scholarly journals Heme Oxygenase-1 in Leukemia Cells Promotes Cancer Associated Fibroblasts Mediating Progression of Acute Lymphoblastic Leukemia

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 3958-3958
Author(s):  
Chengyun Pan ◽  
Dan Ma ◽  
Qin Fang ◽  
Ping Liu ◽  
Jishi Wang
Keyword(s):  
Bone Marrow ◽  
Cell Lines ◽  
Lymphoblastic Leukemia ◽  
Bone Marrow Microenvironment ◽  
Leukemia Cells ◽  
Heme Oxygenase 1 ◽  
Cancer Associated Fibroblasts ◽  
Primary Cells ◽  
Key Factor

Backgroud and objective: Recurrence and resistance is still the biggest challenges for Acute lymphoblastic leukemia (ALL). In recent years, a new concept has been proposed that the interaction between bone marrow microenvironment and leukemia cells could reduce the sensitivity of leukemia cells to chemotherapy. As an important matric element of the bone marrow microenvironment, Cancerassociated fibroblasts (CAFs) can mediate changes in bone marrow microenvironment to promote tumor proliferation and infiltration, but its role in ALL has not been described. Heme Oxygenase-1 (HO-1) is a rate-limiting enzyme in the process of heme catabolism, which is highly expressed in leukemia cells and can promote chemo-resistance by regulating the preservation of hematopoietic stem progenitor cells in the bone marrow microenvironment according to the recent research. In this article, we explored that HO-1 may be a key factor for promoting cancer associated fibroblasts to mediated chemo-resistance of ALL in the bone marrow microenvironment. M ethods : For clinical sample analysis, Bone marrow nucleated cells of 16 ALL patients with complete remission (ALL-CR), 12 ALL with relapse/refractory (ALL-R/R) and 10 normal controls were btained from bone marrow puncture. The expression of HO-1 and CAFs markers which includes α-SMA, FAP and FSP-1 was examined by quantitative reverse transcription-PCR (RT-PCR) and Western blot. In vitro cell experiments, CAFs was obtained from transfusion of bone marrow-derived mesenchymal stem cells (BM-MSCs) with recombinant human TGF-β1 (rhTGF-β1) stimulating. We used ALL primary cells and ALL cell lines (Nalm-6/Super-B15) to culture alone or co-culture with CAFs to detect proliferation, apoptosis and cell cycle of leukemia cells. In addition, we transfected ALL cell lines by constructing siHO-1 lentiviral vector, and co-cultured with CAFs or cultured separately to detect the above index changes in leukemia cells. R esults : The expressions of α-SMA, FAP, FSP-1 and HO-1 were significantly higher in ALL-R/R patients than in ALL-CR group and normal control group (P<0.05), which suggested that CAFs and HO-1 are closely related to recurrence and resistance of ALL. In vitro tests, after rhTGF-β1 stimulated BM-MSCs for 48h, the mRNA and protein expression levels of α-SMA, FAP and FSP-1 were significantly higher than those in BM-MSCs alone (P<0.05), This step was used to obtain CAFs. Then proceed to CAFs and Nalm-6/Super-B15 cell lines or ALL primary cells co-cultured tests. Results showed that CAFs can enhanced the leukemia cells proliferation and decreased apoptosis, and leukemia cells arrested in the G0/G1 phase was increased. Interestingly, this effect could be decreased by silencing HO-1 expression in Nalm-6/Super-B15 cell lines, which suggested that HO-1 may be a key factor mediating the interaction between leukemia cells and CAFs in bone marrow microenvironment. Conclusion:As an important component of the bone marrow microenvironment, CAFs are closely related to the recurrence and resistance of ALL. HO-1 may be a key component mediating the interaction between CAFs and leukemia cells of ALL. This results may provide a new entry point for deepening the mechanism of ALL progression and finding more effective targeted therapies. But further in vitro and in vivo experiments are still needed for verification. Disclosures No relevant conflicts of interest to declare.

Development of a Unique In Vitro and In Vivo Model System of Philadelphia Chromosome-Positive Acute Lymphoblastic Leukemia (Ph-ALL) with Emphasis on Cell to Cell Interaction.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1845-1845 ◽  
Author(s):  
Arinobu Tojo ◽  
Kiyoko Izawa ◽  
Rieko Sekine ◽  
Tokiko Nagamura-Inoue ◽  
Seiichiro Kobayashi
Keyword(s):  
Bone Marrow ◽  
Acute Lymphoblastic Leukemia ◽  
Suspension Culture ◽  
Cell Lines ◽  
Lymphoblastic Leukemia ◽  
Philadelphia Chromosome ◽  
Leukemia Cells ◽  
L2 Cells

Abstract Philadelphia chromosome-positive acute lymphoblastic leukemia (Ph-ALL) is one of the most intractable hematological malignancies, readily acquires resistance to chemotherapeutic drugs including imatinib mesylate (IM), and shows a high relapse rate even after allogeneic stem cell transplantation. Nevertheless, primary blast cells are generally susceptible to apoptotic cell death in sort-term suspension culture after isolation from patients with Ph-ALL. We established two Ph-ALL cell lines and characterized their growth properties supported by adhesive interaction with a murine bone marrow stromal cell line, HESS-5. IMS-PhL1 (L1) cells mainly expressed p210-type BCR-ABL mRNA with wild type sequences in the ABL kinase domain and were weakly positive for p190-type mRNA. IMS-PhL2 (L2) cells exclusively expressed p190-type transcripts with Y253H mutation and showed much lower sensitivity to imatinib than L1 cells. The growth of L1 cells was slowly autonomous in suspension culture, but became more vigorous and their apoptosis was prevented by co-culture with HESS-5 cells. In contrast, the sustained growth and survival of L2 cells was absolutely dependent on direct contact with HESS-5 cells and did not respond to soluble cytokines including SCF, IL3and IL7. Both cell lines adhered to and migrated beneath the HESS-5 cell layer, resulting in the formation of cobblestone areas. This migration was significantly inhibited by the pretreatment of those with a neutralizing antibody against α4-integrin. While non-adherent L1 cells were eradicated by 1 mM IM, a portion of adherent L1 cells could survive even at 10 mM IM. Similarly, adherent L2 cells considerably resisted prolonged exposure to 10 mM IM. Intravenous injection of both cell lines caused leukemia in NOD-SCID mice after distinct latent periods. Leukemia cells appeared in peripheral blood, bone marrow as well as spleen. Interestingly, expression of α5-integrin was significantly down-regulated in both leukemia cells collected from those tissues, but was restored after co-culture with HESS-5. The study of L1 and L2 cells in vitro and in vivo will not only contribute to further insights into microenvironmental regulation of clonal maintenance and progression of Ph-ALL but also provide a unique model for experimental therapeutics against Ph-ALL. Figure Figure

The Novel CXCR4 Antagonist POL5551 Decreases Surface CXCR4 (s-CXCR4) Expression, Inhibits Chemotaxis, and Enhances Chemosensitivity in Acute Lymphoblastic Leukemia (ALL)

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 780-780
Author(s):  
Edward Allan R. Sison ◽  
Daniel Magoon ◽  
Eric Chevalier ◽  
Klaus Dembowsky ◽  
Patrick Brown
Keyword(s):  
Bone Marrow ◽  
T Cell ◽  
B Cell ◽  
Cell Lines ◽  
Cxcr4 Expression ◽  
Leukemia Cells ◽  
The Novel ◽  
Cxcr4 Antagonist ◽  
Induced Apoptosis

Abstract Abstract 780 Background: The interaction between the cell surface receptor CXCR4 and the chemokine SDF-1 (CXCL12) is critical in signaling between leukemic blasts and the bone marrow microenvironment. We previously demonstrated that CXCR4 is an important mediator of chemotherapy resistance, as chemotherapy-induced upregulation of s-CXCR4 in acute myeloid leukemia (AML) cell lines and primary samples led to increased SDF-1-mediated chemotaxis and increased protection by normal human bone marrow stroma from chemotherapy-induced apoptosis. We also showed that stromal protection and chemotherapy resistance could be reversed by treatment with the FDA-approved CXCR4 inhibitor plerixafor, both in vitro in stromal co-cultures of pre-B cell ALL cell lines and in vivo in xenografts of primary samples of infant MLL-rearranged ALL. Therefore, disruption of the CXCR4/SDF-1 axis is a rational means to target extrinsic survival mechanisms in acute leukemia. The novel Protein Epitope Mimetic (PEM) POL5551 is a selective and potent antagonist of CXCR4. Treatment with POL5551 inhibits vascular accumulation of CXCR4+ smooth muscle cells but its effects on ALL have not been reported. We hypothesized that treatment of ALL cell lines with POL5551 would 1) decrease s-CXCR4 expression, 2) inhibit SDF-1-mediated chemotaxis, and 3) reverse stromal-mediated protection from chemotherapy-induced apoptosis. Methods/Results: Pre-B cell ALL (697, HB11;19, NALM-6, SEMK2) and T cell ALL cell lines (CCRF-CEM-1301, Jurkat, Molt-4) were treated with dose ranges of POL5551. Cells were harvested at multiple time points over 72 hours and s-CXCR4 was measured by FACS. S-CXCR4 was potently and markedly reduced in all cell lines, with IC50 levels of <5 nM at 1 hour and IC50 levels of <20 nM at 48 hours. In comparison, 3- to 30-fold higher doses of plerixafor were needed to achieve similar levels of reduction. Simultaneous measurement of cell proliferation using the WST-1 proliferation assay demonstrated that treatment with POL5551 neither increased nor decreased leukemia cell proliferation in a significant manner. To ascertain the functionality of s-CXCR4 inhibition, we performed chemotaxis assays. Leukemia cells were treated with 10 nM POL5551 or vehicle control and placed into hanging cell culture inserts. Migration through a permeable membrane toward an SDF-1 gradient was then measured after 24 hours. Compared to control-treated cells, POL5551-treated cells had significantly decreased SDF-1-induced chemotaxis (average 38% reduction in chemotaxis in pre-B cell lines, p<0.001; average 41% reduction in T cell lines, p=0.05). We also performed co-culture experiments with normal human bone marrow stroma in the presence and absence of POL5551 to further demonstrate the functional effects of s-CXCR4 inhibition. Specifically, we cultured leukemia cells off stroma (O), on stroma (S), or pretreated with POL5551 for 30 minutes prior to plating on stroma (P+S). Cells from each culture condition were then treated with dose ranges of chemotherapy. Following treatment, we measured apoptosis by staining with Annexin V/7-AAD. IC10 through IC90 values were obtained using Calcusyn. To quantify stromal protection, we calculated a Protective Index (PI), defined as the S IC values divided by the O IC values. Thus, PI >1 signified stromal protection, while PI ≤1 signified no stromal protection. To quantify the ability of POL5551 to reverse stromal protection, we calculated a Reversal Index (RI), defined as the P+S IC values divided by the O IC values. Therefore, PI > RI indicated a decrease in stromal protection, while RI ≤1 indicated a reversal of stromal protection. Overall, stroma protected leukemia cells from chemotherapy-induced apoptosis. Importantly, treatment with POL5551 abrogated stromal-mediated protection and restored chemosensitivity (eg, PI 1.182 vs. RI 0.956 for NALM-6 treated with daunorubicin +/− 20 nM POL5551, p<1×10e-9). Conclusions: The novel CXCR4 antagonist POL5551 is a potent inhibitor of CXCR4 in pre-B and T ALL cell lines with activity at nanomolar concentrations in decreasing s-CXCR4 expression, inhibiting SDF-1-induced chemotaxis, and reversing stromal-mediated protection from chemotherapy in vitro. Therefore, if our findings are confirmed in primary samples and in vivo, interruption of leukemia-microenvironment signaling with POL5551 may prove to be an effective strategy in the treatment of pre-B and T cell ALL. Disclosures: Chevalier: Polyphor Ltd: Employment. Dembowsky:Polyphor Ltd: Employment.

Bafilomycin A1 Targets Both Autophagy and Apoptosis Pathways in Pediatric t(1;19) Pre-B Acute Lymphoblastic Leukemia Cells

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 3699-3699
Author(s):  
Na Yuan ◽  
Lin Song ◽  
Suping Zhang ◽  
Weiwei Lin ◽  
Yan Cao ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Bone Marrow ◽  
Acute Lymphoblastic Leukemia ◽  
Western Blotting ◽  
Bone Marrow Cells ◽  
Lymphoblastic Leukemia ◽  
Leukemia Cells ◽  
Primary Cells ◽  
Bafilomycin A1 ◽  
Low Concentration

Abstract The t (1; 19) subtype leukemia accounts for a quarter of pre-B acute lymphoblastic leukemia (ALL) and up to 5% of all ALL patients. Despite plausible remission rate, current treatment regimen on the pediatric pre-B ALL is associated with side effects and CNS relapse, which poses the need for more effective and safer drugs. Bafilomycin A1 (Baf-A1) is known as an inhibitor of late phase of autophagy by inhibiting fusion between autophagosomes and lysosomes as well as by inhibiting lysosomal degradation. Here we show that Baf-A1 of low concentration (1 nM) effectively and specifically inhibits and kills the pre-B ALL cells. E2A/Pbx1 fusion gene positivepre-B ALL 697 cells were used for In vitro experiments. The results of flow cytometry analysis and western blotting showed that Baf-A1 induced cell cycle arrest and proliferation inhibition of ALL cells by upregualting cell cycle negative regulators and downregulating cell cycle positive regulators. In contrast, AML and CML cell lines were insensitive to Baf-A1 treatment. Western blotting and confocal observation on protein LC3 also showed that Baf-A1 at 1 nM blocked basal autophagic flux. Baf-A1 treatment activated mTOR signaling and induced the formation of Becn1–Bcl-2 complex to inhibit the induction of autophagy. Furthermore, apoptosis was induced in ALL cells treated with Baf-A1 for 72 h. However, procaspase-3 and poly-(ADP-ribose) polymerase (PARP) were not cleaved in these cells. We observed that AIF relocalized to the nucleus after 72h Baf-A1 treatment by confocal and immunoblotting. Knockdown of AIF significantly attenuated apoptosis induced by Baf-A1. These data suggest that Baf-A1 targets mitochondria membrane to trigger apoptosis via AIF pathway. In the in vivo experiment, Baf-A1 treatment extended survival and improved pathology of 697 xenograft mice, and significantly reduced the E2A/PBX1 positive leukemia cells in the bone marrow of mice. In vivomouse toxicity assay confirms Baf-A1 as a safe compound. The bone marrow cells of pre-B ALL leukemia patients were sorted against CD133+CD19+ markers, and treatment with Baf-A1 induced a clear inhibition on the CD133+CD19+ primary cells with a significant increased cell death in the sorted B-ALL patient samples. Conversely, Baf-A1 had no inhibitory effect on the bone marrow cells isolated from acute myeloid leukemia patients and healthy people. In summary, Baf-A1 treatment at low concentration effectively and specifically inhibited autophagy by activating mTOR and inducing beclin1-Bcl-2 interaction and induced AIF-dependent apoptosis in t (1; 19) pre-B ALL 697 cells. In the pre-B ALL xenograft mouse model, Baf-A1 specifically targets the leukemia cells while sparing normal cells. More importantly, Baf-A1 potently inhibits and kills the primary cells from pediatric pre-B ALL patients both at initial diagnosis and relapse without compromising normal human hematopoietic cells, all proposing Baf-A1 as a promising drug candidate for this pre-B ALL. Disclosures No relevant conflicts of interest to declare.

scholarly journals Abnormal bone marrow microenvironment: the “harbor” of acute lymphoblastic leukemia cells

Blood Science ◽  
2021 ◽  
Vol 3 (2) ◽  
pp. 29-34
Author(s):  
Zehui Chen ◽  
Yaxin Zheng ◽  
Yaling Yang ◽  
Junnan Kang ◽  
M. James You ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Acute Lymphoblastic Leukemia ◽  
Lymphoblastic Leukemia ◽  
Bone Marrow Microenvironment ◽  
Leukemia Cells ◽  
Abnormal Bone Marrow

scholarly journals Krüppel-like Factor 4 (KLF4) Suppresses T-Cell Acute Lymphoblastic Leukemia By Inhibiting Expression of MAP2K7 and Expansion of Leukemia Initiating Cells

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 3569-3569
Author(s):  
Ye Shen ◽  
Chun Shik Park ◽  
Koramit Suppipat ◽  
Takeshi Yamada ◽  
Toni-Ann Mistretta ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Acute Lymphoblastic Leukemia ◽  
T Cell ◽  
Tumor Suppressor ◽  
Cell Lines ◽  
Conflicts Of Interest ◽  
Lymphoblastic Leukemia ◽  
Leukemia Cells ◽  
Brdu Incorporation ◽  
Jnk Pathway

Abstract Acute lymphoblastic leukemia (ALL) is the most common hematological malignancy in children. Although risk-adaptive therapy, CNS-directed chemotherapy and supportive care have improved the survival of ALL patients, disease relapse is still the leading cause of cancer-related death in children. Therefore, new drugs or novel multi-drug combinations are needed as frontline treatments for high-risk patients and as salvage agents for relapsed disease. T-cell ALL (T-ALL) is a subset of ALL that exhibits activating mutations of NOTCH1 in more than 50% of the patients. However, the use of gamma-secretase inhibitors to reduce NOTCH1 activity has not been successful in patients due to limited response and toxicity. Therefore, identification of genetic factors that cooperate with T-ALL leukemogenesis is needed for the development of alternative therapies. KLF4 is a transcription factor that functions as a tumor suppressor or an oncogene depending on cellular context. Our data showed significant reduction of KLF4 transcripts in lymphoblasts from T-ALL patients compared to blood and bone marrow cells from healthy individuals. In consistent with reduced KLF4 levels, these patients exhibit hyper-methylation of CpG islands located between nt -811 and +1190 relative to KLF4 transcription start site. From these findings we hypothesized that KLF4 has tumor suppressor function in T-ALL leukemogenesis. To test our hypothesis, we transduced 5-FU treated bone marrow (BM) cells from control (Klf4fl/fl), Klf4 null (Klf4fl/fl; Vav-iCre) and Klf4 heterozygous (Klf4fl/+; Vav-iCre) mice with retrovirus carrying a NOTCH1 activating mutant (L1601P-ΔP) and then transplanted these BM cells into irradiated recipient mice. In contrast to controls, mice transplanted with transduced Klf4-null BM cells developed T-ALL with significantly higher penetrance (Klf4 null 76.5% v.s. control 21.3%) and shorter latency (Klf4 null 93 days v.s. control 130 days). Interestingly, Klf4 heterozygous group shows similar survival kinetics as Klf4 null group, suggesting that Klf4 haploinsufficiency is enough to accelerate onset of leukemia. To investigate the effect of Klf4 deletion in established leukemia cells, we transplanted NOTCH1 L1601P-ΔP transduced BM cells from Klf4fl/fl; CreER+ mice to induce leukemia. Post-transplantation deletion of the Klf4 gene by tamoxifen administration was able to accelerate T-ALL development compared to mice injected with vehicle. On the cellular level, loss of KLF4 led to increased proliferation of leukemia cells as assessed by in vivo BrdU incorporation, which correlated with decreased levels of p21 protein. Limited dilution transplantation of primary leukemia cells into secondary recipients showed a 9-fold increase of leukemia initiating cells (LIC) frequency in Klf4null leukemia cells compared to controls, suggesting that KLF4 controls expansion of LIC in T-ALL. To elucidate molecular mechanism underlying KLF4 regulation in T-ALL cells, we performed microarray and ChIP-Seq in control and Klf4 null CD4+CD8+ leukemia cells. Combined analyses revealed 202 genes as KLF4 direct targets, of which 11 genes are also deregulated in human T-ALL cells by comparing with published microarray datasets. One of the top upregulated genes is Map2k7, which encodes a kinase upstream of the JNK pathway. Immunoblots in leukemia cells confirmed increased expression of MAP2K7 protein and enhanced phosphorylation of its downstream targets JNK and ATF2. To further investigate the role of JNK pathway in T-ALL, we tested JNK inhibitor SP600125 in human T-ALL cell lines (KOPTK1, DND41, CCRF-CEM, MOLT3). Interestingly, SP600125 showed dose-dependent cytotoxicity in all human T-ALL cell lines tested regardless of their NOTCH1 status. Overall our results showed for the first time that KLF4 functions as a tumor suppressor in T-ALL by regulating proliferation of leukemia cells and frequency of LIC. Additional study elucidated that KLF4 suppresses the JNK pathway via direct transcriptional regulation of MAP2K7. Moreover, the vulnerability of human T-ALL cell lines to JNK inhibition provides a novel target for future therapy in T-ALL patients. Disclosures No relevant conflicts of interest to declare.

Expansion and Transformation of Primary Acute Lymphoblastic Leukemia Cells into Antigen-Presenting Cells using a Novel Culturing System Enables the Generation of Leukemia-Reactive T Cell Responses that Are Effective in Vivo.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 303-303
Author(s):  
Bart A. Nijmeijer ◽  
Marianke L.J. Van Schie ◽  
Roel Willemze ◽  
J.H. Frederik Falkenburg
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cell Lines ◽  
Lymphoblastic Leukemia ◽  
Primary Cells ◽  
T Cell Anergy ◽  
Cell Anergy ◽  
Antigen Presenting

Abstract Allogeneic cellular immunotherapy is generally ineffective in acute lymphoblastic leukemia (ALL). In vitro studies have suggested that this inefficacy may be the result of a lack of costimulatory molecule expression by ALL cells, resulting in the induction of T cell anergy. Activation of T cells by ALL cells that are transformed into adequate antigen-presenting cells (ALL-APC) may prevent the induction of T cell anergy and result in the generation of competent leukemia-reactive T cell responses for adoptive immunotherapy. However, in vitro modification of ALL cells was hampered by the fact that ALL cells from adult patients could not be cultured in vitro for prolonged periods of time. We have developed a novel serum-free culturing system for B-lineage ALL in which proliferation is initiated and sustained by ALL-cell derived growth factors. Long-term (>2 yrs) proliferation was induced in 12 out of 26 randomly selected primary samples from patients with ALL. The cell cultures ( Leiden cell lines) proliferated with a mean doubling time of 3.0 days (range 2.7–3.6 days). All Leiden cell lines presented the chromosomal abberations observed in the primary cells. The Leiden cell lines displayed an immune phenotype similar to the primary cells, exept for loss of CD34 expression. In vivo characteristics of Leiden cells were evaluated in NOD/scid mice. After intravenous inoculation, Leiden cell lines and primary cells showed identical homing patterns initially involving spleen and bone marrow, followed by the development of overt and progressive leukemia. A comparison of in vivo progression kinetics was performed for one of the Leiden cell lines and the corresponding primary cells. Weekly determination of leukemic cell counts in the blood of engrafted animals revealed that the cell line and the primary cells displayed similar doubling times in vivo of 6.3 and 7.7 days, respectively. To generate cells with improved antigen presentation function, Leiden cell lines were exposed to various activating agents. Stimulation with CpG containing oligonucleotides resulted in induction of CD40 in 9 out of 10 lines. Subsequent ligation of CD40 by culturing CpG-activated Leiden cells on fibroblasts expressing human CD40 ligand resulted in the induction of CD80 or CD86 in 7 of these 10 cell lines. To study the immune stimulatory properties of these Leiden ALL-APC, allogeneic HLA-identical T cells were first activated in vitro by coculturing these cells with either unmodified Leiden cells or with the corresponding Leiden ALL-APC for 3 days, and subsequently infused into groups of 6 leukemic NOD/scid mice. While T cells cocultured with unmodified Leiden cells did not expand in vivo, T cells cocultured with Leiden ALL-APC expanded after infusion in 5 out of 6 animals. This expansion coincided with a 20–75% decrease in leukemic cell numbers in the blood. In conclusion, the novel serum-free culturing system enables long-term culture and manipulation of a significant fraction of primary human ALL. These Leiden cell lines can be modified into ALL-APC that display adequate antigen presenting function, preventing the induction of T cell anergy as demonstrated in vivo in the NOD/scid mouse model.

AT9283, A Novel Aurora Kinase/Jak2 Inhibitor Demonstrates Activity against Refractory Infant Leukemia Cells: Studies On Growth Inhibition, Biological Correlates, Drug Synergy and Effects On Leukemia Stem-Like Cells.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 3078-3078
Author(s):  
Shamim Lotfi ◽  
Aarthi Jayanthan ◽  
Victor A. Lewis ◽  
Greg Guilcher ◽  
Matthew S Squires ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Cell Lines ◽  
Stromal Cells ◽  
Bone Marrow Stromal Cells ◽  
Aurora Kinase ◽  
Marrow Stromal Cells ◽  
Leukemia Cells ◽  
Combination Studies ◽  
Infant Leukemia

Abstract Abstract 3078 Poster Board III-15 Leukemia in children less than 1 year of age confers a poor prognosis, despite intensification of therapy. These leukemias possess unique biologic characteristics including the presence of mixed-lineage leukemia (MLL) gene rearrangement and high expression of Fms-like tyrosine kinase 3 (FLT3). AT9283, a potent inhibitor of Aurora A and B kinases, JAK2, JAK3, and mutant Abl Kinase, has demonstrated inhibition of multiple solid tumor cell lines in vitro and in mouse xenograft models. Aurora kinase inhibition has been shown to inhibit cancer cell growth by interfering with the mitotic apparatus. We investigated the activity of AT9283 against cell lines derived from refractory infant leukemia cells to identify its efficacy in a future treatment protocol. Method Five cell lines derived from infant leukemia cells were used (ALL: BEL1, KOPN8, KCCF2, B1 and AML: TIB202). We also included the cell line SEM that was derived from a 5 year old child with t (4;11) MLL-AF4 preB-ALL. Normal bone marrow stromal cells were used to evaluate cytotoxicity against non-malignant cells. AT9283 was provided by Astex Therapeutics Ltd. (Cambridge, UK). Approximately 1×104 cells per well were seeded in 96-well plates and incubated with increasing concentrations of AT9283, alone or in combination with a panel of conventional and novel therapeutic agents. After four days, cell survival was measured by Alamar blue assay and IC50 values and combination indices were calculated. Stem-like cells were quantified by the distribution of ALDH bright cells by Aldefluor assay (Stem cell technologies) and characterized by conventional clonogenic assays. Alterations in cell-signaling pathways and survival proteins were measured by Western blot analysis using total and phospho-specific antibodies. Results AT9283 inhibited the growth of all five cell lines with a 10 fold variation in IC50 within cell lines (IC50 range, 0.1 to 0.01 μM). There was a corresponding increase in the number of cells displaying a polyploid phenotype, an effect of aurora kinase inhibition. No significant cytotoxicity against bone marrow stromal cells was seen under the experimental conditions used in this study (IC50 > 10 μM). Changes in the activation and expression of a variety of intracellular proteins were noted, including the down regulation of activated ERK1/2, MYC and AKT within 10 minutes of exposure to the agent. An increase in the activated form of RAF and ATF2 was observed immediately after drug exposure. Importantly, a significant decrease in the level of constitutive pFLT-3 was demonstrated. A concurrent increase in cleaved PARP was also noted, indicating the initiation of apoptosis. In combination studies, the HDAC inhibitor Apicidin showed synergy across all cell lines (CI range: 0.07 to 0.62). A decrease in ALDH bright stem-like cells was observed in a dose dependent manner, up to 50% over 24 hours at IC50 concentrations. Conclusions Our in vitro studies show that AT9283 significantly decreases the growth and survival of infant leukemia cell lines. Importantly, AT9283 potently induces FLT3 de-phosphorylation, inhibiting a critical growth stimulatory pathway of infant ALL cells. We have identified changes in a number of signaling and apoptotic molecules that can provide a panel of markers for biological correlative analysis for drug activity in vivo. Also, the drug combination studies demonstrate the potential of HDAC inhibition to synergize with the activity of this agent. Finally, the effect on stem-like cells provides a rationale and critical preclinical data for the formulation of an effective clinical trial for the treatment of infants with refractory ALL. Disclosures Squires: AstexTherapeutics Ltd: Employment.

scholarly journals A Novel Three-Dimensional Co-Culture System to Study Leukemia in the Bone Marrow Microenvironment

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 1864-1864 ◽  
Author(s):  
Zeena Salman ◽  
Juan Carlos Balandrán-Juárez ◽  
Rosana Pelayo ◽  
Monica L. Guzman
Keyword(s):  
Bone Marrow ◽  
Culture System ◽  
Lymphoblastic Leukemia ◽  
Cell Monolayer ◽  
Leukemia Cell ◽  
Leukemia Cells ◽  
Human Leukemia ◽  
Bone Marrow Niche ◽  
3 Dimensional

Abstract The need for novel therapies in acute leukemia has been motivated by sub-optimal 5-year survival rates of 25.4% in acute myeloid leukemia (AML) and approximately 70% in acute lymphoblastic leukemia (ALL). While these rates are higher in the pediatric population, novel approaches are necessary in all age groups to improve outcomes. Pre-clinical studies of novel therapeutics using in vitro and in vivo methods remain suboptimal with frequent lack of correlation with clinical outcomes at the bedside. Recent evidence has shown that human leukemia xenografts into immunodeficient mice yield variable results, indicating that treatment using these methods is not replicable. When using in vitro cell culture methods, the well-documented protective effects of the bone marrow (BM) microenvironment (BMME) on leukemia are not mimicked. Furthermore, these techniques cannot be used to investigate the effects of novel agents on leukemia stem cells (LSC) and their mobilization, which is important in the ablation of leukemia. Thus, we explored a novel 3-dimensional co-culture system to study the effects of drugs on leukemia cells in the presence of stroma in an environment more similar to that of human leukemia in the BMME. We generated a 3-dimensional (3D) spheroid co-culture system using human stromal cell line (HS-5) cells or human mesenchymal stromal cells (hBMSC) from primary AML or ALL BM. To evaluate the dynamics of the 3D system, we labeled the stroma cells with GFP and the leukemia cells with mCherry. We observed rapid homing to the center of the 3D stroma. We evaluated ROS levels, proliferation status, hypoxia and expression of key niche proteins such as CXCL12 in leukemia cells found outside and inside the 3D system. These methods were compared to similar treatments in leukemia cell monolayer culture and 2-dimensional co-culture systems. We treated this system with various drugs such as cytarabine, doxorubicin, TG02 (a multi-kinase inhibitor with LSC mobilization effects), and plerixafor; we then harvested cells from the outer and inner layers and evaluated these separately by multi-parameter flow cytometry for viability and mobilization of LSCs in relation to the stroma and xenotransplant assays. Our studies reveal that the 3D culture system has lower ROS internally, suggesting a similarly hypoxic environment to BMME. Our studies also reveal that, when treated with cytarabine, AML cells closest to the stromal center of the spheroid remain protected, with higher viability compared to those farther from stroma, and even more so than leukemia cells in a 2-D bilayer with stroma or in a monolayer. A lower CXCL12 level was also observed in the stroma of leukemic BM compared to healthy BM within the co-culture system. This culture method possesses many of the characteristics of leukemia cells within the bone marrow niche and should be considered for future in vitro pre-clinical drug testing to model the tumor within its microenvironment. Disclosures No relevant conflicts of interest to declare.

scholarly journals Functional and Genomic Characterization of the Interaction between Acute Lymphoblastic Leukemia Cells and the Microenvironment Identifies Pathways for Therapeutic Intervention

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 1550-1550
Author(s):  
Hiroki Yoshihara ◽  
Michelle L. Churchman ◽  
Jennifer L. Peters ◽  
David B. Finkelstein ◽  
Elisabeth M. Paietta ◽  
...  
Keyword(s):  
Gene Expression ◽  
Bone Marrow ◽  
Cell Lines ◽  
Research Funding ◽  
Lymphoblastic Leukemia ◽  
Time Lapse ◽  
Leukemia Cells ◽  
Adherent Cells ◽  
Bone Marrow Mscs ◽  
Upstream Regulator

Abstract Introduction Residence and interaction with a specialized bone marrow microenvironment is important for normal hematopoietic stem cells and for initiation and progression of myeloid malignancies, but the role of the microenvironment in propagation and therapeutic response of acute lymphoblastic leukemia (ALL) is not well known. Prior work has identified the efficacy of inhibiting FAK signaling, which is deregulated by IKZF1 alterations resulting in induction of THY1-Integrin alpha 5 adhesion in Ph-positive (Ph+) ALL. Here, we hypothesized that this mechanism may be more broadly important in ALL. We applied a systematic integrated genomic/imaging/functional approach to define the nature of interaction and identify changes in leukemic cells upon interaction that may be targetable. Materials and methods Time-lapse confocal imaging was performed to examine how leukemia cells migrate and adhere to mesenchymal stem cells (MSCs). NALM6 (DUX4/ERG), MHH-CALL2 (hypodiploid), 697 (TCF3-PBX1), Reh (ETV6-RUNX1) and SUP-B15 (Ph+) cell lines were cultured with immortalized human bone marrow MSCs transduced with telomerase reverse transcriptase (hTERT) (Mihara, Br J Haematol. 2003;120:846). For RNA-sequencing, non-adherent cell line cells were collected after two days of coculture with hTERT while adherent cells were trypsinized and collected. Both samples were sorted for CD19 positive population. Fresh primary ALL samples were cultured on bone marrow MSCs derived from patients with no hematological disease and collected with the same procedure for RT-PCR. Multicolor immunofluorescence imaging was utilized to observe expression of multiple molecules involved in adherence. Results Time-lapse imaging showed that leukemia cells have a dynamic interaction with MSC monolayers, with temporary adherence, accompanied by dynamic change in their shape. NALM6 cells adherent to MSCs reduced cell cycling, with an increase in the ratio of G0/G1 cells (26.7% to 48.0%) and decrease in S phase (60.7 to 41.8%). Analysis of gene expression showed 138 upregulated genes (log2FC >2 and FDR <0.05) in adherent cells which were common in all five cell lines, with striking upregulation leading to gene expression associated with gene ontology of extracellular matrix organization and collagen fibril organization. Representative genes were validated in adherent NALM6 cells by immunoblotting (FN1, TIMP1, and LGALS1). Pathway analysis showed that transforming growth factor beta 1 (TGFB1) was the top ranked upstream regulator (p-value of overlap 5.26E-35) in that 44 of 65 genes had measurement direction consistent with activation of TGFB1 signaling. In addition, other upstream regulators that may be involved were beta-estradiol, fibroblast growth factor 2, and tumor necrosis factor. Adhesion of leukemia cells to stroma may induce integrin expression and downstream signaling. Our transcriptomic analysis showed that integrins (A5, B1, A3 and B5) and caveolin 1 (CAV1), a main component of the caveolae plasma membranes, are highly transcribed in adherent leukemia cells. As hTERT cells showed unexpectedly low expression of THY1, a common MSC marker, we utilized primary bone marrow MSC for subsequent analysis. Immunoblotting assay showed enhanced expression of CAV1 in all cell lines adhered to MSCs. Multicolor immunofluorescence imaging demonstrated CAV1 and ITGB1 expression on leukemia cells that localized adjacent to stromal cells, which confirms that these molecules were upregulated upon adhesion to MSCs. Moreover, primary ALL cells showed remarkable upregulation of CAV1, ITGB1, ITGA3, and ITGB5 when the cells were adherent to MSCs. Conclusions Our results demonstrate that ALL cells dynamically interact with microenvironment cells, inducing changes in cell morphology, cell cycling, and adhesion, which may facilitate altered responsiveness to therapy. Transcriptional results suggest that TGFβ signaling is an upstream regulator after cell adhesion to MSCs. While integrins and CAV1 mediate the signaling, these pathway and molecules will be candidates for exploring inhibitors of signaling, which may affect their interaction and make them novel therapeutic targets. Figure. Figure. Disclosures Mullighan: Loxo Oncology: Research Funding; Cancer Prevention and Research Institute of Texas: Consultancy; Amgen: Honoraria, Speakers Bureau; Abbvie: Research Funding; Pfizer: Honoraria, Research Funding, Speakers Bureau.

scholarly journals Dynamin inhibition causes context-dependent cell death of leukemia and lymphoma cells

PLoS ONE ◽  
2021 ◽  
Vol 16 (9) ◽  
pp. e0256708
Author(s):  
Christopher von Beek ◽  
Linnéa Alriksson ◽  
Josefine Palle ◽  
Ann-Marie Gustafson ◽  
Mirjana Grujic ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Cell Death ◽  
Acute Leukemia ◽  
Cell Lines ◽  
Lymphoblastic Leukemia ◽  
Leukemia Cell ◽  
Tumor Formation ◽  
Leukemia Cells ◽  
Human Leukemia ◽  
Bone Marrow Biopsies

Current chemotherapy for treatment of pediatric acute leukemia, although generally successful, is still a matter of concern due to treatment resistance, relapses and life-long side effects for a subset of patients. Inhibition of dynamin, a GTPase involved in clathrin-mediated endocytosis and regulation of the cell cycle, has been proposed as a potential anti-cancer regimen, but the effects of dynamin inhibition on leukemia cells has not been extensively addressed. Here we adopted single cell and whole-population analysis by flow cytometry and live imaging, to assess the effect of dynamin inhibition (Dynasore, Dyngo-4a, MitMAB) on pediatric acute leukemia cell lines (CCRF-CEM and THP-1), human bone marrow biopsies from patients diagnosed with acute lymphoblastic leukemia (ALL), as well as in a model of lymphoma (EL4)-induced tumor growth in mice. All inhibitors suppressed proliferation and induced pronounced caspase-dependent apoptotic cell death in CCRF-CEM and THP-1 cell lines. However, the inhibitors showed no effect on bone marrow biopsies, and did not prevent EL4-induced tumor formation in mice. We conclude that dynamin inhibition affects highly proliferating human leukemia cells. These findings form a basis for evaluation of the potential, and constraints, of employing dynamin inhibition in treatment strategies against leukemia and other malignancies.

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