scholarly journals The Transcription Factor PU.1 Controls a Reversible Differentiation Program in Acute Myeloid Leukemia

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 3930-3930
Author(s):  
Mark D McKenzie ◽  
Margherita Ghisi ◽  
Luisa Cimmino ◽  
Michael Erlichster ◽  
Ethan P Oxley ◽  
...  
Keyword(s):  
Acute Myeloid Leukemia ◽  
Transcription Factor ◽  
Myeloid Leukemia ◽  
Conflicts Of Interest ◽  
Significant Loss ◽  
Polymorphonuclear Neutrophil ◽  
Myeloid Differentiation ◽  
Myeloid Lineage ◽  
Acute Myeloid

Abstract Background: Acute myeloid leukemia (AML) is an aggressive malignancy characterized by clonal expansion of transformed myeloid precursors that fail to differentiate into mature cells. Since myeloid lineage maturation curbs self-renewal and is considered irreversible, engaging this process in AML is an attractive therapeutic strategy. Results: Normal myeloid differentiation requires the transcription factor PU.1 (SPI1), which is functionally compromised in several AML subtypes and is directly inhibited by the recurrent fusion oncoproteins AML1-ETO and PML-RARA. To examine the importance of PU.1 suppression in AML maintenance in vivo, we have combined RNAi-mediated PU.1 inhibition with p53 deficiency to drive highly aggressive AML in mice. Using these models we find that restoring endogenous PU.1 activity in established AML in vivo is sufficient to trigger robust transcriptional, immunophenotypic, and morphological differentiation of leukemic blasts, yielding polymorphonuclear, neutrophil-like cells. Maturation of AML is associated with significant loss of cell viability and yields sustained disease clearance in vivo. Although PU.1 restoration is potently anti-leukemic, remarkably we find that subsequent suppression of PU.1 in mature neutrophil-like cells reverts them to a transformed state within several days. While mature AML-derived cells are slower to form blast colonies in methylcellulose cultures, their clonogenic frequency is only reduced four-fold relative to AML blasts suggesting highly efficient de-differentiation. Conclusions: These results demonstrate that triggering myeloid differentiation can effectively resolve a p53-deficient model of treatment resistant AML, but also identify a previously unrecognised ability of AML cells to bidirectionally transition between transformed and differentiated states based on the activity of a single transcription factor. Our findings challenge the concept of 'terminal differentiation' in AML and highlight the importance of therapeutically eradicating leukemia cells at all stages of myeloid lineage maturation. Disclosures No relevant conflicts of interest to declare.

scholarly journals Transcription Factor MEF2D is Required for the Maintenance of MLL-rearranged Acute Myeloid Leukemia

2021 ◽  
Author(s):  
Lianzhong Zhao ◽  
Pengcheng Zhang ◽  
Phillip M Galbo ◽  
Xinyue Zhou ◽  
Sajesan Aryal ◽  
...  
Keyword(s):  
Acute Myeloid Leukemia ◽  
Transcription Factor ◽  
Myeloid Leukemia ◽  
Critical Role ◽  
Leukemia Cell ◽  
Myeloid Differentiation ◽  
Growth Defect ◽  
Strong Negative Correlation ◽  
Acute Myeloid

Acute myeloid leukemia (AML) with MLL-rearrangement (MLL-r) comprises approximately 10% of all AML cases and portends poor outcomes. Much remains uncovered on how MLL-r AML drives leukemia development while preventing cells from normal myeloid differentiation. Here, we identified that transcription factor MEF2D is a super-enhancer-associated, highly expressed gene in MLL-r AML. Knockout of MEF2D profoundly impaired leukemia growth, induced myeloid differentiation, and delayed oncogenic progression in vivo. Mechanistically, MEF2D loss led to robust activation of a CEBPE-centered myeloid differentiation program in AML cells. Chromatin profiling revealed that MEF2D binds to and suppresses the chromatin accessibility of CEBPE cis-regulatory regions. In human acute leukemia samples, MEF2D expression showed a strong negative correlation with the expression of CEBPE. Depletion of CEBPE partially rescued the cell growth defect and myeloid cell differentiation induced by the loss of MEF2D. Lastly, we show that MEF2D is positively regulated by HOXA9, and downregulation of MEF2D is an important mechanism for DOT1L inhibitor-induced anti-leukemia effects. Collectively, our findings suggest that MEF2D plays a critical role in human MLL-r AML and uncover the MEF2D-CEBPE axis as a crucial transcriptional mechanism regulating leukemia cell self-renewal and differentiation block.

Activity of the Hypoxia-Activated Prodrug, TH-302, in Human Acute Myeloid Leukemia Models

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 3611-3611
Author(s):  
Scott Portwood ◽  
Deepika Lal ◽  
Yung-Chun Hsu ◽  
Rodrigo Vargas ◽  
Meir Wetzler ◽  
...  
Keyword(s):  
Overall Survival ◽  
Acute Myeloid Leukemia ◽  
Myeloid Leukemia ◽  
Conflicts Of Interest ◽  
Flow Cytometric Analysis ◽  
Chemotherapeutic Agents ◽  
Scid Mice ◽  
Hypoxic Conditions ◽  
Acute Myeloid

Abstract Abstract 3611 Recent evidence has demonstrated the bone marrow (BM) microenvironment, the principal site of acute myeloid leukemia (AML) initiation and expansion, is characterized by intrinsically low oxygen tension. Theoretically, such microenvironmental changes may lead to the selective outgrowth of AML clones which are “better adapted” to survive within a severely hypoxic microenvironment and/or may confer resistance to chemotherapeutic agents, similar to solid tumor cells. We report here that human AML cells (HL60, ML-2) cultured under chronic hypoxic conditions mimicking the marrow microenvironment (1% O2, 72 hours) exhibited reduced sensitivity to cytarabine-induced apoptosis as compared with normoxic cells, as determined by flow cytometric analysis, western blot analysis, and cell viability assays. Similar results were noted in primary AML samples treated with cytarabine under normoxic and hypoxic conditions in colony formation assays (n=3 samples, p=0.01). In order to improve upon chemotherapy outcomes, we investigated the effects of TH-302, a hypoxia-activated bromo-isophosphoramidate mustard prodrug, which is currently undergoing clinical trial evaluation in multiple tumor types. Treatment of AML cell lines (HL60, HEL) and primary AML samples with TH-302 (at doses ranging from 0.1– 5 mM, p values ranging from <0.05–0.0001) resulted in dose- and hypoxic-dependent inhibition of AML proliferation and apoptosis. In vivo TH-302 treatment significantly decreased disease burden, as measured by total animal bioluminescence, and prolonged overall survival in two systemic human AML xenograft models (HEL-luciferase, HL60-luciferase) (Figure 1). Immunohistochemical studies demonstrated that TH-302 treatment reduced numbers of hypoxic (pimonidazole-positive) cells within the leukemic marrow microenvironment. Because prior data in animal models has shown that AML progression within the marrow is associated with expansion of hypoxic BM areas, we examined the effects of TH-302 treatment on systemic AML growth when initiated early (prior to AML inoculation) or late (several days following AML engraftment) in the disease process. TH-302 was equally effective at both time points. Although anti-vascular therapy has been shown to enhance tumor hypoxia in other cancer types, we noted no synergistic or additive in vivo effects when TH-302 therapy was combined with sorafenib, an inhibitor of vascular endothelial growth factor receptors (VEGFR), in our models. TH-302 therapy administered for two weeks in non-leukemic and leukemia-engrafted mice was not associated with hematologic toxicities. In summary, our results demonstrate the anti-leukemic activity of TH-302 in preclinical AML models and suggest that the efficacy of this and other drugs for AML therapy may be uniquely affected by the BM microenvironment. Further clinical development of TH-302 and other hypoxia-targeted drugs for AML therapy are warranted. Based on our data, higher TH-302 doses and/or chronic drug administration may be needed for optimal in vivo anti-leukemic activity. Figure 1. Effects of TH-302 treatment on systemic AML growth and overall survival in HL60-luciferase engrafted SCID mice. Figure 1. Effects of TH-302 treatment on systemic AML growth and overall survival in HL60-luciferase engrafted SCID mice. Disclosures: No relevant conflicts of interest to declare.

CXCR4 Invalidation Limits the MLL-ENL Induced Leucemogenesis in Vivo

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 4089-4089
Author(s):  
Yanyan Zhang ◽  
Hadjer Abdelouahab ◽  
Aline Betems ◽  
Monika Wittner ◽  
William Vainchenker ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Acute Myeloid Leukemia ◽  
Survival Time ◽  
Median Survival ◽  
Median Survival Time ◽  
Myeloid Leukemia ◽  
Conflicts Of Interest ◽  
Hematopoietic Stem ◽  
Acute Myeloid

Abstract Abstract 4089 The receptor CXCR4 and its ligand SDF-1 play major physiological roles especially on hematopoietic stem cells homing and retention. Many studies have implicated CXCR4 in the invasion by tumor cells of organs that produce SDF-1. In acute myeloid leukemia, the physiological role of CXCR4 is not fully understood. We used retrovirus to express MLL-ENL oncogene in CXCR4+/+ and CXCR4−/− hematopoietic primitive cells (Lin- isolated from fetal liver) and showed that CXCR4 is dispensable for generation of immortalized colonies in vitro. To determine CXCR4 function in vivo, CXCR4+/+ and CXCR4−/− transformed cells were transplanted into lethally irradiated mice. Whatever their phenotype, the recipient developed a myelo-monocytique leukemia characterized by their expression of Gr-1 and Mac-1. As expected, all recipients of MLL-ENL transduced CXCR4+/+ cells were moribund within 35 to 80 days post transplant (median survival time: 50 days). Strikingly, recipients of MLL-ENL transduced CXCR4−/− cells showed significantly increased lifespan, with a median survival time of 90 days. The cellularity of the peripheral blood of recipients of MLL-ENL transduced cells displayed considerable increases over time although this increase was much lower in CXCR4−/− than in CXCR4+/+ chimera. Bone marrow of MLL-ENL transduced CXCR4−/− chimera had moderately decreased numbers of mononuclear cells. There were important numbers of leukemic CD45.2+/Gr1+/Mac1+/c-kit+ cells in spleen from MLL-ENL CXCR4+/+ chimera which suggested that CXCR4 is important for leukemic progenitors cells retention in the bone marrow and especially in the spleen. The homing capacity of transduced CXCR4+/+ cells is comparable to the CXCR4−/− cells. Finally, more DNA damages were found in the BM cells of MLL-ENL CXCR4−/− chimera. All these results were confirmed by treating of MLL-ENL CXCR4+/+ chimera with CXCR4 inhibitor (TN140). These results demonstrated that in absence of CXCR4, the cells transduced by oncogene MLL-ENL are capable of generating leukemia in the recipients. However, mice transplanted with MLL-ENL transduced CXCR4−/− FL cells developed acute myeloid leukemia with reduced aggressiveness and organ infiltration, which is associated with induced differentiation and DNA instability. These results indicated that the MLL-ENL progenitors are dependent on CXCR4 for their maintenance in the BM and spleen suggesting that CXCR4 inhibitors might have potential therapeutic applications. Disclosures: No relevant conflicts of interest to declare.

The Mechanisms of Synergistically Cytotoxicity Induced by Homoharringtonine and Aclarubicin in Acute Myeloid Leukemia Cells

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 4313-4313
Author(s):  
Lei Wang ◽  
Jie Jin
Keyword(s):  
Acute Myeloid Leukemia ◽  
Myeloid Leukemia ◽  
Conflicts Of Interest ◽  
Akt Pathway ◽  
Apparent Difference ◽  
Akt Inhibitor ◽  
Simultaneous Exposure ◽  
Acute Myeloid

Abstract Abstract 4313 Previous studies showed HAA regime [HHT (homoharringtonine), cytarabine and ACR (aclarubicin)] resulted in a high complete remission (CR) rate and a better overall survival (OS) rate in patients with primary acute myeloid leukemia. To confirm if a synergistically cytotoxicity was found in AML cells, we investigated the antitumor effect relationship of HHT and ACR against AML cells. Using in vitro system, we demonstrated that simultaneous exposure to HHT and ACR resulted in strong synergistic anti-proliferative effect and apoptosis inducing in AML cells. In vivo, combination of HHT and ACR may be result in a favorable survival in AML xenograft mice. The assay of microarray gene expressing profiling highlighted apparent difference in expression of PI3K gene and WNT3a gene between cells treated by HHT and cells exposure to ACR. Furthermore, decreased expression of PI3K110 and P-AKT protein were observed in AML cells treated with HHT for 3h while no significant change in the expression of two proteins was observed in 90nM of ACR-treated cells. Western Blot analysis also showed ACR could obviously inhibit WNT3a and β-catenin protein levels in AML cells after 3 hours exposure. Although HHT could not inhibit WNT3a protein, it also could apparently down-regulate expression of β-catenin in AML cells. Simultaneous decrease of PI3K signal and WNT3a signal was induced by the combination of HHT and ACR in AML cell lines and primary AML cells. To explore possible targets of synergistically cytotoxity induced by combined HHT/ACR, we silenced wnt3a expression by RNA interference. Then we found suppression of wnt3a expression could enhance the cytotoxity of HHT and AKT inhibitor. Moreover, combining ACR with AKT inhibitor resulted in a synergistically cytotoxic effect too. β-catenin is a shared molecular in both AKT pathway and WNT pathway. Up-regulating of β-catenin expression failed to reduce cell apoptosis induced by HHT plus ACR while partially decrease the growth inhibition rate caused by combining treatment. β-catenin is required for the self-renewal of AML-LSC. Our study also suggests that combining HHT and ACR may synergistically induce apoptosis in LSC-enriched cells. These results indicate that simultaneously inhibiting activity of PI3K/AKT pathway and WNT/β-catenin pathway is a possible mechanism of synergistically cytotoxity induced combinated HHT/ACR in AML cells. Disclosures: No relevant conflicts of interest to declare.

The BTK Inhibitor Ibrutinib Blocks SDF1/CXCR4 Mediated Migration of Acute Myeloid Leukemia Cells

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 915-915
Author(s):  
Stuart A Rushworth ◽  
Lyubov Zaitseva ◽  
Megan Y Murray ◽  
Matthew J Lawes ◽  
David J MacEwan ◽  
...  
Keyword(s):  
Acute Myeloid Leukemia ◽  
Cell Lines ◽  
Myeloid Leukemia ◽  
Conflicts Of Interest ◽  
Lymphocytic Leukemia ◽  
Cell Trafficking ◽  
Acute Myeloid

Abstract Introduction Despite recent significant progress in the understanding of the biology of acute myeloid leukemia (AML) the clinical outcomes for the majority of patients diagnosed with AML presently remain poor. Consequently, there is an urgent need to identify pharmacological strategies in AML, which are not only effective but can be tolerated by the older, less well patient. Recently our group and others have shown that there is high Bruton’s Tyrosine Kinase (BTK) phosphorylation and RNA expression in AML. Moreover, our recent study described for the first time that ibrutinib and BTK-targeted RNA interference reduced factor-induced proliferation of both AML cell lines and primary AML blasts, as well as reducing AML blast adhesion to bone marrow stromal cells. Inhibition of BTK has been shown to regulate chronic lymphocytic leukemia, mantle cell lymphoma and multiple myeloma cell migration by inhibiting SDF1 (stromal derived factor 1) induced CXCR4 regulated cell trafficking. Here we report that in human AML ibrutinib in addition functions in a similar way to inhibit SDF1/CXCR4-mediated AML migration at concentrations achievable in vivo. Methods To investigate the role of BTK in regulating AML migration we used both pharmacological inhibitor ibrutinib and genetic knockdown using a lentivirus mediated BTK targeted miRNA in primary AML blasts and AML cell lines. We examined migration of AML blasts and AML cells to SDF-1 using Transwell permeable plates with 8.0µM pores. Western blotting was used to examine the role of SDF-1 in regulating BTK, AKT and MAPK activation in primary AML blasts. Results We initially examined the expression of CXCR4 in human AML cell lines and found that 4/4 cell lines were positive for CXCR4 expression. Next we examined the effects of ibrutinib on the migration of the AML cell lines U937, MV4-11, HL60 and THP-1 in response to SDF1. We found that ibrutinib can inhibit the migration of all AML cell lines tested. We tested the in-vitro activity of ibrutinib on SDF-1 induced migration in a spectrum of primary AML blasts from a wide age spectrum of adult patients and across a range of WHO AML subclasses and found that ibrutinib significantly inhibits primary AML blast migration (n=12). Next we found that ibrutinib can inhibit SDF-1 induced BTK phosphorylation and downstream MAPK and AKT signalling in primary AML blast. Finally to eliminate the problems associated with off target ibrutinib activity we evaluated migration of AML cells lines using genetic inhibition of BTK. The introduction of BTK-specific miRNA dramatically inhibited the expression of BTK in THP-1 and HL60 and reduced SDF1 mediated migration confirming that BTK is involved in regulating AML migration in response to SDF1. Conclusions These results reported here provide a molecular mechanistic rationale for clinically evaluating BTK inhibition in AML patients and suggests that in some AML patients the blasts count may initially rise in response to ibrutinib therapy, analgous to similar clinical observations in CLL. Disclosures No relevant conflicts of interest to declare.

Protease-Activated Receptor 1 (PAR-1) Inhibits Proliferation but Enhances Leukemia Stem Cell Activity in Acute Myeloid Leukemia

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 2730-2730 ◽  
Author(s):  
Susumu Goyama ◽  
Mahesh Shrestha ◽  
Janet Schibler ◽  
Leah Rosenfeldt ◽  
Whitney Miller ◽  
...  
Keyword(s):  
Acute Myeloid Leukemia ◽  
Myeloid Leukemia ◽  
Conflicts Of Interest ◽  
Clinical Investigation ◽  
Cell Activity ◽  
Stem Cell Activity ◽  
Acute Myeloid Leukemia Cells ◽  
Transplantation Assay ◽  
Acute Myeloid

Abstract Leukemic stem cells (LSCs) are capable of limitless self-renewal and indefinitely propagating leukemia. Eradication of LSCs is the ultimate goal of treating acute myeloid leukemia (AML). Using a mouse model of AML induced by the MLL-fusion protein MLL-AF9, we recently showed that the combined loss of Runx1/Cbfb inhibited the development of leukemia in vivo (Goyama S…Mulloy JC. Transcription factor RUNX1 promotes survival of acute myeloid leukemia cells. Journal of Clinical Investigation 123(9): 3876-3888, 2013). However, LSC-enriched cells with immature surface phenotype (cKit+Gr1-) remained viable in Runx1/Cbfb-deleted MLL-AF9 cells, indicating that RUNX targeting may not eradicate the most immature LSCs. Gene expression analyses of Runx1/Cbfb-deleted MLL-AF9 cells revealed the upregulation of thrombin pathway genes including a thrombin-activatable receptor PAR-1. Interestingly, both overexpression and knockout of PAR-1 inhibit leukemogenesis but do so through distinct mechanisms. Similar to the effect of Runx1/Cbfb-depletion, PAR-1 overexpression induced p21 expression and attenuated proliferation in MLL-AF9 cells. To our surprise, PAR-1-deficiency also prevented leukemia development induced by a small number of MLL-AF9 LSCs in vivo. Re-expression of PAR-1 in PAR-1-deficient cells combined with a limiting-dilution transplantation assay demonstrated the cell-dose dependent role of PAR-1 in MLL-AF9 leukemia: PAR-1 inhibited rapid leukemic proliferation when there are a large number of LSCs, while a small numbers of LSCs required PAR-1 for their growth. Mechanistically, PAR-1 increased adhering properties of MLL-AF9 cells and promoted their engraftment to bone marrow. PAR-1-deficiency also reduced leukemogenicity of AML1-ETO-induced leukemia. Together, these data reveal a multifaceted role for PAR-1 in leukemogenesis, and highlight this receptor as a potential target to eradicate primitive LSCs in AML. Disclosures No relevant conflicts of interest to declare.

SRPK1 Is a Therapeutic Vulnerability in Acute Myeloid Leukemia through Its Effects on Alternative Isoforms of Epigenetic Regulators Including BRD4

Blood ◽  
2017 ◽  
Vol 130 (Suppl_1) ◽  
pp. 781-781
Author(s):  
Konstantinos Tzelepis ◽  
Etienne De Braekeleer ◽  
Isaia Barbieri ◽  
Vijay Baskar ◽  
Demetrios Aspris ◽  
...  
Keyword(s):  
Acute Myeloid Leukemia ◽  
Myeloid Leukemia ◽  
Conflicts Of Interest ◽  
Fusion Gene ◽  
Ectopic Expression ◽  
Pharmacological Inhibition ◽  
Epigenetic Regulators ◽  
Acute Myeloid

Abstract Acute myeloid leukemia (AML) is an aggressive cancer with a poor prognosis, for which the therapeutic landscape has changed little for decades. Aberrant mRNA splicing plays an important role in cancer development and genes coding for several of the major components of the spliceosome are targeted by somatic mutations in several cancers including myelodysplastic syndromes and AML. Recently, myeloid neoplasms harbouring spliceosome gene mutations were shown to be preferentially susceptible to pharmacological disruption of the spliceosome. Here we report that targeting particular pathways of the spliceosome machinery can also be an effective therapeutic strategy in other types of AML. Recently, we generated a comprehensive catalogue of genetic vulnerabilities in AML using CRISPR-Cas9 genome-wide recessive screens and reported several novel intuitive and non-intuitive therapeutic candidates. Amongst these we identified SRPK1, the gene coding for a serine-threonine kinase that phosphorylates the major spliceosome protein SRSF1. Here, we demonstrate that targeted genetic disruption of SRPK1 in MLL-rearranged AMLs leads to differentiation and apoptosis (Fig. 1A). Additionally, the survival of immunocompromised mice transplanted with human AML cell lines carrying the MLL-AF9 fusion gene, namely MOLM-13 and THP-1, was significantly prolonged by genetic disruption of SRPK1 with CRISPR-Cas9. Similar effects were seen with pharmacological inhibition of SRPK1 in vitro and in vivo, using the novel SRPK1-specific kinase inhibitor SPHINX31 (Fig. 1B-C). Importantly, we go on to demonstrate that, while the SRPK1 kinase activity is required for AML cell survival, it is dispensable for normal hematopoiesis. At the molecular level, we show that genetic or pharmacological inhibition of SRPK1 was associated with widespread changes in the splicing of multiple genes including several with roles in leukemogenesis such as MYB, BRD4 and MED24 . We focused on BRD4 as its splicing isoforms have distinct molecular properties and found that SRPK1 inhibition led to a substantial switch from the short (BRD4S) to the long (BRD4L) isoform at the mRNA and protein levels (Fig. 1D-E). This was associated with BRD4 eviction from genomic loci involved in myeloid leukemogenesis including BCL2 and MYC. Notably, ectopic expression of the short (BRD4S) isoform rescued the phenotype of SRPK1 inhibition suggesting that the observed BRD4 splicing switch mediates at least part of the anti-leukemic effects of SRPK1 inhibition. Furthermore, we show that the BRD inhibitor iBET-151 synergizes with SRPK1 inhibition to kill human MLL-AF9 -driven AMLs in vitro and in vivo. Collectively our findings reveal that SRPK1 is required for normal splicing of key epigenetic regulators including BRD4 and represents a novel therapeutic vulnerability in AML that can be used alone or in combination with clinically relevant epigenetic drugs to enhance their anti-leukemic effects. Disclosures No relevant conflicts of interest to declare.

Preclinical Efficacy of MEK Inhibition in Nras Mutant Acute Myeloid Leukemia

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 3753-3753
Author(s):  
Michael R. Burgess ◽  
Eugene Hwang ◽  
Ari J Firestone ◽  
Tannie Huang ◽  
Jin Xu ◽  
...  
Keyword(s):  
Acute Myeloid Leukemia ◽  
Myeloid Leukemia ◽  
Conflicts Of Interest ◽  
Ras Signaling ◽  
Hematopoietic Stem ◽  
Mek Inhibitors ◽  
Mek Inhibition ◽  
Primary Mouse ◽  
Acute Myeloid

Abstract Oncogenic NRAS mutations are highly prevalent in hematologic malignancies. In acute myeloid leukemia (AML), genetic analysis supports the hypothesis that NRAS mutations cooperate with antecedent molecular lesions in leukemogenesis. Furthermore, NRAS mutations identified at diagnosis may disappear at relapse, raising questions regarding the potential clinical benefits of inhibiting oncogenic N-Ras in AML. To directly investigate the consequences of Nras inactivation in normal hematopoiesis, we used the Mx1-Cre transgene to inactivate a conditional mutant Nras allele and analyzed hematopoiesis and hematopoietic stem and progenitor cells (HSPC) under normal and stressed conditions. We show that HSPCs lacking Nras expression are functionally equivalent to normal HSPCs in the adult mouse. Importantly, shRNA-mediated knockdown in human AML cell lines and primary mouse leukemias with oncogenic NRAS/Nras mutations revealed dependence on continued oncogene expression in vitro and in vivo. Next, we interrogated the functional consequences of pharmacologic inhibition of the canonical Ras effector pathways, the mitogen-activated protein kinase (MAPK) and phosphatidylinositol 3-kinase (PI3K) pathways,alone and in combination. Recipient mice transplanted with five independent primary mouse AMLs generated by infecting NrasG12D “knock in” mice with the MOL4070LTR retrovirus (Li et al, Blood 2011; 117:2022) were treated with the allosteric MEK inhibitors PD0325901 (PD901) or trametinib or the PI3K inhibitor GDC-0941. Both MEK inhibitors significantly prolonged survival and reduced proliferation and blast colony formation, but did not induce apoptosis, differentiation, or promote clonal evolution. PI3K inhibition alone was ineffective in vivo and combinations of MEK and PI3K inhibitors were no better than MEK inhibition alone. All mice ultimately succumbed from progressive leukemia. These data, along with observations that Nras is dispensable for normal hematopoiesis, validate oncogenic N-Ras signaling as a therapeutic target in AML and support testing combination regimens that include MEK inhibitors in leukemias harboring NRAS mutations. Disclosures No relevant conflicts of interest to declare.

Vgamma9Vdelta2 T Cells-Based Immunotherapy Targeting BTN3 Molecules in Acute Myeloid Leukemia

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 3726-3726
Author(s):  
Daniel Olive ◽  
Audrey Benyamine ◽  
Aude Le Roy ◽  
Rémy Castellano ◽  
Julie Gertner-Dardenne ◽  
...  
Keyword(s):  
T Cells ◽  
Acute Myeloid Leukemia ◽  
Cell Lines ◽  
Myeloid Leukemia ◽  
Mevalonate Pathway ◽  
Conflicts Of Interest ◽  
Tumor Burden ◽  
Acute Myeloid

Abstract As they can kill Acute Myeloid Leukemia (AML) blasts in vitro and in vivo, Vg9Vd2T cells are key players in the design of new strategies of immunotherapy. AminoBisphonates (NBP) can enhance their activation in vitro and in vivo. Their combination with low-dose IL2 has shown promising results in 2 patients with AML who underwent partial remission. NBP treatment of blasts inhibits the Mevalonate pathway. The subsequent accumulation of Isopentenyl Diphosphate sensitize AML blasts to Vg9Vd2T cells killing but some AML cell lines blasts are resistant to this TCR mediated-lysis. Butyrophilin 3 A1 (BTN3A1) has been shown to be involved in IPP recognition and Vg9Vd2 T cells activation. Agonist monoclonal antibodies (mAb) recognizing the 3 isoforms of BTN3, can trigger BTN3 on tumor cell lines and sensitize them to Vg9Vd2 T cells lysis. We show that primary AML blasts from patient at diagnosis are heterogeneously killed by allogenic-IL-2-NBP-expanded Vg9Vd2 T. Some are resistant to this lysis and/or poorly sensitized by NBP. BTN3 molecules are highly expressed by blasts of AML cell lines and primary AML samples. We show that treatment of primary AML blasts with agonist anti-BTN3 mAb can overcome the resistance to Vg9Vd2 cells lysis in vitro. We assess this effect in vivo, showing that the addition of agonist anti-BTN3 mAb to Vg9Vd2 cells infusion decreased the tumor burden and increased the survival of NOG mice xenografted with luciferase-transduced U937 cell line. We confirm this effect in a model of mice xenografted with primary AML blasts, showing that treatment with anti-BTN3 mAb added to Vg9Vd2 cells infusion can decrease the number of blastic cells in the spleen, bone marrow and the blood, without requiring additional cytokine infusion. This drastic effect on sensitization of primary AML blasts to Vg9Vd2T cells killing could be of great interest especially in cases of refractory or relapsing AML. Disclosures No relevant conflicts of interest to declare.

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