scholarly journals Characterizing the Motility of Chemotherapeutics-Treated Acute Lymphoblastic Leukemia Cells by Time-Lapse Imaging

Cells ◽  
2020 ◽  
Vol 9 (6) ◽  
pp. 1470
Author(s):  
Hsiao-Chuan Liu ◽  
Eun Ji Gang ◽  
Hye Na Kim ◽  
Yongsheng Ruan ◽  
Heather Ogana ◽  
...  
Keyword(s):  
Drug Resistance ◽  
Acute Lymphoblastic Leukemia ◽  
Physical Properties ◽  
Lymphoblastic Leukemia ◽  
Leukemia Cell ◽  
Time Lapse ◽  
Leukemia Cells ◽  
Integrin Α4 ◽  
Chemotherapeutic Treatment ◽  
Time Lapse Imaging

Drug resistance is an obstacle in the therapy of acute lymphoblastic leukemia (ALL). Whether the physical properties such as the motility of the cells contribute to the survival of ALL cells after drug treatment has recently been of increasing interest, as they could potentially allow the metastasis of solid tumor cells and the migration of leukemia cells. We hypothesized that chemotherapeutic treatment may alter these physical cellular properties. To investigate the motility of chemotherapeutics-treated B-cell ALL (B-ALL) cells, patient-derived B-ALL cells were treated with chemotherapy for 7 days and left for 12 h without chemotherapeutic treatment. Two parameters of motility were studied, velocity and migration distance, using a time-lapse imaging system. The study revealed that compared to non-chemotherapeutically treated B-ALL cells, B-ALL cells that survived chemotherapy treatment after 7 days showed reduced motility. We had previously shown that Tysabri and P5G10, antibodies against the adhesion molecules integrins α4 and α6, respectively, may overcome drug resistance mediated through leukemia cell adhesion to bone marrow stromal cells. Therefore, we tested the effect of integrin α4 or α6 blockade on the motility of chemotherapeutics-treated ALL cells. Only integrin α4 blockade decreased the motility and velocity of two chemotherapeutics-treated ALL cell lines. Interestingly, integrin α6 blockade did not affect the velocity of chemoresistant ALL cells. This study explores the physical properties of the movements of chemoresistant B-ALL cells and highlights a potential link to integrins. Further studies to investigate the underlying mechanism are warranted.

scholarly journals Overcoming Microenvironment-Mediated Chemoprotection through Stromal Galectin-3 Inhibition in Acute Lymphoblastic Leukemia

2021 ◽  
Vol 22 (22) ◽  
pp. 12167
Author(s):  
Somayeh S. Tarighat ◽  
Fei Fei ◽  
Eun Ji Joo ◽  
Hisham Abdel-Azim ◽  
Lu Yang ◽  
...  
Keyword(s):  
Drug Resistance ◽  
Acute Lymphoblastic Leukemia ◽  
Stromal Cells ◽  
Lymphoblastic Leukemia ◽  
High Specificity ◽  
Leukemia Cells ◽  
Cell Precursor ◽  
Cell Responses ◽  
Galectin 3 ◽  
Dose Dependent

Environmentally-mediated drug resistance in B-cell precursor acute lymphoblastic leukemia (BCP-ALL) significantly contributes to relapse. Stromal cells in the bone marrow environment protect leukemia cells by secretion of chemokines as cues for BCP-ALL migration towards, and adhesion to, stroma. Stromal cells and BCP-ALL cells communicate through stromal galectin-3. Here, we investigated the significance of stromal galectin-3 to BCP-ALL cells. We used CRISPR/Cas9 genome editing to ablate galectin-3 in stromal cells and found that galectin-3 is dispensable for steady-state BCP-ALL proliferation and viability. However, efficient leukemia migration and adhesion to stromal cells are significantly dependent on stromal galectin-3. Importantly, the loss of stromal galectin-3 production sensitized BCP-ALL cells to conventional chemotherapy. We therefore tested novel carbohydrate-based small molecule compounds (Cpd14 and Cpd17) with high specificity for galectin-3. Consistent with results obtained using galectin-3-knockout stromal cells, treatment of stromal-BCP-ALL co-cultures inhibited BCP-ALL migration and adhesion. Moreover, these compounds induced anti-leukemic responses in BCP-ALL cells, including a dose-dependent reduction of viability and proliferation, the induction of apoptosis and, importantly, the inhibition of drug resistance. Collectively, these findings indicate galectin-3 regulates BCP-ALL cell responses to chemotherapy through the interactions between leukemia cells and the stroma, and show that a combination of galectin-3 inhibition with conventional drugs can sensitize the leukemia cells to chemotherapy.

Targeting STAT5 in Leukemia Through Inhibition of Bromodomain Proteins

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 399-399
Author(s):  
Suhu Liu ◽  
Sarah Walker ◽  
Erik Nelson ◽  
Robert Cirulli ◽  
Michael Xiang ◽  
...  
Keyword(s):  
Gene Expression ◽  
Acute Lymphoblastic Leukemia ◽  
Tyrosine Kinases ◽  
Transcriptional Activity ◽  
Therapeutic Strategy ◽  
Target Gene ◽  
Lymphoblastic Leukemia ◽  
Leukemia Cell ◽  
Leukemia Cells ◽  
Bromodomain Proteins

Abstract Abstract 399 Introduction: The transcription factor STAT5 is constitutively activated in many forms of hematologic malignancies, including chronic myeloid leukemia (CML) and acute lymphoblastic leukemia (ALL). STAT5 can be activated by constitutively activated tyrosine kinases or autocrine and paracrine secretion of cytokines signaling through Jak kinases. STAT5 is essential for the pathogenesis of neoplasms induced by BCR-ABL1 and Jak2V617F, as well as for leukemia stem cell self-renewal. Development of tyrosine kinases inhibitors (TKIs), such as imatinib, has greatly improved the outcome of patients with leukemias harboring aberrantly activated oncogenic tyrosine kinases. However, TKIs used as a single agent only achieve significant success in CML, with very limited benefit in the more aggressive ALL. Moreover, patients with CML who initially respond well may acquire resistance to TKIs with the progression of their disease. In fact, increased activity of STAT5 is often associated with CML progression and may underlie resistance to TKIs. Importantly, leukemia cells that are resistant to TKIs remain sensitive to STAT5 inhibition, and dual inhibition of both tyrosine kinases and STAT5 leads to more efficient reduction of leukemia cell viability. Thus targeting STAT5 alone or in combination is a promising therapeutic strategy for many hematological malignancies. While many strategies directly inhibit STAT5, we considered the possibility that STAT5 association with co-regulatory proteins is essential for STAT5 function and therefore targeting this association may be a suitable therapeutic strategy. Methods and Results: Given the importance of BET bromodomain proteins in chromatin remodeling necessary for transcription, we tested the activity of the BET bromodomain inhibitor JQ1 on STAT5-dependent transcriptional activity. Using both heterologous reporter systems and endogenous STAT5 target genes, we found that JQ1, but not its inactive enantiomer, potently and specifically inhibited STAT5-dependent gene expression. Inhibition of STAT5 dependent gene regulation was also replicated by another BET bromodomian inhibitor, iBET, further demonstrating that BET inhibition inhibits STAT5. Since JQ1 inhibits BET family members Brd2, Brd3, Brd4, and BrdT, we asked which BET family member is specifically associated with STAT5 transcriptional function. To do this, we utilized shRNA to knock-down each bromodomain protein and determined the effect on STAT5 activity. We found that knocking-down Brd2, but not Brd3 or Brd4, reduces STAT5 target gene expression, indicating that Brd2 is specifically involved in regulating STAT5 transcriptional function. JQ1 can reduce STAT5 transcriptional activity without inhibiting STAT5 phosphorylation or STAT5 binding to its genomic binding sites. Similarly, knocking-down Brd2 can reduce STAT5 target gene expression without influencing STAT5 phosphorylation. We hypothesize that Brd2 regulates STAT5 transcriptional function by acting as a co-activator for STAT5. Thus through blocking Brd2, JQ1 can inhibit STAT5 transcriptional function without directly targeting STAT5 itself. In a group of aggressive T cell acute lymphoblastic leukemia (T-ALL) cell lines, where constitutively activated STAT5 contributes to leukemia cell survival, knocking-down Brd2 renders leukemia cells more sensitive to TKI induced apoptosis. In addition, combined treatment with TKIs and JQ1 showed strong synergy in inducing T-ALL leukemia cells apoptosis and reducing viability. Overexpressing a constitutively active form of STAT5 rescues these leukemia cells from death induced by TKIs and JQ1, indicating an important role of STAT5 as a target for TKI and JQ1 induced cell death in T-ALL cells. Conclusion: We found that the BET bromodomain inhibitor JQ1 can reduce STAT5 transcriptional function by blocking Brd2 without reducing STAT5 phosphorylation or STAT5 DNA binding. In addition, the combination of TKIs and JQ1 induces T-ALL leukemia cell apoptosis and reduces survival in a synergistic manner, and represents a rational drug combination for treating this sub-group of highly aggressive leukemias. Disclosures: Bradner: Tensha Therapeutics: Consultancy, Equity Ownership, Scientific founder Other.

Gene Delivery to Human B-Precursor Acute Lymphoblastic Leukemia Cells

Blood ◽  
1998 ◽  
Vol 92 (10) ◽  
pp. 3537-3545 ◽  
Author(s):  
Leo Mascarenhas ◽  
Renata Stripecke ◽  
Scott S. Case ◽  
Dakun Xu ◽  
Kenneth I. Weinberg ◽  
...  
Keyword(s):  
Acute Lymphoblastic Leukemia ◽  
Gene Transfer ◽  
Gene Delivery ◽  
Cell Lines ◽  
Lymphoblastic Leukemia ◽  
Leukemia Virus ◽  
Leukemia Cell ◽  
Leukemia Cells ◽  
Reh Cells ◽  
Hiv 1

Autologous leukemia cells engineered to express immune-stimulating molecules may be used to elicit antileukemia immune responses. Gene delivery to human B-precursor acute lymphoblastic leukemia (ALL) cells was investigated using the enhanced green fluorescent protein (EGFP) as a reporter gene, measured by flow cytometry. Transfection of the Nalm-6 and Reh B-precursor ALL leukemia cell lines with an expression plasmid was investigated using lipofection, electroporation, and a polycationic compound. Only the liposomal compound Cellfectin showed significant gene transfer (3.9% to 12% for Nalm-6 cells and 3.1% to 5% for Reh cells). Transduction with gibbon-ape leukemia virus pseudotyped Moloney murine leukemia virus (MoMuLV)-based retrovirus vectors was investigated in various settings. Cocultivation of ALL cell lines with packaging cell lines showed the highest transduction efficiency for retroviral gene transfer (40.1% to 87.5% for Nalm-6 cells and 0.3% to 9% for Reh cells), followed by transduction with viral supernatant on the recombinant fibronectin fragment CH-296 (13% to 35.5% for Nalm-6 cells and 0.4% to 6% Reh cells), transduction on human bone marrow stroma monolayers (3.2% to 13.3% for Nalm-6 cells and 0% to 0.2% Reh cells), and in suspension with protamine sulfate (0.7% to 3.1% for Nalm-6 cells and 0% for Reh cells). Transduction of both Nalm-6 and Reh cells with human immunodeficiency virus–type 1 (HIV-1)–based lentiviral vectors pseudotyped with the vesicular stomatitis virus-G envelope produced the best gene transfer efficiency, transducing greater than 90% of both cell lines. Gene delivery into primary human B-precursor ALL cells from patients was then investigated using MoMuLV-based retrovirus vectors and HIV-1–based lentivirus vectors. Both vectors transduced the primary B-precursor ALL cells with high efficiencies. These studies may be applied for investigating gene delivery into primary human B-precursor ALL cells to be used for immunotherapy.

scholarly journals Targeting Mutant p53 in Pediatric Acute Lymphoblastic Leukemia

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 903-903
Author(s):  
Salih Demir ◽  
Galina Selivanova ◽  
Eugen Tausch ◽  
Lisa Wiesmüller ◽  
Stephan Stilgenbauer ◽  
...  
Keyword(s):  
Cell Death ◽  
Acute Lymphoblastic Leukemia ◽  
High Risk ◽  
Cell Lines ◽  
Therapeutic Intervention ◽  
High Performance ◽  
Lymphoblastic Leukemia ◽  
Leukemia Cell ◽  
Therapy Resistance ◽  
Leukemia Cells

Abstract Mutations of the tumor suppressor gene TP53 have been described to be associated with aggressive disease and inferior prognosis in different types of cancer, including hematological malignancies. In acute lymphoblastic leukemia (ALL), TP53 alterations are infrequently found at diagnosis but have recently been described in about 12% of patients at relapse. This suggests an association with therapy resistance in high risk/relapsed ALL and patients with TP53 mutated ALL have in fact an inferior outcome. Small molecule compounds targeting mutated TP53 such as APR-246, initially described as PRIMA-1MET (p53-dependent reactivation and induction of massive apoptosis) leading to apoptosis induction have shown activity in several types of malignancies with mutated TP53. In ALL, however, mutant TP53 has so far not been addressed as a target for therapeutic intervention. In this study, we investigated a large cohort of patient-derived pediatric B cell precursor (BCP)-ALL primograft samples to identify cases with mutated TP53. Further, we analyzed the effects of APR-246 and evaluated its activity on BCP-ALL cell lines and primografts with mutated (mut) orwild type (wt) TP53. Altogether, 62 BCP-ALL primograft samples established from patients at diagnosis (n=53) or relapse (n=9) by transplantation of primary ALL cells onto NOD/SCID mice were screened for TP53 mutations by denaturating high-performance liquid chromatography (dHPLC) followed by Sanger sequencing of exons 4 to 10 to confirm detected mutations. We identified 4 cases with TP53 mut, 3 obtained from diagnosis (5.6%) and one at relapse (11.1%), corresponding to frequencies described in clinical studies. Mutated cases were further analyzed by fluorescence in situ hybridization (FISH), revealing a 17p deletion in one TP53 mut sample. Similarly, we analyzed 6 BCP-ALL cell lines and identified 2 TP53 mut and 4 TP53 wt lines. Exposure of BCP-ALL primograft (TP53 mut n=4, TP53 wt n=4) and cell line (TP53 mut n=2, TP53 wt n=4) samples to the DNA damaging agent doxorubicin showed, as expected, resistance of TP53 mut leukemia cells for cell death induction, reflected by significantly higher half maximal inhibitory concentrations (IC50; TP53 mut 49 and 143 ng/ml, TP53 wt mean 12 ng/ml) and lower induction of cell death (TP53 mut 16 to 23%, TP53 wt 10 to 60%) in TP53 mut ALL, corroborating the tumor-suppressive function of p53 in ALL. We then investigated the sensitivity of BCP-ALL cell lines for cell death induction by APR-246 (kindly provided by Aprea, Stockholm, Sweden). We observed high sensitivity for APR-246 in TP53 mut (IC50: 5 µM for both cell lines) as compared to TP53 wt ALL (mean IC50: 58 µM). DNA fragmentation and Annexin-V/propidium-iodide (PI) positivity revealed apoptosis as mechanism of APR-246 mediated cell death. Reactive oxygen species (ROS) have recently been described to mediate APR-246 induced cell death in multiple myeloma cells. Therefore, we investigated ROS levels by detection of oxidation-specific fluorescence of dichlorodihydrofluorescein diacetate (DCFDA) in ALL cells. Interestingly, ROS quenching by N-acetyl cysteine abolished induction of cell death in TP53 mut but not TP53 wt ALL cells indicating ROS as a mediator of APR-246 induced cell death in TP53 mut ALL. Furthermore, we addressed p53 activation in response to APR-246 by assessing phosphorylation of p53 (p53pSer15) using phosphoflow cytometry. Most interestingly, APR-246 led to 6-fold increased p53pSer15 levels in TP53 mut compared to no activation in TP53 wt leukemia cells, indicating restoration of p53function upon APR-246treatment in BCP-ALL. Based on these findings, we addressed the effectivity of APR-246on primary, patient-derived primografts and compared sensitivities for cell death induction in TP53 mut (n=4) and TP53 wt (n=4) samples. Importantly, the pattern of responsiveness of TP53 mut ALL was also identified in TP53 mut patient-derived ALL samples with induction of significantly higher cell death rates in TP53 mut ALL (TP53 mut 48%, TP53 wt 18%, 5 µM APR-246, 24 h). Taken together, we showed that TP53 mut BCP-ALL can be targeted by APR-246 leading to re-activation of p53, induction of ROS dependent apoptosis and effective leukemia cell killing. Thus, targeting and re-activation of mutated p53 provides a promising novel strategy for therapeutic intervention in this high-risk subtype of BCP-ALL. Disclosures Selivanova: Aprea: Patents & Royalties: APR-246. Tausch:Gilead: Other: Travel support. Stilgenbauer:Gilead: Honoraria, Research Funding.

scholarly journals Overcoming microenvironment-mediated chemoprotection through stromal Galectin-3 inhibition in acute lymphoblastic leukemia

2021 ◽  
Author(s):  
Nora Heisterkamp ◽  
Somayeh S Tarighat ◽  
Fei Fei ◽  
Eun ji Joo ◽  
Hisham Abdel-Azim ◽  
...  
Keyword(s):  
Drug Resistance ◽  
Acute Lymphoblastic Leukemia ◽  
Stromal Cells ◽  
Lymphoblastic Leukemia ◽  
High Specificity ◽  
Leukemia Cells ◽  
Cell Precursor ◽  
Cell Responses ◽  
Galectin 3 ◽  
Dose Dependent

Environmentally-mediated drug resistance in B-cell precursor acute lymphoblastic leukemia (BCP-ALL) significantly contributes to relapse. Stromal cells in the bone marrow environment protect leukemia cells by secretion of chemokines as cues for BCP-ALL migration towards, and adhesion to, stroma. Stromal cells and BCP-ALL cells communicate through stromal Galectin-3. Here, we investigated the significance of stromal Galectin-3 to BCP-ALL cells. We used CRISPR/Cas9 genome editing to ablate Galectin-3 in stromal cells and found that Galectin-3 is dispensable for steady-state BCP-ALL proliferation and viability. However, efficient leukemia migration and adhesion to stromal cells are significantly dependent on stromal Galectin-3. Importantly, loss of stromal Galectin-3 production sensitized BCP-ALL cells to conventional chemotherapy. We therefore tested novel carbohydrate-based small molecule compounds (Cpd14 and Cpd17) with high specificity for Galectin-3. Consistent with results obtained using Galectin-3-knockout stromal cells, treatment of stromal-BCP-ALL co-cultures inhibited BCP-ALL migration and adhesion. Moreover, these compounds induced anti-leukemic responses in BCP-ALL cells including a dose-dependent reduction of viability and proliferation, induction of apoptosis and, importantly, inhibition of drug resistance. Collectively, these findings indicate Galectin-3 regulates BCP-ALL cell responses to chemotherapy through the interactions between leukemia cells and the stroma, and show that a combination of Galectin-3 inhibition with conventional drugs can sensitize the leukemia cells to chemotherapy.

Single-Cell Phospho-Profiling in Pediatric Acute Lymphoblastic Leukemia (ALL) Reveals Constitutive and Cytokine Induced Specific Signatures Involving Stat5

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 2511-2511
Author(s):  
Manon Queudeville ◽  
Hannah Kunze ◽  
Sarah M. Eckhoff ◽  
Klaus-Michael Debatin ◽  
Lueder H. Meyer
Keyword(s):  
Acute Lymphoblastic Leukemia ◽  
B Cells ◽  
Single Cell ◽  
B Lymphocytes ◽  
Cell Lines ◽  
Lymphoblastic Leukemia ◽  
Leukemia Cell ◽  
Leukemia Cells ◽  
Leukemia Cell Lines ◽  
Significant Difference

Abstract Oncogenesis and tumor progression are supported by alterations in cellular signaling. We used phospho-specific antibodies in flow cytometry to analyze specific signaling profiles of leukemia cells at a single cell level in 7 B cell precursor (BCP)-ALL leukemia cell lines and 7 primary pediatric BCP-ALL xenograft samples. Peripheral blood lymphocytes gated on CD19-positive B cells were used as normal nonmalignant controls. Cells were stimulated by different stimulants and cytokines (PMA, anisomycin, IL-4, IL-6, IL-7, IL-10 and IFN-α) and activation of various phosphoepitopes (pERK, pp38, pJNK, pStat1, pStat3, pStat5, pStat6) was analyzed and compared to the basal state of unstimulated samples. Signaling profiles of normal B-lymphocytes were compared to those of the BCPALL cell lines as well as to the BCP-ALL xenograft samples. Significance of differences was assessed by the nonparametric Mann-Whitney U-test. Basal phosphorylation was significantly higher in the leukemia cell lines than in normal lymphocytes. Similarly, basal phosphorylation of all analyzed epitopes in xenografts exceeded the phosphorylation state of normal B-lymphocytes (with the exception of p38 phosphorylation, where there was no significant difference). Interestingly, the BCP-leukemia cell lines also had significantly higher basal phosphorylation levels than the primary BCP-ALL xenografts. However, when comparing the amounts of phosphorylation before and after stimulation mature normal B-cells displayed significantly higher profiles compared to the leukemia cell lines e.g. for pp38 and pJNK after stimulation with PMA (P= .001), for pStat3 after stimulation with IL-6 (P= .002) and IL-10 (P= .037) and for pStat6 (P= .001) after stimulation with IL-4. Conversely, the leukemia cell lines showed increased phosphorylation of p38 after stimulation with anisomycin (P= .021) as well as higher Stat5 phosphorylation after stimulation with IL-7 (P= .021) compared to normal lymphocytes. In normal B-cells compared to xenografts higher levels were found after stimulation with PMA for pp38 (P= .007), for pJNK after PMA stimulation (P= .001), for pStat3 after IL-6 (P=.003) and for pStat6 after IL-4 (P= .002) stimulation while the xenograft samples displayed stronger reaction to stimulation with anisomycin for pp38 (P= .037) and to stimulation with IL-7 for pStat5 (P= .028). The level of phosphorylation after treatment with different stimulants in the xenografted leukemia samples was similar to that of the leukemia cell lines although the cell lines displayed higher basal phosphorylation values. The BCP-leukemia cell lines and the BCP xenograft samples both displayed high levels of constitutive phosphorylation in general reducing their ability to react to a given stimulus compared to normal B-lymphocytes. With the most important exception of Stat5: we consistently found that Stat5 phosphorylation is increased in acute lymphoblastic leukemia cell lines and primary xenografts after stimulation with IL-7 compared to normal B-lymphocytes. Stat5 is known to enhance proliferation and protect from apoptosis and our data now strongly suggest that Stat5 and Stat5 dependent pathways are critically involved in leukemogenesis. Since we could identify significant and specific phosphorylation signatures characteristic for leukemia cells, this provides a strategy to define pathways important for continued survival, proliferation and resistance of leukemia and allows identification of therapeutic targets and novel biomarkers associated with clinical outcome.

Functional Modulation of VLA6 in BCR-ABL1+ Pre-B Acute Lymphoblastic Leukemia.

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 2565-2565
Author(s):  
Eun Ji Gang ◽  
Yao-Te Hsieh ◽  
Huimin Geng ◽  
Jennifer Pham ◽  
Markus Muschen ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Flow Cytometry ◽  
Drug Resistance ◽  
Acute Lymphoblastic Leukemia ◽  
Lymphoblastic Leukemia ◽  
Conditional Knockout ◽  
Leukemia Cells

Abstract Abstract 2565 Chemotherapy drug resistance in acute lymphoblastic leukemia (ALL) remains a major problem, resulting in reduced treatment efficacy and relapse. The bone marrow environment (BME) has been shown to promote resistance of leukemia cells towards chemotherapy, which has been attributed to several proteins, including integrins. Our analysis of 207 children with high-risk (BCR/ABL1−) pre-B ALL revealed that high expression of the laminin-binding integrin VLA6 (alpha6beta1) portends poor clinical outcomes in patients with minimal residual disease (MRD+) on day 29 of induction. In addition, our comparative analysis of pre-B leukemia and normal B-cells revealed that VLA6 is preferentially upregulated on BCR/ABL1+ pre-B ALL blasts. Alterations in adhesion properties have been described for BCR/ABL1+ (p210) chronic myeloid leukemia. The role of integrins and integrin VLA6 in particular for cell adhesion-mediated drug resistance (CAM-DR) in BCR/ABL1+ (p210) ALL has not been addressed. With respect to its role for normal immature hematopoietic cells, contradictory observations have been reported. Therefore, we hypothesized that VLA6-mediated adhesion of ALL cells to the bone marrow stromal niche contributes to drug resistance. We evaluated the role of VLA6 in BCR-ABL1+ leukemia using two of our established models of leukemia, a conditional knockout model of VLA6 in murine BCR-ABL1+ leukemia and a xenograft model of human BCR-ABL1+ leukemia. VLA6fl/fl cells were oncogenically transformed using BCR-ABL1 (p210) and cultured under lymphoid-skewing conditions. Induction of pre- B (B220+ CD19+) ALL was confirmed by flow cytometry. Subsequent transduction with CreERT2 or EmptyERT2 generated leukemia cells in which VLA6 ablation could be induced (CreERT2) or not (EmptyERT2) by addition of Tamoxifen. Conditional ablation of VLA6 in vitro decreased adhesion significantly compared to undeleted controls (19.7%±8.1% vs. 87.7%±6.0%; p=0.00041) and increased apoptosis of murine BCR-ABL1+ leukemia cells as determined by analysis of Annexin V−/7-AAD− viable cells by flow cytometry (VLA6 deleted vs. undeleted: 35.3%±1.1% vs. 75.1%±1.2%; p=0.0001). Moreover, VLA6 deletion sensitized murine ALL to a tyrosine kinase inhibitor (TKI), Nilotinib (p=0.022, 45.6%±2.4% vs. 73.3%±13.0%). To test the effect of VLA6 deletion on leukemic progression in vivo, VLA6 BCR/ABL1+ pre-B (B220+ CD19+) CreERT2+ or control transduced ALL cells were transferred into NOD/SCID mice. 3 days thereafter, VLA6 deletion was induced by Tamoxifen administration to all animals in 2 cycles for 5 days. In vivo deletion of VLA6 in delayed leukemia progression compared to VLA6 competent controls from a median survival time (MST) of 30 days post-leukemia injection to a MST of 43 days post-leukemia injection (p=0.008 Log-rank test). In vivo deletion of VLA6 in combination with Nilotinib treatment delayed leukemia progression compared to VLA6 competent, as Nilotinib-treated control animals have uniformly died of leukemia with a MST of 39.5 days, however the Nilotinib treated VLA6 deleted group is completely alive and is still being monitored (p=0.0025). When VLA6 was ablated before transfer and recipients were observed for leukemia progression, the recipients of VLA6–deficient murine leukemia cells also showed attenuated leukemia progression compared to recipients of VLA6 competent cells. Moreover, we show that VLA6 blockade de-adheres primary ALL cells from their cognate counter receptor laminin in vitro, and sensitizes primary ALL cells to TKI Taken together, modulating the function of VLA6 in ALL offers a new approach to overcome drug resistance in ALL. Given that VLA6 is probably largely redundant for normal immature hematopoiesis, this approach may be preferable over targeting of other integrins in BCR/ABL1+ leukemias on which VLA6 is expressed. Disclosures: No relevant conflicts of interest to declare.

Myosin-IIa Is Required for Leukemia Cell Extravasation and Its Inhibition Reduces Leukemia Dissemination and Prolongs Survival in a Mouse Model of Acute Lymphoblastic Leukemia

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 967-967
Author(s):  
Jordan Jacobelli ◽  
Eric Wigton ◽  
Scott B Thompson
Keyword(s):  
Cell Migration ◽  
Acute Lymphoblastic Leukemia ◽  
Mouse Model ◽  
Lymphoblastic Leukemia ◽  
Leukemia Cell ◽  
Leukemia Cells ◽  
Transfer Model ◽  
Endothelial Migration ◽  
Myosin Iia

Abstract Background: Leukemia affects approximately 45,000 people each year in the USA with more than 20,000 fatalities. Many leukemia patients experience initial remission but often relapse, with the relapsing leukemia affecting organs such as the central nervous system (CNS). The CNS acts as a sanctuary site allowing leukemia cells to escape treatments such as tyrosine kinase inhibitors (TKIs) and chemotherapy. Leukemia dissemination is a complex process requiring leukemia cells to exit the blood circulation by extravasation and invade target tissues. To extravasate, leukemia cells cross through vascular endothelial walls in a process called trans-endothelial migration, which requires cytoskeletal remodeling. However, the specific cytoskeletal effectors of leukemia extravasation are not fully known. Notably, leukemia dissemination correlates negatively with survival rates. Goal: Our goal was to validate Myosin-IIA, a class II myosin motor protein, as a molecular target to inhibit lymphoid leukemia cell extravasation and hinder leukemia dissemination, particularly infiltration into the CNS. Myosin-IIA has been shown to play a role in cell migration. We recently showed that this myosin is more profoundly required for activated lymphocyte entry into the CNS than for homeostatic entry into secondary lymphoid organs. This suggests that Myosin-IIA may be a promising candidate to prevent leukemia infiltration into the CNS without completely inhibiting homeostatic lymphocyte trafficking. Results: For this study, we used a mouse model of Bcr-Abl driven B cell acute lymphoblastic leukemia (B-ALL), which closely recapitulates Philadelphia chromosome positive human leukemias. We used shRNA interference to inhibit Myosin-IIA protein expression in the leukemia cells. Myosin-IIA depletion did not affect baseline apoptosis of the leukemia cells but did result in a small but significant reduction in their growth rate. Myosin-IIA was key in promoting leukemia cell migration in response to the chemokine CXCL12. Expression of Myosin-IIA was also critical for leukemia cells to complete trans-endothelial migration through brain-derived endothelial cells in an in vitro model of extravasation. In addition, our data suggested that inhibition of Myosin-IIA reduces the ability of leukemia cells to disseminate in vivo, including reducing leukemia infiltration into the CNS. Finally, compared to control leukemia cells, inhibition of Myosin-IIA significantly prolonged survival in an in vivo leukemia transfer model. Conclusion: Our data suggest that Myosin-IIA regulates leukemia migration thus making this myosin a promising target to inhibit leukemia dissemination in vivo, particularly into the CNS. Given the serious side-effects of cranial irradiation and (intrathecal or systemic) chemotherapy, the ability to inhibit CNS infiltration of leukemia cells can be a valuable therapeutic tool to improve the efficacy of current therapies by reducing the relapse frequency following TKI and chemotherapy treatments. Disclosures No relevant conflicts of interest to declare.

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