scholarly journals Amlexanox As a Possible Breakthrough Drug for MLL/AF4-Positive Acute Lymphoblastic Leukemia

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 1590-1590
Author(s):  
Hayato Tamai ◽  
Hiroki Yamaguchi ◽  
Koichi Miyake ◽  
Miyuki Takatori ◽  
Tomoaki Kitano ◽  
...  
Keyword(s):  
Stem Cell ◽  
Acute Lymphoblastic Leukemia ◽  
Caspase 3 ◽  
Lymphoblastic Leukemia ◽  
Control Diet ◽  
Hematopoietic Stem ◽  
Tnf Α ◽  
Breakthrough Drug

Abstract Background: MLL/AF4-positive acute lymphoblastic leukemia (ALL) is associated with poor prognosis even after allogeneic hematopoietic stem cell transplantation. Previously, we reported that this ALL shows resistance to TNF-α, which is the factor involved in the graft versus leukemia (GVL) effect or tumor immunity, by upregulation of S100A6 expression followed by interference with the p53-caspase pathway. Amlexanox, an anti-allergic drug, was reported to inhibit the translocation pathway of endogenous S100A6 in endothelial cells. Aims: This study was performed to examine the effects of Amlexanox on MLL/AF4-positive ALL. Methods: In vitro analysis, cell growth of MLL/AF4-positive ALL cell lines ( SEM and RS4;11) were analyzed with TNF-α (10 ng/mL) and Amlexanox (0, 10, and 100 µg/mL).The effect of Amlexanox on S100A6 and p53-caspase pathways were examined by Western blotting (WB) analysis. In vivo analysis MLL/AF4-positive transgenic mice model, which show CD45R/B220+leukemia by 12 months of age we established and human peripheral blood mononuclear cell (Hu-PBMC) NOD/SCID mice transplanted with SEM-Luc were examined to compare mice fed diet containing Amlexanox (0.02%) with mice fed control diet. Results: There were no significant differences between the growth of SEM or RS4;11 cells in the absence or presence of 10 µg/mL of Amlexanox in vitro under 10 ng/mL of TNF-α. However, both cells showed significant growth inhibition by 10 ng/mL of TNF-α in the presence of 100 µg/mL of Amlexanox (P = 0.0085 for SEM, P = 0.0196 for RS4;11) WB analysis showed that S100A6 was activated in the presence of 10 ng/mL TNF-α, and activated S100A6 was decreased and both acetyl-p53/p53 ratio and cleaved caspase 3/caspase 3 ratio were increased in cells treated with 100 µg/mL of Amlexanox under 10 ng/mL of TNF-α in the MLL/AF4-positive human ALL cell lines. In vivo, MLL/AF4-positive transgenic mice fed a diet containing Amlexanox (0.02%) developed significantly less volume of CD45R/B220+ leukemia at the age of 1 year in comparison with mice fed control diet (P<0.001 for BM and .P<0.001 for spleen). Hu-PBMC NOD/SCID mice transplanted with SEM-Luc in the Amlexanox group showed significantly longer survival than those in the control group (P < 0.014). Conclusions: Amlexanox may be a breakthrough drug for MLL/AF4-positive ALL because it inhibits the resistance of MLL/AF4-positive ALL to TNF-α by downregulating S100A6 expression followed by upregulating the p53-caspase pathway.Specifically, allogeneic hematopoietic stem cell transplantation is expected to show beneficial effects in combination with Amlexanox. Disclosures No relevant conflicts of interest to declare.

scholarly journals BRD4 PROTAC degrader ARV-825 inhibits T-cell acute lymphoblastic leukemia by targeting 'Undruggable' Myc-pathway genes

2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Shuiyan Wu ◽  
You Jiang ◽  
Yi Hong ◽  
Xinran Chu ◽  
Zimu Zhang ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Proliferation ◽  
Acute Lymphoblastic Leukemia ◽  
Caspase 3 ◽  
Lymphoblastic Leukemia ◽  
Rna Seq ◽  
Cell Acute Lymphoblastic Leukemia ◽  
Bet Protein

Abstract Background T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive disease with a high risk of induction failure and poor outcomes, with relapse due to drug resistance. Recent studies show that bromodomains and extra-terminal (BET) protein inhibitors are promising anti-cancer agents. ARV-825, comprising a BET inhibitor conjugated with cereblon ligand, was recently developed to attenuate the growth of multiple tumors in vitro and in vivo. However, the functional and molecular mechanisms of ARV-825 in T-ALL remain unclear. This study aimed to investigate the therapeutic efficacy and potential mechanism of ARV-825 in T-ALL. Methods Expression of the BRD4 were determined in pediatric T-ALL samples and differential gene expression after ARV-825 treatment was explored by RNA-seq and quantitative reverse transcription-polymerase chain reaction. T-ALL cell viability was measured by CCK8 assay after ARV-825 administration. Cell cycle was analyzed by propidium iodide (PI) staining and apoptosis was assessed by Annexin V/PI staining. BRD4, BRD3 and BRD2 proteins were detected by western blot in cells treated with ARV-825. The effect of ARV-825 on T-ALL cells was analyzed in vivo. The functional and molecular pathways involved in ARV-825 treatment of T-ALL were verified by western blot and chromatin immunoprecipitation (ChIP). Results BRD4 expression was higher in pediatric T-ALL samples compared with T-cells from healthy donors. High BRD4 expression indicated a poor outcome. ARV-825 suppressed cell proliferation in vitro by arresting the cell cycle and inducing apoptosis, with elevated poly-ADP ribose polymerase and cleaved caspase 3. BRD4, BRD3, and BRD2 were degraded in line with reduced cereblon expression in T-ALL cells. ARV-825 had a lower IC50 in T-ALL cells compared with JQ1, dBET1 and OTX015. ARV-825 perturbed the H3K27Ac-Myc pathway and reduced c-Myc protein levels in T-ALL cells according to RNA-seq and ChIP. In the T-ALL xenograft model, ARV-825 significantly reduced tumor growth and led to the dysregulation of Ki67 and cleaved caspase 3. Moreover, ARV-825 inhibited cell proliferation by depleting BET and c-Myc proteins in vitro and in vivo. Conclusions BRD4 indicates a poor prognosis in T-ALL. The BRD4 degrader ARV-825 can effectively suppress the proliferation and promote apoptosis of T-ALL cells via BET protein depletion and c-Myc inhibition, thus providing a new strategy for the treatment of T-ALL.

scholarly journals Adoptive Transfers of Plasmacytoid Dendritic Cells after Hematopoietic Stem Cell Transplantation Decrease the Risk Acute Lymphoblastic Leukemia Relapse without Increasing the Risk of Graft-Versus-Host-Disease

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 2053-2053 ◽  
Author(s):  
Sabine Herblot ◽  
Valérie Paquin ◽  
Paulo Cordeiro ◽  
Michel Duval
Keyword(s):  
T Cells ◽  
Dendritic Cells ◽  
Stem Cell ◽  
Acute Lymphoblastic Leukemia ◽  
Nk Cell ◽  
Lymphoblastic Leukemia ◽  
Hematopoietic Stem ◽  
Graft Versus Host ◽  
Antigen Presenting

Abstract Despite advances in chemotherapy and hematopoietic stem cell transplantation (HSCT), the outcome of children with relapsed acute lymphoblastic leukemia (ALL) has not significantly improved over the last 2 decades. About 50% of children with relapsed leukemia still die from their disease and ALL is still the first cause of death by cancer in children. A new hope of cure for patients with chemo-resistant cancers has emerged with the development of cancer immunotherapy. However, the major risk of post-transplant immunotherapy is the exacerbation of life-threatening Graft-versus-Host Disease (GvHD) mediated by donor-derived T cells. We therefore explored the avenue of innate immune stimulation. Several reports have demonstrated that activated Natural Killer (NK) cells can control acute myeloid leukemia (AML) in transplanted patients, whereas ALL is deemed to be resistant to NK cell killing. We recently challenged this paradigm and demonstrated that the stimulation of NK cells with third-party activated plasmacytoid dendritic cells (pDC) killed most ALL cell lines and patient-derived ALL blasts. We further demonstrated the efficacy of pDC adoptive transfers to cure ALL in a humanized mouse model of HSCT. Collectively, these results uncovered for the first time the unique therapeutic potential of activated pDC as immunotherapeutic tools to stimulate NK cell anti-leukemic activity early after HSCT. The next step toward the clinical translation of pDC-based post-transplant immunotherapy is to verify that adoptive transfers of pDC do not stimulate T cells nor exacerbate GvHD in the presence of mature T cells. We designed a GMP-compliant method for in vitro expansion and differentiation of cord blood progenitors giving rise to sufficient numbers of pDC for adoptive transfers in patients. We showed that after Toll-like receptor (TLR) stimulation, these in vitro differentiated pDC displayed a phenotype of interferon producing cells (CD80neg PDL-1+) but not of antigen presenting cells (CD80+PDL-1neg). Accordingly, in vitro mixed lymphocyte reactions with purified allogeneic T cells demonstrated that TLR-activated pDC induced very low allogeneic T cell proliferation as compared with bona fide antigen presenting cells such as myeloid dendritic cells (mDC - CD11c+) or monocyte-derived dendritic cells (mo-DC) (Figure 1A). To test whether activated pDC could exacerbate GvHD in the presence of mature T cells, we used a xenoGvHD model in which human peripheral blood mononuclear cells (PBMC) were injected in immune-deficient mice (Nod/Scid/gRc-/-, NSG). We monitored GvHD 3-times a week according to a GvHD-assessment scale as previously described. Overt GvHD was characterized by cutaneous and intestinal lesions, weight loss and high numbers of human CD3+ cells in peripheral blood. Mice were sacrificed when endpoints were reached and GvHD was confirmed by immunohistochemistry and flow cytometry. Five weekly injections of TLR-activated in vitro differentiated pDC did not accelerate the GvHD onset and the severity of the lesions were not increased. We did not either observe any difference in survival between control and pDC-treated groups (Figure 1B). Collectively, our results indicate that TLR-activated pDC do not stimulate allogeneic T cells and do not increase the risk of acute GvHD in a mouse model of xenoGvHD. We therefore expect this novel pDC-based immunotherapy to be safe for transplanted patients. These data open the way for the next step: a Phase I clinical trial of in vitro differentiated pDC after transplantation for leukemia. Disclosures No relevant conflicts of interest to declare.

Rac2 GTPase Activation Is Necessary for Development of p190-BCR-ABL-Induced B-Cell Acute Lymphoblastic Leukemia

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 3790-3790
Author(s):  
Abel Sanchez-Aguilera ◽  
Ami tava Sengupta ◽  
Joseph P Mastin ◽  
Kyung H Chang ◽  
David A Williams ◽  
...  
Keyword(s):  
Acute Lymphoblastic Leukemia ◽  
B Cells ◽  
Tyrosine Kinase ◽  
B Cell ◽  
Rho Gtpases ◽  
Lymphoblastic Leukemia ◽  
Hematopoietic Stem ◽  
Cell Acute Lymphoblastic Leukemia

Abstract The fusion gene BCR-ABL, resulting from t(9;22) reciprocal chromosomal translocations, encodes a constitutively active tyrosine kinase. Two different isoforms of BCR-ABL, p190 and p210, are associated to two completely different diseases. In the tyrosine kinase inhibitor (TKI) era, while p210-BCR-ABL-induced CML is highly responsive to TKI, p190-BCR-ABL still induces a poor prognosis B-cell acute lymphoblastic leukemia (B-ALL). The only difference between these two forms of BCR-ABL is the existence of a DH/Cdc24/PH domain in p210-BCR-ABL, which acts as a guanine nucleotide exchange factor (GEF) able to activate Rho GTPases. Rac is a subfamily of Rho GTPases with regulatory activity on hematopoietic stem cell and progenitor (HSC/P) functions. We have previously shown that Rac2 and further the combination of Rac1 and Rac2 mediate downstream signals in p210 BCR-ABL-induced myeloproliferation (Thomas EK, et al., Cancer Cell, 2007). Interestingly, despite the absence of a GEF domain in p190-BCR-ABL, Rac is activated, suggesting the activation of other GEF(s). Here we have analyzed whether Vav and Rac family members are involved in p190-BCR-ABL-induced B-ALL. We have used a combination of in vitro (Ba/F3 pro-B cells transduced with p190 or p210 BCR-ABL) and in vivo (murine transduction-transplantation model of p190 BCR-ABL-induced B-ALL) approaches. In Ba/F3 cells, both p190 BCR-ABL and p210 BCR-ABL activated Rac and the Rac effector p21 activated kinase (PAK), and their proliferation and survival appeared severely decreased in response to the Rac activation inhibitor NSC23766. Stat3, Stat5 and Jnk, but not ERK, p38 or NF-kB, were constitutively hyperactivated in p190 BCRABL-expressing Ba/F3 cells and primary murine B-ALL cells. Intracellular flow cytometry analysis demonstrated that Stat5 was specifically activated in the pro/pre-B leukemic cell population, compared to normal B cells. In the murine model of B-ALL, loss of Rac2, but not Rac3, prolonged survival and impaired leukemia development. Like in Ba/F3 cells, primary B-CFU and outgrowth in Witte-Whitlock assays of leukemic primary cells from mice was severely decreased by the addition of NSC23766 to the culture. Although Vav was activated by both p190- and p210-BCR-ABL, since NSC23766 does not block the activation by Vav1, we hypothesized that other GEFs were involved. Indeed, the loss of Vav1 or even combined loss of Vav1 and Vav2 did not impair BCR-ABL-mediated lymphoid leukemogenesis in vivo. Vav3, another member in the Vav family which uses a different mechanism of activation of Rac GTPases was a likely candidate. In fact, loss of Vav3 alone was able to significantly prolong the survival and attenuate development of p190 BCR-ABL-driven B-ALL. In conclusion, the results of this study indicate that Rac activation is necessary for the development of B-ALL induced by p190-BCR-ABL in vitro and in vivo, and validate a new signaling pathway as a therapeutic target for BCR-ABL-induced B-ALL.

scholarly journals Germline RUNX1 Variation and Predisposition to Childhood Acute Lymphoblastic Leukemia

2021 ◽  
Author(s):  
Yizhen Li ◽  
Meenakshi Devidas ◽  
Wentao Yang ◽  
Stuart S Winter ◽  
Wenjian Yang ◽  
...  
Keyword(s):  
Acute Lymphoblastic Leukemia ◽  
T Cell ◽  
Lymphoblastic Leukemia ◽  
Ectopic Expression ◽  
Genetic Alterations ◽  
Dominant Negative ◽  
Transcription Activator ◽  
Hematopoietic Stem

RUNX1 is a transcription factor critical for definitive hematopoiesis and genetic alterations in RUNX1 have been implicated in both benign and malignant blood disorders, particularly of the megakaryocyte and myeloid lineages. Somatic RUNX1 mutations are reported in B- and T-cell acute lymphoblastic leukemia (B-ALL and T-ALL), but germline genetic variation of RUNX1 in these lymphoid malignancies have not been comprehensively investigated. Sequencing 4,836 children with B-ALL and 1,354 cases of T-ALL, we identified 31 and 18 unique germline RUNX1 variants in these two ALL subtypes, respectively. RUNX1 variants in B-ALL were predicted to have minimal impact. By contrast, 54.5% of variants in T-ALL result in complete or partial loss of RUNX1 activity as a transcription activator in vitro, with dominant negative effects for 4 variants. Ectopic expression of dominant negative deleterious RUNX1 variants in human CD34+ cells repressed differentiation into erythroid, megakaryocytes, and T cells, while promoting differentiation towards myeloid cells. We then performed chromatin immunoprecipitation profiling in isogenic T-ALL models with variants introduced by genome editing of endogenous RUNX1. We observed highly distinctive patterns of DNA binding and target genomic loci by RUNX1 proteins encoded by the truncating vs missense variants. The p.G365R RUNX1 variant resulted in a novel methylation site in RUNX1 and alteration in its interaction with CBFβ. Further whole genome sequencing showed that JAK3 mutation was the most frequent somatic genomic abnormality in T-ALL with germline RUNX1 variants. Consistently, co-introduction of RUNX1 variant and JAK3 mutation in hematopoietic stem and progenitor cells in mouse gave rise to T-ALL with early T-cell precursor phenotype in vivo, compared to thymic T-ALL seen in mice with JAK3 mutation alone. Taken together, these results indicated that RUNX1 is an important predisposition gene for ALL, especially in T-ALL and also pointed to novel biology of RUNX1-mediated leukemogenesis in the lymphoid lineages.

scholarly journals AAV8 vector expressing IL24 efficiently suppresses tumor growth mediated by specific mechanisms in MLL/AF4-positive ALL model mice

Blood ◽  
2012 ◽  
Vol 119 (1) ◽  
pp. 64-71 ◽  
Author(s):  
Hayato Tamai ◽  
Koichi Miyake ◽  
Hiroki Yamaguchi ◽  
Miyuki Takatori ◽  
Kazuo Dan ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Caspase 3 ◽  
Lymphoblastic Leukemia ◽  
C2c12 Cell ◽  
Caspase 8 ◽  
Apoptotic Pathway ◽  
High Relapse Rate ◽  
Tnf Α

Abstract Mixed-lineage leukemia (MLL)/AF4-positive acute lymphoblastic leukemia (ALL) is a common type of leukemia in infants, which is associated with a high relapse rate and poor prognosis. IL24 selectively induces apoptosis in cancer cells and exerts immunomodulatory and antiangiogenic effects. We examined the effects of adeno-associated virus type 8 (AAV8) vector-mediated muscle-directed systemic gene therapy in MLL/AF4-positive ALL using IL24. In a series of in vitro studies, we examined the effects of AAV8-IL24–transduced C2C12 cell-conditioned medium. We also examined the effects of AAV8-IL24 in MLL/AF4 transgenic mice. The results revealed the effects of AAV8-IL24 in MLL/AF4-positive ALL both in vitro and in vivo. With regard to the mechanism of therapy using AAV8-IL24 in MLL/AF4-positive ALL, we demonstrated the antiangiogenicity and effects on the ER stress pathway and unreported pathways through inhibition of S100A6 and HOXA9, which is specific to MLL/AF4-positive ALL. Inhibition of S100A6 by IL24 was dependent on TNF-α and induced acetylation of p53 followed by activation of the caspase 8–caspase 3 apoptotic pathway. Inhibition of HOXA9 by IL24, which was independent of TNF-α, induced MEIS1 activation followed by activation of the caspase 8–caspase 3 apoptotic pathway. Thus, gene therapy using AAV8-IL24 is a promising treatment for MLL/AF4-positive ALL.

CD8+/TCR− Graft Facilitating Cells Enhance HSC Engraftment and Survival Via TNF-α Mediated Prevention of Apoptosis.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 1352-1352
Author(s):  
Suzanne T. Ildstad ◽  
Francine Rezzoug ◽  
Yiming Huang ◽  
Marcin Wysoczynski ◽  
Carrie L. Schanie ◽  
...  
Keyword(s):  
Stem Cell ◽  
Regulatory Effect ◽  
Hematopoietic Stem ◽  
Accessory Cells ◽  
Tnf Α ◽  
Apoptotic Effect ◽  
Low Levels ◽  
First Time

Abstract The use of accessory cells to enhance hematopoietic stem cell (HSC) engraftment could have a significant therapeutic impact, especially when stem cell numbers are limited. The bone marrow (BM) microenvironment is involved in regulation of HSC, allowing production of mature blood cells while maintaining HSC self renewal. To date, the precise identity of specific cells in the microenvironment that exert this regulatory effect on HSC has not been defined. We recently reported that CD8+/TCR− facilitating cells (FC), a subpopulation of BM cells containing predominantly B220+/CD11c+/CD11b− tolerogenic precursor-plasmacytoid dendritic cells, enhance HSC engraftment in allogeneic recipients. Additionally, FC significantly enhance engraftment of limiting numbers of HSC in syngeneic recipients. In the present studies, we investigated the mechanism of FC-mediated enhancement of HSC engraftment. We show for the first time that FC significantly increase HSC survival in vitro and exert an anti-apoptotic effect on HSC via TNF-α. Co-culture of FC with HSC induces production of physiologically relevant low levels of TNF-α by FC. FC from TNF-α−/− mice are impaired in function in vitro and in facilitating HSC engraftment in vivo. Furthermore, neutralization of TNF-α on FC using anti-TNF antibody results in loss of FC function in vitro, confirming a major role for TNF-α in FC function. Notably, co-culture of FC with HSC prevents HSC apoptosis and is associated with significant upregulation of the anti-apoptotic I-κB family member Bcl-3 in HSC. Blocking of TNF-α on FC abrogates the anti-apoptotic effect of FC on HSC and prevents upregulation of Bc1-3 in HSC. Taken together, these findings demonstrate that TNF-α-induced in FC affects highly primitive HSC and identify Bcl-3 as a possible pathway for TNF-α in regulating HSC survival.

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