scholarly journals Inhibitor of Multi-cyclin-dependent Kinases (AT7519) Reduced Survival of U937 Leukemic Cells and Enhanced Anti-leukemic Effect of Vincristine: A Highlight to CDK Inhibition Efficacy in Acute Leukemia

2021 ◽  
Vol In Press (In Press) ◽  
Author(s):  
Fateme Moradi Moraddahande ◽  
Mona Shameli Houjghan ◽  
Amir-Mohammad Yousefi ◽  
Ava Safaroghli-Azar ◽  
Atieh Pourbagheri-Sigaroodi ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Acute Myeloid Leukemia ◽  
Acute Leukemia ◽  
Myeloid Leukemia ◽  
Leukemic Cells ◽  
Mrna Levels ◽  
U937 Cells ◽  
Cyclin Dependent Kinases ◽  
The Impact ◽  
Acute Myeloid

Background: The conservative character of the cell cycle outlined that any dysregulation in the regulatory components of this process in normal cells opens a gate toward neoplastic transformation. Objectives: Given the critical role of cyclin-dependent kinases (CDKs) in cancer pathogenesis and based on their frequent aberrancy in human leukemia, the present study aimed at evaluating the suppressive effect of a multi-CDK inhibitor AT7519 on acute myeloid leukemia-derived U937 cells. Methods: To assess the anti-leukemic effects of the inhibitor on acute myeloid leukemia (AML) cells, we used MTT and trypan blue assays. Flow cytometric analysis and q-RT-PCR were also applied to evaluate the impact of AT7519 on cell cycle and apoptosis. Results: The results suggested that suppression of CDK in U937 cells hampered the proliferation of leukemic cells through a G2/M arrest mediated by p21 gene. Additionally, the anti-survival impact of AT7519 on these cells was shown to be along with the apoptosis initiation not only through the increment of pro-apoptotic gene expression but also through diminishing the mRNA levels of both Pin1 and Survivin. Notably, the potent anti-leukemic property of this agent has become more prominent when we found that the blockage of CDKs in AML cells could synergize with the cytotoxic effect of vincristine (VCR). To the best of our knowledge, little is known about the molecular mechanisms of resistance to AT7519 and we proposed that the effectiveness of this agent was partially attenuated through either c-Myc or autophagy activation in U937 cells. Conclusions: This study suggests that the pharmacological targeting of CDKs could probably unwind the complexity of therapeutic obstacles on the way of acute leukemia, either in the context of mono- or combined-modal strategy.

scholarly journals Pan-RAF Inhibition Shows Anti-Leukemic Activity in RAS-Mutant Acute Myeloid Leukemia Cells and Potentiates the Effect of Sorafenib in Cells with FLT3 Mutation

Cancers ◽  
2020 ◽  
Vol 12 (12) ◽  
pp. 3511
Author(s):  
Joseph D. Khoury ◽  
Mehrnoosh Tashakori ◽  
Hong Yang ◽  
Sanam Loghavi ◽  
Ying Wang ◽  
...  
Keyword(s):  
Acute Myeloid Leukemia ◽  
Cell Signaling ◽  
Myeloid Leukemia ◽  
Critical Role ◽  
Cyclin B1 ◽  
Leukemic Cells ◽  
Phase Array ◽  
Flt3 Mutations ◽  
The Impact ◽  
Acute Myeloid

RAF molecules play a critical role in cell signaling through their integral impact on the RAS/RAF/MEK/ERK signaling pathway, which is constitutively activated in a sizeable subset of acute myeloid leukemia (AML) patients. We evaluated the impact of pan-RAF inhibition using LY3009120 in AML cells harboring mutations upstream and downstream of RAF. LY3009120 had anti-proliferative and pro-apoptotic effects and suppressed pERK1/2 levels in leukemic cells with RAS and FLT3 mutations. Using reverse protein phase array analysis, we identified reductions in the expression/activation of cell signaling components downstream of RAF (activated p38) and cell cycle regulators (Wee1/cyclin B1, Cdc2/Cdk1, activated Rb, etc.). Notably, LY3009120 potentiated the effect of Ara-C on AML cells and overcame bone marrow mesenchymal stromal cell-mediated chemoresistance, with RAS-mutated cells showing a notable reduction in pAKT (Ser473). Furthermore, the combination of LY3009120 and sorafenib resulted in significantly higher levels of apoptosis in AML cells with heterozygous and hemizygous FLT3 mutations. In conclusion, pan-RAF inhibition in AML using LY3009120 results in anti-leukemic activity, and combination with Ara-C or sorafenib potentiates its effect.

Detection, isolation, and stimulation of quiescent primitive leukemic progenitor cells from patients with acute myeloid leukemia (AML)

Blood ◽  
2003 ◽  
Vol 101 (8) ◽  
pp. 3142-3149 ◽  
Author(s):  
Yinghui Guan ◽  
Brigitte Gerhard ◽  
Donna E. Hogge
Keyword(s):  
Cell Cycle ◽  
Acute Myeloid Leukemia ◽  
Myeloid Leukemia ◽  
Active Cell ◽  
Leukemic Cells ◽  
Gm Csf ◽  
Macrophage Colony Stimulating Factor ◽  
Macrophage Colony ◽  
Acute Myeloid

Abstract Although many acute myeloid leukemia (AML) colony-forming cells (CFCs) and long-term culture–initiating cells (LTC-ICs) directly isolated from patients are actively cycling, quiescent progenitors are present in most samples. In the current study,3H-thymidine (3H-Tdr) suicide assays demonstrated that most NOD/SCID mouse leukemia-initiating cells (NOD/SL-ICs) are quiescent in 6 of 7 AML samples. AML cells in G0, G1, and S/G2+M were isolated from 4 of these samples using Hoechst 33342/pyroninY staining and cell sorting. The progenitor content of each subpopulation was consistent with the 3H-Tdr suicide results, with NOD/SL-ICs found almost exclusively among G0 cells while the cycling status of AML CFCs and LTC-ICs was more heterogeneous. Interestingly, after 72 hours in serum-free culture with or without Steel factor (SF), Flt-3 ligand (FL), and interleukin-3 (IL-3), most G0 AML cells entered active cell cycle (percentage of AML cells remaining in G0 at 72 hours, 1.2% to 37%, and 0% to 7.6% in cultures without and with growth factors [GFs], respectively) while G0 cells from normal lineage-depleted bone marrow remained quiescent in the absence of GF. All 4 AML samples showed evidence of autocrine production of 2 or more of SF, FL, IL-3, and granulocyte-macrophage colony-stimulating factor (GM-CSF). In addition, 3 of 4 samples contained an internal tandem duplication of theFLT3 gene. In summary, quiescent leukemic cells, including NOD/SL-ICs, are present in most AML patients. Their spontaneous entry into active cell cycle in short-term culture might be explained by the deregulated GF signaling present in many AMLs.

scholarly journals ABCB1 Expression in Acute Myeloid Leukemia (AML): A Possible Predictive Value for Treatment Resistance?

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 3618-3618
Author(s):  
Stephany Corrêa ◽  
Eliana Abdelhay ◽  
Peter Paschka ◽  
Verena I. Gaidzik ◽  
Rocio Hassan ◽  
...  
Keyword(s):  
Acute Myeloid Leukemia ◽  
Induction Therapy ◽  
Myeloid Leukemia ◽  
Induction Treatment ◽  
Mrna Levels ◽  
Complex Karyotype ◽  
Hematopoietic Stem ◽  
Expression Levels ◽  
The Impact ◽  
Acute Myeloid

Abstract Introduction: Over the last years, there has been a tremendous increase in understanding acute myeloid leukemia (AML) biology and a great effort has been taken in order to improve AML chemotherapy strategies. However, the growing knowledge of leukemia associated molecular mechanisms just started to translate into improved outcome. With regard to conventional chemotherapy multidrug resistance (MDR) is a persisting problem and the impact of ABCB1 (MDR1) expression is still controversially discussed. Methods: In this study we evaluated the ABCB1 expression using qRT-PCR and gene expression profiling (Affymetrix U133plus2.0 arrays) in 250 diagnostic AML samples derived from patients enrolled on a prospective treatment trial of the German-Austrian AML Study Group (AMLSG 07-04 trial; NCT00151242), in which patients were treated with an intensive anthracycline/cytarabine-based induction therapy. Findings were also evaluated in 154 TCGA AML cases receiving a 7+3 induction treatment (data available at http://cancergenome.nih.gov/) and put into perspective with previous reports. Furthermore, we investigated ABCB1 expression associated gene signatures and examined epigenetic regulation mechanisms by COBRA and methyl-CpG immunoprecipitation sequencing (MCIp-seq) in selected cases. Results: Our global analysis showed that patients who obtained a complete response (CR) following double induction therapy had lower ABCB1 mRNA levels compared to patients with refractory disease (RD) (p=0.07). Regarding cytogenetic AML subtypes, ABCB1 mRNA levels varied among the different cytogenetic groups with the complex karyotype group showing the highest ABCB1 and the inv(16) group the lowest ABCB1 expression levels. A comparison of CR versus RD cases within the cytogenetically determined prognostic groups showed that in the intermediate [CN-AML, t(11q23), and other intermediate risk cytogenetic aberrations (othersinter)] and poor risk groups (complex karyotype and othershigh), RD patients presented with significantly higher ABCB1 mRNA levels (p=0.02). Similarly, patients with favorable risk cytogenetics [t(8;21) and inv(16)], who achieved a CR, presented with lower ABCB1 levels compared to the ones, who were refractory. Patients with the lowest ABCB1 expression quartile (ABCB1low) showed significantly longer event-free survival (EFS) times than patients in the highest quartile cohort (ABCB1high) (median EFS 322 vs 105 days; p=0.02), while no differences were observed with regard to overall survival. In accordance, there was a significant enrichment of RD cases in the ABCB1high patient group (p=0.03). Next, in order to better understand the regulation of ABCB1 in AML, we specifically evaluated the DNA methylation level of a previously identified GC box important for ABCB1 expression regulation in CML and we performed global analyses of the entire ABCB1 5' region. While both analyses did not reveal significant differences, further investigation of an ABCB1 associated gene pattern showed a correlation with CD34 and KIT expression (p<0.001). This suggests that like in CML, ABCB1 might be regulated by WNT, and in line, normal CD34+ hematopoietic stem cells also showed high ABCB1 expression levels. Conclusions: In summary, our data provide further evidence for a potential impact of ABCB1 deregulation on the response to AML chemotherapy, especially in more stem cell like leukemia cohorts as well as cytogenetically high risk AML. While we are currently further investigating the involvement of the Wnt/β-catenin pathway in the regulation of ABCB1 transcription in AML, further integration of molecular findings are warranted to better decipher the underlying drug resistance mechanisms. Ultimately, these analyses will improve patient management by adding valuable predictive biomarkers. Disclosures No relevant conflicts of interest to declare.

scholarly journals Essential Role for Phospholipase C Gamma 1 (PLC-γ1) in the Survival of t(8;21) Acute Myeloid Leukemia

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 1699-1699 ◽  
Author(s):  
Hasan Mahmud ◽  
Frank JG Scherpen ◽  
Tiny Meeuwsen-de Boer ◽  
Harm-Jan Lourens ◽  
Eveline S. de Bont
Keyword(s):  
Cell Cycle ◽  
Acute Myeloid Leukemia ◽  
Phospholipase C ◽  
Cell Line ◽  
Myeloid Leukemia ◽  
Genetic Alterations ◽  
Therapeutic Strategies ◽  
Mrna Levels ◽  
Phospholipase C Gamma ◽  
Acute Myeloid

Abstract The t(8;21) (q22;q22) chromosomal translocation is one of the most frequent genetic alterations in acute myeloid leukemia (AML) and it remains a significant clinical problem especially for children which indicates the need for improved therapeutic strategies. Recently, we showed that peptide derived phospholipase C gamma 1 (PLC-γ1) was highly phosphorylated in pediatric t(8;21) AML. In this study, we determined PLC-γ1 phosphorylation and mRNA levels showing that PLC-γ1 expression was significantly higher in t(8;21) AML compared to other AML karyotypes and normal bone marrow (NBM) (peptide phosphorylation: p<0.01 compared to NBM, mRNA: p<0.001, compared to other AML karyotypes). This was confirmed by PLC-γ1 protein phosphorylation using primary AML samples and AML cell lines. PLC-γ1 is known to play a role in cancer progression, however, the impact of PLC-γ1 in AML is currently unknown. Therefore, we aimed to study the functional role of PLC-γ1 by investigating the cellular growth, survival and its underlying mechanism in a t(8;21) AML cell line (Kasumi-1) . ShRNA-mediated knockdown of PLC-γ1 in kasumi-1 cells significantly blocked leukemic cell growth at day 8 after transduction (p<0.05). The percentage of apoptosis in PLC-γ1 suppressed kasumi-1 cells at day 4 after transduction was two-fold higher compared to the scrambled control (p<0.01). The inhibition of cell proliferation and the induction of apoptosis upon PLC-γ1 suppression could be explained by cell cycle arrest and by increased activation of apoptotic related and cell cycle regulatory protein expressions (BAX, BCL2, p53 and Chk2). As the multidrug resistance is one of the major cause of relapse and poor prognosis in t(8;21) AML, therefore, we demonstrated, if PLC-γ1 suppression increased the sensitivity of kasumi-1 leukemia cells to cytotoxic chemotherapeutic agents (methotrexate, amsacrine and etoposide). PLC-γ1 knockdown cells at day 4 after transduction were shown to significantly reduced cell viability to the genotoxic agents, methotrexate (p<0.05, p<0.001), amsacrine (p<0.01, p<0.001) and etoposide (p<0.05, p<0.01 and p<0.001) in kasumi-1 in a dose-dependent manner. These results provide a strong rationale for the development of PLC-γ1-based therapeutic strategies for the enhancement of efficacy in t(8;21) AML treatment. Additionally, PLC-γ1 suppressed kasumi-1 cells showed significantly less proliferation upon hypoxic stress. Taken together, these results strongly support an important role for PLC-γ1 in the survival of t(8;21) AML mimicking kasumi-1 cell line and identify PLC-γ1 as a potential target for t(8;21) AML treatment. Disclosures No relevant conflicts of interest to declare.

scholarly journals PROGNOSTIC VALUE OF BRAIN AND ACUTE LEUKEMIA CYTOPLASMIC GENE EXPRESSION IN EGYPTIAN CHILDREN WITH ACUTE MYELOID LEUKEMIA

2015 ◽  
Vol 7 ◽  
pp. e2015033 ◽  
Author(s):  
Adel Abd elhaleim Hagag
Keyword(s):  
Gene Expression ◽  
Bone Marrow ◽  
Acute Myeloid Leukemia ◽  
Complete Remission ◽  
Acute Leukemia ◽  
Myeloid Leukemia ◽  
Egyptian Children ◽  
Significant Difference ◽  
The Impact ◽  
Acute Myeloid

Abstract      Background: Acute myeloid leukemia (AML) accounts for 25%-35% of the acute leukemia in children. BAALC (Brain and Acute Leukemia, Cytoplasmic gene) is a recently identified gene on chromosome 8q22.3 that has prognostic significance in AML.  The aim of this work was to study the impact of BAALC gene expression on prognosis of AML in Egyptian children. Patients and methods: This study was conducted on 40 patients of newly diagnosed AML who were subjected to the following: Full history taking, clinical examination, laboratory investigations including: complete blood count, LDH, bone marrow aspiration, cytochemistry and immunophenotyping, assessment of BAALC Gene by real time PCR in bone marrow aspirate mononuclear cells before the start of chemotherapy. Results: BAALC gene expression showed positive expression in 24 cases (60%) and negative expression in 16 cases (40%). Patients who showed positive BAALC gene expression included 10 patients achieved complete remission, 8 patients died and 6 relapsed patients, while patients who showed negative expression include 12 patients achieved complete remission, 1 relapsed patient and 3 patients died. There was significant association between BAALC gene expression and FAB classification of patients of AML patientsas positive BAALC expression is predominantly seen in FAB subtypes M1 and M2 compared with negative BAALC gene expression that was found more in M3 and M4 (8 cases with M1, 12 cases with M2, 1 case with M3 and 3 cases with M4 in positive BAALC expression versus 2 cases with M1, 3 cases with M2, 4 cases with M3 and 7 cases with M4 in BAALC gene negative expression group with significant difference regarding FAB subtypes). As regard age, sex, splenomegaly, lymphadenopathy, pallor, purpura, platelets count, WBCs count, and percentage of blast cells in BM, the present study showed no significant association with BAALC. Conclusion: BAALC expression is an important prognostic factor in AML patients and its incorporation into novel risk-adapted therapeutic strategies will improve the currently disappointing cure rate of this group of patients.

scholarly journals Direct relationship between remission duration in acute myeloid leukemia and cell cycle kinetics: a leukemia intergroup study

Blood ◽  
1990 ◽  
Vol 76 (11) ◽  
pp. 2191-2197 ◽  
Author(s):  
A Raza ◽  
HD Preisler ◽  
R Day ◽  
Z Yasin ◽  
M White ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Acute Myeloid Leukemia ◽  
Myeloid Leukemia ◽  
Leukemic Cell ◽  
Leukemic Cells ◽  
Remission Induction ◽  
Cell Cycle Time ◽  
Resistant Disease ◽  
Remission Duration ◽  
Acute Myeloid

Abstract Cell cycle characteristics including labeling indices (LI), duration of S-phase (Ts), and total cell cycle time (Tc) were determined in 54 standard-risk, newly diagnosed patients with acute myeloid leukemia following an infusion of bromodeoxyuridine. Remission induction therapy consisting of cytosine arabinoside and daunomycin was then administered to all patients, followed by three courses of consolidation to those who achieved complete remissions (CR). Older patients appeared to have more rapidly cycling cells (P = .003). No unique cell cycle characteristics were identified for patients who achieved remission versus those who had resistant disease. However, the pretherapy cell cycle characteristics were a strong prognosticator for remission duration. CR patients were divided into those whose leukemic cell Tc were above median (A) and below median (B). Among 14 B patients, median duration of response was 211 days, and all relapsed by day 600. Among 18 A patients, the median has not as yet been reached, with nine patients in continuous complete remission (log rank P = .007, Wilcoxon P = .04). We conclude that cell cycle characteristics of leukemic cells play a role in determining remission duration, perhaps because the leukemic cells of the former patients regrow slowly between courses of chemotherapy.

scholarly journals Direct relationship between remission duration in acute myeloid leukemia and cell cycle kinetics: a leukemia intergroup study

Blood ◽  
1990 ◽  
Vol 76 (11) ◽  
pp. 2191-2197
Author(s):  
A Raza ◽  
HD Preisler ◽  
R Day ◽  
Z Yasin ◽  
M White ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Acute Myeloid Leukemia ◽  
Myeloid Leukemia ◽  
Leukemic Cell ◽  
Leukemic Cells ◽  
Remission Induction ◽  
Cell Cycle Time ◽  
Resistant Disease ◽  
Remission Duration ◽  
Acute Myeloid

Cell cycle characteristics including labeling indices (LI), duration of S-phase (Ts), and total cell cycle time (Tc) were determined in 54 standard-risk, newly diagnosed patients with acute myeloid leukemia following an infusion of bromodeoxyuridine. Remission induction therapy consisting of cytosine arabinoside and daunomycin was then administered to all patients, followed by three courses of consolidation to those who achieved complete remissions (CR). Older patients appeared to have more rapidly cycling cells (P = .003). No unique cell cycle characteristics were identified for patients who achieved remission versus those who had resistant disease. However, the pretherapy cell cycle characteristics were a strong prognosticator for remission duration. CR patients were divided into those whose leukemic cell Tc were above median (A) and below median (B). Among 14 B patients, median duration of response was 211 days, and all relapsed by day 600. Among 18 A patients, the median has not as yet been reached, with nine patients in continuous complete remission (log rank P = .007, Wilcoxon P = .04). We conclude that cell cycle characteristics of leukemic cells play a role in determining remission duration, perhaps because the leukemic cells of the former patients regrow slowly between courses of chemotherapy.

scholarly journals Lin28A/CENPE Promoting the Proliferation and Chemoresistance of Acute Myeloid Leukemia

2021 ◽  
Vol 11 ◽  
Author(s):  
Mingyue Shi ◽  
Junwei Niu ◽  
Xiaona Niu ◽  
Honggang Guo ◽  
Yanliang Bai ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Acute Myeloid Leukemia ◽  
Myeloid Leukemia ◽  
Cell Cycle Progression ◽  
Rna Binding ◽  
Leukemic Cells ◽  
Luciferase Reporter ◽  
Rip Assay ◽  
Acute Myeloid

The prognosis of chemoresistant acute myeloid leukemia (AML) is still poor, mainly owing to the sustained proliferation ability of leukemic cells, while the microtubules have a major role in sustaining the continuity of cell cycle. In the present study, we have identified CENPE, a microtubular kinesin-like motor protein that is highly expressed in the peripheral blood of patients with chemoresistant AML. In our in vitro studies, knockdown of CENPE expression resulted in the suppression of proliferation of myeloid leukemia cells and reversal of cytarabine (Ara-C) chemoresistance. Furthermore, Lin28A, one of the RNA-binding oncogene proteins that increase cell proliferation and invasion and contribute to unfavorable treatment responses in certain malignancies, was found to be remarkably correlated with CENPE expression in chemoresistance AML. Overexpression of LIN28A promoted the proliferation and Ara-C chemoresistance of leukemic cells. RIP assay, RNA pull-down, and dual luciferase reporter analyses indicated that LIN28A bound specifically to the promoter region GGAGA of CENPE. In addition, the impacts of LIN28A on cell growth, apoptosis, cell cycle progression, and Ara-C chemoresistance were reverted by the knockdown of CENPE. Hence, Lin28A/CENPE has enhanced the proliferation and chemoresistance of AML, and therefore, it could be a prospective candidate for AML treatment.

scholarly journals Synergy between FLT3-ITD and p53 Haploinsufficiency or Loss in the Development of Acute Myeloid Leukemia

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 771-771
Author(s):  
Zengkai Pan ◽  
Min Yang ◽  
Kezhi Huang ◽  
Guntram Buesche ◽  
Gudrun Göhring ◽  
...  
Keyword(s):  
Acute Myeloid Leukemia ◽  
Acute Leukemia ◽  
Median Survival ◽  
Proteasome Inhibitor ◽  
Myeloid Leukemia ◽  
P53 Mutation ◽  
Leukemic Cells ◽  
Efficient Treatment ◽  
Acute Myeloid

Abstract FLT3-ITD (internal tandem duplication) is a late event in the pathogenesis of acute myeloid leukemia (AML). Identification of early cooperating events for FLT3 mutations may improve our understanding of the pathogenesis of AML and lead to a more efficient treatment and improved outcome for AML patients. p53 alteration can be found in up to 70% of AML patients with complex karyotypes, while studies from our group and others show a relatively low incidence of p53 mutation (generally around 10%) in other AML patients. Dysfunction of p53 pathway resulting from overexpressed MDM2/MDM4 is more often found than p53 mutations in patients with de novo AML. An early mouse study identified the FLT3 gene to be preferentially mutated by insertional mutagenesis in p53 knock-out but not in p53 wild-type tumors. Importantly, p53 mutation appears to be an early event in the pathogenesis of AML. In this study, we investigated whether p53 haploinsufficiency or loss cooperates with FLT3-ITD in the induction of AML. To this end, we crossed FLT3-ITD knock-in mice with p53 knockout mice to generate mice harboring both ITD/ITD and p53 knockout mutations. ITD/ITD; p53+/- (FLT3-ITD homozygous and p53 heterozygous) mice became moribund much faster than mice with ITD/ITD alone or p53+/- alone. The median survival of ITD/ITD;p53+/- mice (n=69) was 313 days, which was significantly shorter than that of ITD/ITD mice (littermates of ITD/ITD; p53+/- mice, 583 days, n=16), p53+/- mice (521 days, n=30), and WT mice (862 days, n=10) (p<0.0001). Interestingly, the median survival ITD/ITD;p53-/- mice (FLT3-ITD homozygous and p53 homozygous, littermates of ITD/ITD; p53+/- mice, n=11) was further reduced (125 days), and significantly shorter than ITD/ITD; p53+/- mice and p53-/- mice (148 days, n=15) (p<0.05). Moreover, cooperation of FLT3-ITD and p53 inactivation also significantly altered disease spectrum in mice. While AML was observed only in 25% ITD/ITD mice and p53+/- mice mainly developed solid tumors, 88% ITD/ITD; p53+/- mice developed acute leukemia including AML (58%). The incidence of AML was further increased in ITD/ITD; p53-/- mice (73%, 8/11). All ITD/ITD; p53-/- mice developed acute leukemia, while 72% p53-/- mice developed lymphoma/lymphoblastic leukemia. Interestingly, >50% ITD/ITD; p53-/- mice and ITD/ITD; p53+/- mice with acute leukemia demonstrated a bi-clone disease, with co-existence of AML and ALL. Unexpectedly, all analyzed murine AMLs with complete or heterozygous loss of p53 (n=9) showed normal karyotypes. p53 haploinsufficiency or loss did not increase self-renewal of hematopoietic stem/progenitor cells as defined by serial replanting assays in vitro and limiting dilution transplantation of leukemic cells in vivo. However, animals with only AML in the ITD/ITD; p53+/- group showed a significant increase of CMPs in comparison with ITD/ITD mice with CMML, P53+/- and WT mice, e.g. a 66-fold increase of CMPs in #1376 mouse compared with WT mice. This suggests that the block of differentiation from CMPs to GMPs may have contributed to the development of AML. P53 haploinsufficiency or loss reduced the sensitivity of murine FLT3-ITD leukemia to crenolanib in vitro (IC50: 313.3nM, 461.4nM, and 185.8nM for ITD/ITD;p53+/-, ITD/ITD;p53-/-, and ITD/ITD leukemia, respectively), but not their sensitivity to midostaurin (IC50: 117.3nM, 110.2nM, and 201.3nM). To develop an efficient therapy for p53-mutated AML, we firstly tried to combine FLT3 inhibitors and MDM2 antagonist idasanutlin, FLT3 inhibitor and Bcl2 inhibitor venetoclax. Unfortunately, these did not enhance induction of apoptosis in leukemic cells from ITD/ITD;p53+/- mice. However, exposure to the proteasome inhibitor carfilzomib had a strong cytotoxic effect against ITD/ITD; p53+/- and ITD/ITD;p53-/-, leukemic cells (IC50: 5.2nM and 17.1nM vs. 6.8nM for ITD/ITD leukemia). In addition, the combination of carfilzomib with midostaurin showed an additive cytotoxicity in murine ITD/ITD; p53+/- leukemia as well as in primary leukemic cells from most patients with AML (n=10). Taken together, our data indicate a strong cooperating effect of FLT3-ITD and p53 haploinsufficiency or loss in the induction of AML and ALL. Our data emphasize more careful analysis of p53 deregulation in AML. Targeting FLT3 in combination with drugs working independently of p53 status, such as proteasome inhibitor carfilzomib, might improve outcomes of AML patients. Disclosures Ganser: Novartis: Membership on an entity's Board of Directors or advisory committees.

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