scholarly journals ASPP2 k, a Dominant-Negative Splicing Variant of the Apoptosis-Stimulating Protein of p53-2 (ASPP2), Modulates Treatment Response Towards BCL-Signaling Inhibitors in Acute Myeloid Leukemia

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 3343-3343
Author(s):  
Marcus M. Schittenhelm ◽  
Vasileia Tsintari ◽  
Rebecca Fröhlich ◽  
Kerstin Maria Kampa-Schittenhelm
Keyword(s):  
Bone Marrow ◽  
Drug Resistance ◽  
Clinical Investigation ◽  
Annexin V ◽  
High Sensitivity ◽  
Advisory Board ◽  
Dominant Negative ◽  
Leukemia Cells ◽  
Predictive Tool ◽  
Splicing Variant

Abstract In recent years, it has become increasingly apparent that BCL-2 inhibition in AML is a clinically highly effective approach - and the first BCL-2 inhibitor, venetoclax, has gained FDA and EMA approval. However, drug resistance frequently occurs and mechanisms or biological markers to predict response to BCL-2 inhibition are urgently needed. ASPP2 plays a central role to orchestrate induction of apoptosis via binding of p53 - but also via binding of antiapoptotic BCL-2, allowing release of proapoptotic proteins. We recently described an alternatively spliced oncogenic ASPP2 isoform (ASPP2k) (Schittenhelm et al., 2019), which is characterized by truncation of the C-terminus allocating the BCL2- as well as the p53 binding sites (in analogy to major TP53 mutations lacking the ASPP2 binding motifs). We therefore hypothesized that expression of ASPP2k attenuates efficacy of pro-apoptotic compounds: AML cell line models, including MOLM13/14 (FLT3 ITD+), OCI-AML3 (NPM1 A+) and HL60, as well as freshly isolated native leukemia blasts (n=40) are used in dose-dilution assays to assess for pro-apoptotic efficacy in annexin V-based assays. Bone marrow donors served as a control population. A lentiviral approach was used for isoform-specific ASPP2k-shRNA transduction. A HisMax vector was used to forcely express ASPP2k. Several compounds targeting BCL-2 signaling, which are under clinical investigation, were tested (BCL-2: venetoclax, BCL-2/Xl: AZD4320, MCL-1: AZD5991 and CDK9: AZD4573). To summarize, leukemia cells demonstrated variable and preferential sensitivity profiles towards the tested compounds: MOLM cells were sensitive towards all tested compounds. In contrast, OCI-AML3 proofed reduced sensitivity towards BCL-2/Xl inhibition - whereas MCL-1 and CDK9 inhibition (indirectly targeting MCL-1 signaling) showed IC50s in the nanomolar range (AZD5991: 307nM, AZD4573: 16nM). Furthermore, HL60 were relatively resistant towards both BCL-2/Xl and MCL-1 inhibition - however remained high sensitivity towards CDK9 inhibition (IC50 25nM). Priming with a hypomethylating agent (HMA, decitabine) resulted in additive (CI close to 1) to synergistic (CI 0.25 - 0.7) proapoptotic effects in isobologram analysis and led to a release of drug resistance in primary resistant cell lines. Exposure of native leukemia cells towards all inhibitors confirmed (individually differing) sensitivity in the nanomolar ranges - whereas bone marrow donor controls were relatively resistant towards the tested compounds, which argues for a therapeutic clinical window. In a last step, we addressed, whether ASPP2k functionally impedes the proapoptotic efficacy observed for the tested compounds. Indeed, we demonstrate that isoform-specific ASPP2k interference similarly results in a significant increase of pro-apoptotic capacities of all tested BCL-2, BCL-2/XL, MCL-1 and CDK9 inhibitors, (resp. attenuation thereof after forcely expressing the dominant-negative splicing variant). To summarize, we show that inhibition of BCL-2 signaling is a promising approach to target acute leukemia - as a monoagent as well as in combination with HMA: Further, we provide a path for exploration of ASPP2k as a predictive tool as well as a therapeutic sensitizer of pro-apoptotic drugs, which will be addressed in future studies. Disclosures Schittenhelm: BMS: Other: advisory board; Astellas: Other: advisory board; Takeda: Other: advisory board; University of Tuebingen: Patents & Royalties: patent for ASPP2k. Kampa-Schittenhelm: University of Tuebingen: Patents & Royalties: patent related to ASPP2k.

Thymic Stromal Lymphopoeitin Signaling Contributes To Increased Risk Of Relapse Of Pediatric Acute Lympoblastic Leukemia Through Enhanced Survival Of Leukemic Cells That Overexpress Thymic Stromal Lymphopoeitin Receptor

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 2543-2543
Author(s):  
Christopher D Chien ◽  
Matthew Kreitman ◽  
Haiying Qin ◽  
Terry J Fry
Keyword(s):  
Bone Marrow ◽  
Annexin V ◽  
Thymic Stromal Lymphopoietin ◽  
Leukemic Cells ◽  
Leukemia Cells ◽  
Early Stages ◽  
Increased Risk ◽  
Significant Difference ◽  
Risk Of Relapse

Abstract Pediatric acute lymphoblastic leukemia (ALL) is the most common childhood malignancy. Although the cure rate for this disease is greater than 85%, ALL remains the number one cause of cancer-related deaths in children due to relapse of ALL. Therefore, there is a great need to identify new therapies for patients who have recurrent disease. Recently, a subset of pediatric ALL patients whose leukemic cells express high levels of thymic stromal lymphopoietin receptor (TSLPR/CRLF2) have been shown to have an increased risk of relapse and shorter disease free and poorer overall survival. Overexpression of TLSPR occurs in 8% of unscreened pediatric precursor B ALL and occurs by genomic rearrangement of the TSLPR gene, which fuses the unmutated TSLPR gene to altered transcriptional control or by other yet to be described means. The mechanism by which Thymic Stromal Lymphopoietin (TLSP) signaling contributes to increased risk of relapse is unknown. Studies have shown aberrant signaling in high TSLPR expressing ALL patient derived cell lines relying heavily on in vitro experiments. As no pre-clinical model of high TSLPR ALL has been published, we created a model of high TSLPR expressing leukemia to study TSLPR overexpressing leukemia progression. We have created a high TSLPR expressing leukemia cell line through retroviral transduction of a transplantable syngeneic mouse leukemia model in which the leukemic progression can be studied with physiologic levels of TSLP. This high TSLPR leukemia has levels of expression of TSLPR comparable to what is found on human leukemia that overexpress TSLPR. The TSLPR is functional in these cells and we see increased phosphorylation of STAT5 protein in response to IL-7 or TSLP stimulation. When we introduce the leukemia into mice and look at disease progression, we observed an 8 fold difference in the numbers of cells in the bone marrow 5 days after intravenous injection corresponding to an early stage of leukemia progression (Figure 1. high TSLPR 1.61%+/-0.95 vs. low TSLPR 0.20%+/-0.16). Interestingly we find no significant difference in long term survival of mice injected with either low or high TSLPR leukemia lines. The increased numbers of leukemic cells in the bone marrow at early stages of leukemic progression could be due to an increased rate of proliferation or better survival/engraftment. Low and high TSLPR expressing cells show no significant difference in growth rate in vitro or in vivo in dye dilution assays (Figure 2) suggesting that the increase in leukemic cells in the bone marrow is through enhanced survival. To test this, we treated low and high TSLPR leukemia lines with the steroid dexamethasone in the absence or presence of TSLP. We found that the addition of TSLP significantly reduced the Annexin V positive relative to cells not treated with TSLP in the high TSLP expressing leukemia cells, while in low TSLPR expressing cells we observed no decrease in Annexin V positive cells (Figure 3). This suggests that high TSLPR expression sensitizes leukemia cells to TSLP in the leukemia microenvironment. To confirm that this is the case we have found by gene expression analysis that we can detect TSLP in mouse bone marrow. We hypothesize that therapies targeting the TSLP signaling axis in ALL would decrease the risk of relapse. To test this hypothesis we have generated TSLPR-Fc conjugates to block TSLP signaling. We plan on using these reagents to block TSLP signaling to see if we can reverse the increased amounts of leukemia we find in mice at early stages of leukemic progression as well as the eliminate the survival advantage provided by TSLP to high TSLPR expressing leukemic cells in response to chemotherapeutic agents. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Expression of MPL in Leukemia Stem Cells and Its Role in Stemness Maintainance

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 1723-1723 ◽  
Author(s):  
Huan Li ◽  
Qing Rao ◽  
Pei Yu ◽  
Shuying Chen ◽  
Zheng Li ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Flow Cytometry ◽  
Drug Resistance ◽  
Colony Formation ◽  
Leukemia Cells ◽  
Leukemia Stem Cells ◽  
Self Renewal ◽  
Cd34 Cells ◽  
Clonogenic Potential

Abstract Osteoblast cells play an important role in bone marrow niche. The interaction between osteoblast and leukemia cells promotes leukemia development, which is mediated by some cytokines including TPO. It has become evident that TPO-MPL signaling is essential for the quiescence and self-renewal of hematopoietic stem cells, however, its expression pattern and the role in leukemia stem cells have not been reported. This study was aimed to determine the expression of MPL in acute myeloid leukemia (AML) and investigate the role of MPL in the leukemia stem cells' quiescence, drug resistance and self renewal. The expression levels of CD34, CD38 and MPL were detected by flow cytometry in bone marrow cells from 57 newly diagnoses AML patients. The correlation between MPL and CD34, CD38 expression in AML patients were analyzed. The results showed that expression of MPL in AML patients was higher significantly than that in 13 normal donors (P<0.05). Expression of MPL in CD34 positive AML patients was obviously higher than that in CD34 negative AML patients (P<0.01). MPL was higher expressed in CD34+ cells than that in CD34- cells significantly (P<0.0001). We also detected the expression of MPL in different populations of leukemia cells in AML1-ETO9a mouse leukemia model established in our lab. We found that the ratios of MPL positive cells in Lin-c-kit+ and Lin-c-kit+sca-1+ populations were significantly higher than that in total leukemia cells. In addition, in chemotherapy treated AML1-ETO9a mice, the proportion of Lin-c-kit+MPL+ leukemia cells were increased 23.5 folds than that in untreated leukemia mice, which indicates that MPL+Lin-c-kit+ LSCs population could be enriched by chemotherapy. Furthermore, MPL+ and MPL- cells in Lin-c-kit+ leukemia population were sorted by flow cytometry and the colony formation and quiescence state were determined. The results showed that MPL+Lin-c-kit+ cells produced significantly more colonies in the second round of colony formation (p<0.05) than MPL-Lin-c-kit+ cells. The G0 phase accumulation of MPL+Lin-c-kit+ cells was significantly higher than that of MPL-Lin-c-kit+ cells (p<0.01). Above results indicate that MPL+ leukemia cells display more clonogenic potential and maintain quiescence. These data demonstrate that as a receptor of TPO, MPL is highly expressed in leukemia stem cells and MPL positive leukemia stem cells could be enriched by chemotherapy. MPL positive leukemia stem cells exhibit more clonogenic potential, quiescence and drug resistance. It suggests TPO-MPL mediated interaction of osteoblast and leukemia cells take a role in the stemness of leukemia stem cells. Disclosures No relevant conflicts of interest to declare.

BRD4 Proteolysis Targeting Chimera (PROTAC) Leads to Sustained Degradation of BRD4 with Broad Activity Against Acute Leukemias and Overcomes Stroma Mediated Resistance By Modulating Surface Expression of CXCR4

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 675-675
Author(s):  
Sujan Piya ◽  
Teresa McQueen ◽  
Duncan Mak ◽  
Marina Konopleva ◽  
Patrick Zweidler-McKay ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Drug Resistance ◽  
Trypan Blue ◽  
Research Funding ◽  
Annexin V ◽  
Blue Staining ◽  
Employment Equity ◽  
Acute Leukemias ◽  
Lower Chamber ◽  
Equity Ownership

Abstract Background: Hematological neoplasms are characterized by aberrant epigenetic events that modify the chromatin regulatory machinery to enhance oncogene expression (Zuber et al. Nature 2011). BRD4, a member of the bromodomain and extra terminal domain (BET) family, is critical in the assembly of a 'super enhancer complex' that drives expression of oncogenes MYC, CCND1, SOX2 and NF-kB and anti-apoptotic proteins e.g. Bcl-2 and BCL-XL (Klapproth et al. Br J Haematol 2010; Wang et al. Cancer Res 2014; Zou et al. Oncogene 2014). Small molecule BRD4 inhibitors lead to rapid accumulation of BRD4 that may partially account for their moderate suppression of MYC (Lu et al. Chem Biol 2015). ARV-825 is hetero-bifunctional PROTAC (Proteolysis Targeting Chimera) that recruits BRD4 to the E3 ubiquitin ligase cereblon, leading to fast, efficient, and prolonged degradation of BRD4 and sustained down-regulation of MYC (Lu et al. Chem Biol 2015). Aim: We investigated the activity of ARV-825 against leukemia cell lines representing acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL) including gamma secretase inhibitor (GSI) resistant T-ALL and against primary AML blasts and compared it to that of the BRD4 inhibitor, JQ1. As stroma renders leukemia resistant, we tested if and through what mechanism ARV-825 can overcome stroma mediated drug resistance. Results: The IC50s for all tested cell lines at 72 hours were in very low nanomolar range; AML-OCIAML3-8.3 nM, HL60-2.4nM; ALL-KOPT-K1/SUPTI-1.3nM (GSI resistant T-ALL), LOUCY-2.3nM (early T-cell phenotype ALL), MOLT4-3.12nM and HPB-ALL-4.6nM (T- ALL) while the same for JQ1 was 102.6 nM, 5 nM, 1.5nM/8.8 nM, 4.5nM, 9.4 and 8.4 nM respectively. Consistent with sustained degradation of BRD4, the apoptotic effect of ARV-825 is sustained longer than that of JQ1 after the removal of compounds following a 24-hr treatment at IC50 (Annexin V positive 70% vs. 4% at 48 hrs post-washout respectively) (Fig. 1). Importantly, IC50 values for primary AML samples for ARV-825 were approximately 6-100 times lower than those for JQ1 (Fig. 2). ARV-825 led to sustained decreases of BRD4 and MYC proteins with corresponding increase in cleaved PARP, cleaved Caspase-3 and γH2AX in MOLT4 and OCIAML3 cells. While Bcl-2 level remained suppressed with ARV-825, it recovered at 48 hrs of treatment with JQ1. We co-cultured OCI-AML3 cells with normal bone marrow derived mesenchymal stromal cells (MSCs) to mimic the bone marrow microenvironment. While MSC protected OCI-AML3 cells from cytarabine (25 % reduction in cell death), there was no stromal protection for ARV-825. To gain mechanistic insight, we used a Mass cytometry (CyTOF) based approach to analyze the changes in apoptotic, cell adhesion and signaling proteins (panel of 24 antibodies) in OCI-AML3 cells after 12 and 24-hour treatment with ARV-825. MYC and surface CXCR4 were the two most down-regulated proteins. The reduction of surface CXCR4 was confirmed by conventional flow cytometry (Fig. 3A) and the functional relevance was confirmed in migration assays against the CXCR4 ligand SDF-1 (29.2% Control vs. 9.03% ARV-825) (Fig. 3B). Conclusion: ARV-825, a BRD4 PROTAC, has substantial anti-leukemia activity across a range of acute leukemias and importantly can overcome stroma-mediated drug resistance. ARV-825 promises to be an exciting molecule in the quest towards efficient BRD4 inhibition. Figure 1. OCI-AML3 cells treated for 24 hours, re-suspended without drug, stained for Annexin V at post 48 hours. Primary AML cells treated with ARV-825 or JQ1 and cell viability tested with Cell Titer-Glo 2.0® PROMEGA. (A) OCI-AML3 treated for 24 hrs and stained with CXCR4-APC antibody. (B) OCI-AML3 cells treated for 24 hrs and percentage of cells migrating at 4 hrs to the lower chamber containing recombinant SDF-1 was counted using Vi-Cell (Trypan blue staining assay). Figure 1. OCI-AML3 cells treated for 24 hours, re-suspended without drug, stained for Annexin V at post 48 hours. / Primary AML cells treated with ARV-825 or JQ1 and cell viability tested with Cell Titer-Glo 2.0® PROMEGA. / (A) OCI-AML3 treated for 24 hrs and stained with CXCR4-APC antibody. (B) OCI-AML3 cells treated for 24 hrs and percentage of cells migrating at 4 hrs to the lower chamber containing recombinant SDF-1 was counted using Vi-Cell (Trypan blue staining assay). Disclosures Konopleva: Novartis: Research Funding; AbbVie: Research Funding; Stemline: Research Funding; Calithera: Research Funding; Threshold: Research Funding. Lu:Arvinas, LLC: Employment, Equity Ownership. Qian:Arvinas, LLC: Employment, Equity Ownership.

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.

Combining ABL1 Kinase Inhibitor, Imatinib and the Jak Kinase Inhibitor TG101348: A Potential Treatment for Residual BCR-ABL Positive Leukemia Cells

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 3737-3737
Author(s):  
Seiichi Okabe ◽  
Tetsuzo Tauchi ◽  
Seiichiro Katagiri ◽  
Yuko Tanaka ◽  
Kazuma Ohyashiki
Keyword(s):  
Bone Marrow ◽  
Drug Resistance ◽  
Cell Growth ◽  
Growth Inhibition ◽  
Intracellular Signaling ◽  
Kinase Inhibitor ◽  
K562 Cells ◽  
Leukemia Cells ◽  
Dependent Manner ◽  
Cell Growth Inhibition

Abstract Abstract 3737 ABL kinase inhibitor, imatinib is highly effective therapy against chronic myeloid leukemia (CML) patients and eliminates disease progression and transformation. However, imatinib is not curative for most CML patients. Residual CML cells are present in bone marrow microenvironment. Bone marrow microenvironment is a source of soluble factors and regulates the proliferation of leukemia cells. These leukemia cells are contained within a niche in the bone marrow and are often impervious to current treatments, thus maintaining their proliferative activity when the treatment is ceased, suggests that the new therapeutic strategies designed to override stroma-associated drug resistance are required to treat against Philadelphia (Ph)-positive leukemia patients. The hematopoietic cytokine receptor signaling is mediated by tyrosine kinases termed Janus kinases (Jaks) and downstream transcription factors, signal transducers and activators of transcription (STATs). Jak-STAT signaling is also activated in CML cells. One of the Jak kinase inhibitor, TG101348 (SAR302503) is an orally available inhibitor of Jak2 and developed for the treatment of patients with myeloproliferative diseases. Therefore, combination therapy using a BCR-ABL tyrosine kinase inhibitors and a Jak inhibitor, TG101348 may help prevent stroma-associated drug resistance and these approaches may be expected to improve the outcomes of CML patients. In this study, we investigated the ABL tyrosine kinase inhibitor, imatinib and TG101348 efficacy by using the BCR-ABL positive cell lines, K562 and primary CML samples when leukemic cells were protected by the feeder cell lines (HS-5 and S9). 72 hours treatment of imatinib exhibits cell growth inhibition and induced apoptosis against K562 cells in a dose dependent manner. However, the treatment of imatinib exhibits cell growth inhibition partially against K562 cells in the presence of HS-5 conditioned media. We found that the treatment of TG101348 did not exhibit cell growth inhibition against K562 cells directly, but the combination treatment with imatinib and TG101348 abrogated the protective effects of HS-5 conditioned media in K562 cells. We next investigated the intracellular signaling of imatinib and TG101348. Phosphorylation of BCR-ABL, Crk-L was not reduced after TG101348 treatment. However, phosphorylation of BCR-ABL, Crk-L was significantly reduced and increased apoptosis after combination treatment with imatinib and TG101348. We next investigated the efficacy between imatinib and TG101348 by using CD34 positive primary CML samples. The treatment of imatinib exhibits cell growth inhibition partially against CD34 positive CML samples in the presence of feeder cells. Combined treatment of CD34 positive primary samples with imatinib and TG101348 caused significantly more cytotoxicity and induced apoptosis. We also found that mitogen-activated protein kinase (MAPK) was also inhibited by imatinib and TG101348 treatment. We next investigated the intracellular signaling of imatinib and TG101348 by using the CD34 positive primary samples. Phosphorylation of BCR-ABL, Crk-L was significantly reduced and increased apoptosis after treatment with imatinib and TG101348. Moreover, combination of imatinib and TG101348 inhibited the colony growth of Ph-positive primary samples. We also investigated the TG101348 activity against feeder cell. Phosphorylation of STAT5 was reduced by TG101348 in a dose dependent manner. The cytokine production was analyzed by using cytokine array systems. The cytokine production such as granulocyte macrophage colony-stimulating factor (GM-CSF) from HS-5 was also reduced by TG101348 treatment. Data from this study suggested that administration of the imatinib and Jak inhibitor, TG101348 may be a powerful strategy against stroma-associated drug resistance of Ph-positive cells and enhance cytotoxic effects of imatinib in those residual CML cells. Disclosures: No relevant conflicts of interest to declare.

AML Depends on VEGFR1 and Not VEGFR2 for Proliferation and Protection From Chemotherapy,

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 3598-3598
Author(s):  
Swarna Bais ◽  
Elizabeth Wise ◽  
Eric M. Harris ◽  
Amy Meacham ◽  
Christopher R Cogle
Keyword(s):  
Bone Marrow ◽  
Cell Proliferation ◽  
Cell Lines ◽  
Myeloid Leukemia ◽  
Conflicts Of Interest ◽  
Colorimetric Assay ◽  
Leukemia Cell ◽  
Annexin V ◽  
Leukemia Cells ◽  
Nsg Mice

Abstract Abstract 3598 Background: Vascular endothelial growth factor (VEGF) and its cognate receptors (VEGFRs) are known to serve important roles in normal hematopoiesis, but the importance of the VEGF/VEGFR axis in malignant hematopoiesis, namely acute myeloid leukemia (AML), is poorly defined. Methods: Bone marrow from AML patients were analyzed for VEGFRs on the malignant myeloblast populations. Human myeloid leukemia cell lines, KG-1, HL60 and K562, were also evaluated for VEGFR expression. Knockdown of VEGFR1 and VEGFR2 receptors in AML cells expressing these receptors were performed using lentivirus transfection of shRNA. Cell proliferation was quantified using XTT colorimetric assay and apoptosis was evaluated by Annexin V and PI staining. Results: Bone marrow from 12 consecutive AML patients showed that 33% of patients had malignant myeloblasts that expressed VEGFR1. Of the AML cell lines tested, only KG-1 cells expressed VEGFR1 and VEGFR2. Knockdown of VEGFR1 and VEGFR2 in KG-1 cells were confirmed by Western blot. When VEGFR1 was knocked down in KG-1 leukemia cells, cell proliferation was significant decreased (Figure 1). In addition, knocking down VEGFR1 resulted in enhanced sensitivity to cytarabine chemotherapy (Figure 1). In contrast, knockdown of VEGFR2 did not result in changes in leukemia cell proliferation or sensitivity to cytarabine chemotherapy (Figure 2). When KG-1 leukemia cells knocked down for VEGFR1 expression were transplanted into sublethally, irradiated NOD/scid/IL2R□−/− (NSG) mice (n=10), none of the mice showed engraftment of VEFR1 deficient cells. Conclusions: The VEGF/VEGFR axis is an important determinant in AML pathobiology. In specific, AML cells depend upon VEGFR1 and this receptor represents a promising target for future therapeutic intervention. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Distinct Roles of GATA2 in Development and Evolution of CBFB-MYH11 AML

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 770-770
Author(s):  
Satoshi Saida ◽  
Tao Zhen ◽  
Erika Mijin Kwon ◽  
Guadalupe Lopez ◽  
Paul P Liu
Keyword(s):  
Bone Marrow ◽  
Transcription Factor ◽  
Median Survival ◽  
Bone Marrow Cells ◽  
Annexin V ◽  
Expression Level ◽  
Leukemia Cells ◽  
Rna Seq ◽  
Qrt Pcr ◽  
Gata2 Deficiency

Abstract Introduction. Core binding factor acute myeloid leukemia (CBF-AML) is caused by the dysfunction of a heterodimeric protein complex composed of the transcription factor RUNX1 and its partner CBFb. An inversion of chromosome 16 generates a fusion between CBFB and MYH11. The encoded fusion protein, CBFb-smooth muscle myosin heavy chain (SMMHC), contributes to the pathogenesis of CBF-AML. Our previous reports suggest that CBFB-MYH11 contributes to leukemogenesis by up-regulation of genes such as Gata2, which is an essential hematopoietic transcription factor. On the other hand, we recently identified recurrent monoallelic deletions of GATA2 on chromosome 3 in relapsed CBF-AML patients (Sood et al., Leukemia 30:501-504, 2016). From these findings we propose two hypotheses; 1) up-regulation of GATA2 contributes to leukemogenesis by CBFB-MYH11 in the initiation phase; 2) GATA2 deficiency contributes to the relapse of CBF-AML. Methods. Two datasets (GSE19194 and GSE102388) from microarray and RNA-Seq were used to determine Gata2 expression level in Cbfb-MYH11 preleukemic murine hematopoietic cells. Cbfb-MYH11 conditional knock-in (Cbfb+/56M), Gata2 conditional knockout (Gata2+/f), and Mx1-Cre transgenic mice were crossed to generate Gata2+/fCbfb+/56MMx1-Cre mice. Mice were injected with pIpC to induce the expression of Cbfb-MYH11 and/or knockout of Gata2 through Cre-recombinase activation. For transplantation assays, spleen cells obtained from leukemic mice were injected into irradiated recipient mice through tail vein. For in vitro colony forming assays, colonies were counted after 10 days in culture. Cell apoptosis was determined by Annexin V and 7AAD staining. Results. To test the first hypothesis, we determined the expression level of Gata2 in preleukemic cells in the Cbfb-MYH11 expressing mice. Data from both microarray and RNA-seq experiments revealed that Gata2 was highly expressed in the preleukemic hematopoietic cells of the Cbfb-MYH11 mice, as compared to those of the WT mice, and this finding was confirmed by qRT-PCR. Based on published ChIP-seq data, Gata2 is likely a direct transcriptional target of CBFb-SMMHC. Next, we determined the impact of Gata2 deficiency on leukemogenesis by Cbfb-MYH11. qRT-PCR showed reduced Gata2 expression in bone marrow cells from Gata2+/fCbfb+/56MMx1-Cre mice 12 days after pIpC injection (0.029±0.0092 vs 0.076±0.014; p=0.0089). Colony forming ability was decreased for the pre-leukemic bone marrow cells in Gata2+/fCbfb+/56MMx1-Cre mice when compared to Cbfb+/56MMx1-Cre mice (mean 37.2±6.35 vs. 74.23±8.335; p=0.0002). In addition, the Gata2+/fCbfb+/56MMx1-Cre mice had a smaller abnormal myeloid population in the bone marrow, which is capable of inducing leukemia, when compared with Cbfb+/56MMx1-Cre mice (mean 0.43±0.14% vs. 1.42±0.34%; p=0.0092). Most significantly, Gata2+/fCbfb+/56MMx1-Cre mice developed leukemia with a much longer latency than Cbfb+/56MMx1-Cre mice (median survival 215 days vs 125 days; p=0.0007). To test hypothesis 2, we compared the phenotype of the end stage mice for each genotype. Gata2+/fCbfb+/56MMx1-Cre mice had higher WBC count in peripheral blood than Cbfb+/56MMx1-Cre mice (mean 92,000±20,429 cells/ul vs. 35,644±12,001 cells/ul; p=0.0243), which is a poor prognostic marker in human leukemia. Leukemic cells from Gata2+/fCbfb+/56MMx1-Cre mice also had lower percentage of Annexin V positive cells than Cbfb+/56MMx1-Cre mice in short term culture (31.0±7.1 vs. 68.9±6.5%; p=0.0117). More importantly, upon transplantation, the recipient mice transplanted with Gata2+/fCbfb+/56MMx1-Cre leukemia cells developed leukemia much faster than recipient mice transplanted with equal numbers of Cbfb+/56MMx1-Cre leukemia cells (median survival 35.5 vs. 91.0 days; p<0.0001). Conclusions. Our findings suggest that Gata2 plays important but distinct roles in two different stages of Cbfb-MYH11 leukemia. Reduction of Gata2 activity delays leukemia development in primary Cbfb-MYH11 knockin mice, while contributing to a more aggressive phenotype in leukemic phase as shown in primary leukemic mice and transplanted recipients, which may be correlated with leukemia relapse in human patients. We are analyzing data from whole exome sequencing and RNA-seq to understand the mechanism underlying the observed phenotypes, and the findings will be presented at the annual meeting. Disclosures No relevant conflicts of interest to declare.

Sign in / Sign up

Export Citation Format

Share Document