 (50 nM) and the FLT3-antagonist quizartinib or ponatinib (100 to 200 nM), versus each agent alone, caused more reduction in the nuclear levels and binding of β-catenin to TBL1 (by confocal immunofluorescence analysis). This was associated with greater decline in the expression of c-Myc, cyclin D1 and survivin, but increase in the levels of p21 and BIM. Compared to each agent alone, co-treatment with BC2059 and quizartinib or ponatinib also synergistically induced apoptosis of the FLT3-ITD expressing cultured (MOLM13 and MV4-11) and pAML BPCs (combination indices of < 1.0, by isobologram analyses) but not of normal CD34+ progenitor cells. Treatment with BC2059 (25 to 100 nM) also significantly increased the apoptosis observed by the shRNA mediated incomplete knockdown of TBL1 or β-catenin (approximately 70%) in MOLM13 cells (p < 0.01). Collectively, our findings support that targeted inhibition of the levels and binding of β-catenin to TBL by BC2059 and FLT3-antagonist is a promising approach to exert lethal activity against AML BPCs expressing FLT3-ITD. Further pre-clinical development of this combination therapy against FLT3-ITD expressing AML is progressing. Disclosures No relevant conflicts of interest to declare.

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Triterpenoid Methyl-CDDO Is a Potent Inducer of Apoptosis in CD34+ AML Progenitor Cells Via Activation of SAPK Pathways and Inhibition of MAPK Cascades.

Blood ◽

10.1182/blood.v104.11.2533.2533 ◽

2004 ◽

Vol 104 (11) ◽

pp. 2533-2533

Author(s):

Marina Konopleva ◽

Peter Ruvolo ◽

Rooha Contractor ◽

Svitlana Kurinna ◽

Yue-Xi Shi ◽

...

Keyword(s):

Cell Death ◽

Progenitor Cells ◽

Leukemic Cell ◽

Mek Inhibitor ◽

U937 Cells ◽

Potent Inducer ◽

Induction Of Apoptosis ◽

Induced Apoptosis

Abstract Outcome results in AML are a continued challenge for the development of novel therapeutic strategies. C-28 methyl ester of 2-cyano-3,12-dioxoolen-1,9-dien-28-oic acid, CDDO-Me, a novel triterpenoid, induces apoptosis in myeloid and lymphoid leukemic cell lines and in primary AML samples in sub-micromolar concentrations. We reported previously that CDDO-Me inhibits the activation of ERK1/2, blocks Bcl-2 phosphorylation, and promoted apoptosis in AML-derived U937 cells (Blood 2002, 99(1):326–35). Here, we examined the effects of CDDO-Me on CD34+ AML progenitor cells in vitro. Exposure to 1μM CDDO-Me induced apoptosis in all but one AML sample (46±4% annexin(+) CD34+ cells, n=20). To assess the anti-leukemia activity of CDDO-Me in vivo, scid mice intravenously injected with U937 cells were treated with liposomally-delivered CDDO-Me (20mg/kg/day IV every other day, starting at day 7, for a total of 9 injections). While CDDO-Me was not toxic to the mice, pathological and flow cytometry analysis revealed reduced (55–86%) leukemia burden in the bone marrow, liver, and spleen of mice. Since we had shown that CDDO-Me inhibits phosphorylation of pERK in U937 cells, a further goal of this study was to assess the role of ERK in CDDO-Me-induced cell death in primary AML samples. ERK was expressed and phosphorylated in all (n=15) patients’ samples studied. CDDO-Me inhibited ERK phosphorylation in 9 of 15 patient samples and promoted apoptosis. However, CDDO-Me still induced apoptosis in 5/6 samples that displayed no significant changes in pERK levels in response to the drug. This finding suggests that ERK is not the sole target of the compound. The stress-activated protein kinases JNK and p38 are related to ERK but in general activate pathways that are opposed to ERK. By Western blot analysis, CDDO-Me induced early (30 min) phosphorylation of JNK and p38, which preceded induction of cell death. Pre-treatment of U937 cells with JNK and p38 inhibitors SP600125 and SB203580 partially abrogated induction of apoptosis, while MEK inhibitor PD-98059 significantly enhanced cytotoxicity. CDDO-Me induced p38 phosphorylation in 5 of 6 primary AML samples tested. Collectively, these finding indicate that CDDO-Me markedly shifts signaling toward the JNK and p38 MAPK stress-related pathways and away from the cytoprotective MAPK cascade. In summary, the triterpenoid CDDO-Me is a potent inducer of apoptosis in primary AML including CD34+ AML progenitor cells. Induction of apoptosis is in part mediated via inhibition of ERK signaling combined with JNK/p38 activation. These studies in primary AML samples and the anti-leukemia activity of the compound in vivo suggest potential utility of CDDO-Me for the treatment of AML patients.

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Decreased PU.1 and Enhanced CITED2 Cooperate To Maintain Self-Renewal In Hematopoietic Stem/Progenitors

Blood ◽

10.1182/blood.v122.21.2411.2411 ◽

2013 ◽

Vol 122 (21) ◽

pp. 2411-2411

Author(s):

Hein Schepers ◽

Patrick M. Korthuis ◽

Jan Jacob Schuringa ◽

Edo Vellenga

Keyword(s):

Progenitor Cells ◽

Conflicts Of Interest ◽

Ectopic Expression ◽

Fold Increase ◽

Negative Regulator ◽

Self Renewal ◽

Hematopoietic Stem ◽

Cd34 Cells

Abstract The transcriptional co-activator CITED2 has a conserved role in the maintenance of normal adult hematopoiesis. We have shown before that CD34+ cells from a subset of acute myeloid leukemia (AML) patients display enhanced CITED2 expression and that interfering with this expression is detrimental for leukemia maintenance. Ectopic expression of CITED2 in normal CD34+ stem and progenitor cells (HSPCs) resulted in increased proliferation and skewed myelo-erythroid differentiation in vitro. Long-Term Culture-Initiating Cell assays (LTC-IC) revealed a 5-fold increase in the number of Cobblestone Area Forming Cells (CAFCs), as a result of an increase in the number of phenotypically defined CD34+CD38- HSCs. CFC frequencies were also enhanced 5-fold upon CITED2 overexpression. To further substantiate these observations in vivo, we transplanted CITED2-transduced CD34+ cells into NSG mice. CD34+ cells with increased CITED2 expression displayed a >10x higher engraftment at week 12, as compared to control cells, confirming the higher frequency of CD34+CD38- HSCs, while myelo-lymphoid differentiation of these cells was comparable to control transplanted cells. Till date we have not observed leukemia development in these transplanted mice, suggesting that CITED2 as a single hit is not sufficient to transform human CB CD34+ cells. We recently identified the myeloid transcription factor PU.1 as a strong negative regulator of CITED2 and enhanced CITED2 expression in AML samples correlates with low PU.1 expression. We therefore investigated whether high CITED2 and low PU.1 expression would collaborate in maintaining self-renewal of HSCs. We combined lentiviral downregulation of PU.1 with overexpression of CITED2 (PU.1Low-CITED2High) and performed LTC-IC cultures on MS5 stroma. These experiments revealed that combined loss of PU.1 and enhanced CITED2 expression was sufficient to induce a strong proliferative advantage compared to control cells. Furthermore, a 3-fold increase of progenitor numbers was observed in CFC assays. While overexpression of CITED2 alone was not sufficient to allow 2nd CFC formation, additional downregulation of PU.1 now led to colony formation upon serial replating. This replating capacity of PU.1Low-CITED2High cells was limited to CD34+CD38- HSCs, as replating of CD34+CD38+ progenitor cells did not yield CFCs. This suggests that the combined loss of PU.1 and enhanced CITED2 expression is sufficient to maintain self-renewal properties of HSC, but this combination is not sufficient to reinforce self-renewal in committed progenitor cells. To more stringently assess self-renewal, cells were first cultured for 4 weeks on MS5 under myeloid differentiating conditions (G-CSF, IL3 and TPO) and subsequently plated into CFC assays, followed by secondary and tertiary replating experiments. Only PU.1Low-CITED2High cells were able to form CFCs after 10 weeks of culture, indicating that this combination indeed preserves self-renewal. Current experiments focus on the in vivo engraftment and self-renewal properties of these PU.1Low-CITED2High cells. Preliminary data indicate that these PU.1Low-CITED2High cells contribute ∼3-fold more to the myeloid lineage at week 12, compared to control and CITED2 only cells, and AML development is currently being investigated in these mice. Together, these data suggest that CITED2 is sufficient to increase LTC-IC and CFC frequencies, to skew myeloid differentiation, and to enhance engraftment of CB CD34+ cells in xenograft mice. Furthermore, CITED2 overexpression together with reduced PU.1 levels is necessary to maintain stem cell self-renewal. Disclosures: No relevant conflicts of interest to declare.

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