scholarly journals Inhibitors of class I HDACs and of FLT3 combine synergistically against leukemia cells with mutant FLT3

2021 ◽  
Author(s):  
Vanessa Wachholz ◽  
Al-Hassan M. Mustafa ◽  
Yanira Zeyn ◽  
Sven J. Henninger ◽  
Mandy Beyer ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Kinase Inhibitor ◽  
Class I ◽  
Leukemic Cells ◽  
Cell Cycle Distribution ◽  
Checkpoint Kinases ◽  
Downstream Signaling ◽  
Cyclin Dependent Kinase Inhibitor ◽  
Drug Dependent ◽  
Class I Hdacs

AbstractAcute myeloid leukemia (AML) with mutations in the FMS-like tyrosine kinase (FLT3) is a clinically unresolved problem. AML cells frequently have a dysregulated expression and activity of epigenetic modulators of the histone deacetylase (HDAC) family. Therefore, we tested whether a combined inhibition of mutant FLT3 and class I HDACs is effective against AML cells. Low nanomolar doses of the FLT3 inhibitor (FLT3i) AC220 and an inhibition of class I HDACs with nanomolar concentrations of FK228 or micromolar doses of the HDAC3 specific agent RGFP966 synergistically induce apoptosis of AML cells that carry hyperactive FLT3 with an internal tandem duplication (FLT3-ITD). This does not occur in leukemic cells with wild-type FLT3 and without FLT3, suggesting a preferential toxicity of this combination against cells with mutant FLT3. Moreover, nanomolar doses of the new FLT3i marbotinib combine favorably with FK228 against leukemic cells with FLT3-ITD. The combinatorial treatments potentiated their suppressive effects on the tyrosine phosphorylation and stability of FLT3-ITD and its downstream signaling to the kinases ERK1/ERK2 and the inducible transcription factor STAT5. The beneficial pro-apoptotic effects of FLT3i and HDACi against leukemic cells with mutant FLT3 are associated with dose- and drug-dependent alterations of cell cycle distribution and DNA damage. This is linked to a modulation of the tumor-suppressive transcription factor p53 and its target cyclin-dependent kinase inhibitor p21. While HDACi induce p21, AC220 suppresses the expression of p53 and p21. Furthermore, we show that both FLT3-ITD and class I HDAC activity promote the expression of the checkpoint kinases CHK1 and WEE1, thymidylate synthase, and the DNA repair protein RAD51 in leukemic cells. A genetic depletion of HDAC3 attenuates the expression of such proteins. Thus, class I HDACs and hyperactive FLT3 appear to be valid targets in AML cells with mutant FLT3.

The Transcription Factor ELF4 Promotes Survival of Myeloid Leukemic Stem Cells.

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 1209-1209
Author(s):  
Chun Shik Park ◽  
Koramit Suppipat ◽  
H. Daniel Lacorazza
Keyword(s):  
Stem Cells ◽  
Transcription Factor ◽  
Kinase Inhibitor ◽  
Conflicts Of Interest ◽  
Blast Crisis ◽  
Philadelphia Chromosome ◽  
Lymphocytic Leukemia ◽  
Leukemic Cells ◽  
Leukemic Stem Cells ◽  
Hematopoietic Stem

Abstract Abstract 1209 Chronic myeloid leukemia (CML) is a myeloproliferative disease that originate in hematopoietic stem cells (HSCs) as a result of the t(9;22) translocation, giving rise to the Ph (Philadelphia chromosome) and BCR-ABL oncoprotein. Although treatment of CML patients with tyrosine kinase inhibitor can efficiently eliminate most leukemic cells, chemoresistant leukemic stem cells (LSCs) can survive and drive recurrence of CML in these patients. A number of genes have been described to promote or inhibit proliferation of LSCs. Some of them have similar roles in normal HSCs. The transcription factor ELF4 promotes cell cycle entry of quiescent HSCs during homeostasis (Lacorazza et al., 2006). Thus, to investigate the function of ELF4 in CML initiation and maintenance, we developed a BCR-ABL-induced CML-like disease using retroviral transfer of BCR-ABL in Elf4-null bone marrow (BM) cells. We first investigated whether ELF4 is required for the induction of CML. Recipient mice of BCR-ABL-transduced WT BM cells developed CML and died with a latency 16–23 days, whereas recipient mice of BCR-ABL-transduced Elf4-/- BM cells showed longer latency of 45–47 days (n=20; p<0.0005). Progression of leukemia was monitored in peripheral blood, BM and spleen by flow cytometry. In mice transplanted with BCR-ABL-transduced Elf4-null BM cells, Gr-1+ leukemic cells expanded the first two weeks after BM transplantation followed by a decline at expense of a secondary expansion of B220+ cells. In contrast, Gr-1+ leukemic cells continuously expanded in mice receiving BCR-ABL-transduced WT BM cells. These results suggest that loss of ELF4 causes a profound abrogation in BCR-ABL-induced CML, while allowing progression of B-cell acute lymphocytic leukemia. Since loss of Elf4 led to impaired maintenance of myeloid leukemic cells, we postulated that ELF4 may affect survival of LSCs. Thus, we analyzed the frequency of Lin-c-Kit+Sca-1+ (LSK) cells that are BCR-ABL positive in BM and spleen. We found that BCR-ABL+ LSK cells were significantly reduced in recipients of BCR-ABL-transduced Elf4-/- BM cells. These studies indicate that ELF4 is essential to maintain the LSC pool in CML acting as a molecular switch between myeloid and lymphoid blast crisis. Disclosures: No relevant conflicts of interest to declare.

Small Interfering RNA against Bcr-Abl Transcripts Sensitized Mutated T315I Cells to Nilotinib.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 2188-2188
Author(s):  
Michael Koldehoff ◽  
Ahmet H Elmaagacli
Keyword(s):  
Tyrosine Kinase ◽  
Cell Lines ◽  
Small Interfering Rna ◽  
Kinase Inhibitor ◽  
Conflicts Of Interest ◽  
Leukemic Cells ◽  
Cell Cycle Distribution ◽  
Multidrug Resistance Gene ◽  
Inhibition Of Proliferation ◽  
Interfering Rna

Abstract 2188 Poster Board II-165 Background: Selective inhibition of the BCR-ABL tyrosine kinase by RNA interference has been demonstrated in leukemic cells. Therefore, we evaluated the specific bcr-abl small interfering RNA (siRNA) silencing in BCR-ABL positive cell lines, including those resistant to imatinib (IM) and particularly those with the T315I mutation. Design and Methods: The factor-independent 32Dp210 bcr-abl oligoclonal cell lines and in human IM-resistant bcr-abl positive cells from different patients with leukemia disorders were investigated. The effects of bcr-abl siRNA or the combination of bcr-abl siRNA with both IM and nilotinib were compared with those of the ABL inhibitors IM and nilotinib. Results: Coadministration of bcr-abl siRNA with IM or nilotinib dramatically reduced the .{/MAIN;133}BCR-ABL expression in wild-type (wt) and mutated bcr-abl cells and increased the lethal capacity. The bcr-abl siRNA significantly induced apoptosis and inhibited proliferation in wt (p<0.0001) and mutated cells (H396P, T315I, p<0.0001) versus controls. Cotreatment of bcr-abl siRNA with IM or nilotinib resulted in an increased inhibition of proliferation and induction of apoptosis as compared to IM or nilotinib (p<0.0001) in T315I cells. Furthermore, the combination of bcr-abl siRNA with IM or nilotinib significantly (p<0.01) reversed multidrug resistance gene 1-dependent resistance of mutated cells. In T315I cells bcr-abl siRNA with nilotinib has shown powerful effect on the cell-cycle distribution. Conclusions: Our data suggest that silencing by bcr-abl siRNA with IM or nilotinib may be associated with an additive antileukemic activity against tyrosine kinase inhibitor-sensitive and –resistant BCR-ABL cells, and might be an alternative approaches to overcome BCR-ABL mutations. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Down-Regulation of CK2α Leads toUp-Regulation of the Cyclin-Dependent Kinase Inhibitor p27KIP1 in Conditions Unfavorable for the Growth of Myoblast Cells

2020 ◽  
Vol 54 (6) ◽  
pp. 1177-1198
Keyword(s):  
Cell Cycle ◽  
Transcription Factor ◽  
Cell Growth ◽  
Knockout Mouse ◽  
Cell Cycle Progression ◽  
Molecular Mechanisms ◽  
Kinase Inhibitor ◽  
Cyclin Dependent Kinase ◽  
Cycle Progression ◽  
Cyclin Dependent Kinase Inhibitor

BACKGROUND/AIMS: Compelling evidence indicates that CK2α, which is one of the two catalytic isoforms of protein kinase CK2, is required for cell viability and plays an important role in cell proliferation and differentiation. While much is known on CK2 in the context of disease states, particularly cancer, its critical role in non-cancerous cell growth has not been extensively investigated. METHODS: In the present study, we have employed a cell line derived from rat heart with inducible down-regulation of CK2α and CK2α-knockout mouse tissue to identify CK2-mediated molecular mechanisms regulating cell growth. For this, we have performed Incucyte® live-cell analysis and applied flow cytometry, western blot, immunoprecipitation, immunohistochemistry, RT-qPCR and luciferase-based methods. RESULTS: Here, we show that lack of CK2α results in significantly delayed cell cycle progression through G1, inhibition of cyclin E-CDK2 complex, decreased phosphorylation of Rb protein at S795, and inactivation of E2F transcription factor. These events are accompanied by nuclear accumulation and up-regulation of the cyclin-dependent kinase inhibitor p27KIP1 in cells and CK2α-knockout mouse tissues. We found that increased levels of p27KIP1 are mainly attributable to post-translational modifications, namely phosphorylation at S10 and T197 amino acid residues catalyzed by Dyrk1B and AMPK, respectively, as silencing of FoxO3A transcription factor, which activates CDKN1B the gene coding for p27KIP1, does not result in markedly decreased expression levels of the corresponding protein. Interestingly, simultaneous silencing of CK2α and p27KIP1 significantly impairs cell cycle progression without increasing cell death. CONCLUSION: Taken together, our study sheds light on the molecular mechanisms controlling cell cycle progression through G1 phase when myoblasts proliferation potential is impaired by CK2α depletion. Our results suggest that elevated levels of p27KIP1,which follows CK2α depletion, contribute to delay the G1-to-S phase transition. Effects seen when p27KIP1 is down-regulated are independent of CK2α and reflect the protective role exerted by p27KIP1 under unfavorable cell growth conditions.

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