Mechanistic insights into p53‐regulated cytotoxicity of combined entinostat and irinotecan against colorectal cancer cells

Introduction: Posttranslational modification of proteins by reversible acetylation regulates key biological processes. Histone deacetylases (HDACs) catalyze protein deacetylation and are frequently dysregulated in tumors. This has spurred the development of HDAC inhibitors (HDACi). Such epigenetic drugs modulate protein acetylation, eliminate tumor cells, and are approved for the treatment of blood cancers. Objectives: We aimed to identify novel, nanomolar HDACi with increased potency over existing agents and selectivity for the cancer-relevant class I HDACs (HDAC1/-2/-3/-8). Moreover, we wanted to define how such drugs control the apoptosis-autophagy interplay. As test systems, we used human leukemic cells and embryonic kidney-derived cells. Methods: We synthesized novel pyrimidine-hydroxamic acid HDACi (KH9/KH16/KH29) and performed in vitro activity assays and molecular modeling of their direct binding to HDACs. We analyzed how these HDACi affect leukemic cell fate, acetylation, and protein expression with flow cytometry and immunoblot. The publicly available DepMap database of CRISPR-Cas9 screenings was used to determine sensitivity factors across human leukemic cells. Results: Novel HDACi show nanomolar activity against class I HDACs. These agents are superior to the clinically used hydroxamic acid HDACi vorinostat. Within the KH-series of compounds, KH16 (yanostat) is the most effective inhibitor of HDAC3 (IC50 = 6 nM) and the most potent inducer of apoptosis (IC50 = 110 nM; p<0.0001) in leukemic cells. KH16 though spares embryonic kidney-derived cells. Global data analyses of knockout screenings verify that HDAC3 is a dependency factor in human blood cancer cells of different lineages, independent of mutations in the tumor suppressor p53. KH16 alters pro- and anti-apoptotic protein expression, stalls cell cycle progression, and induces a caspase-dependent processing of the autophagy proteins ULK1 and p62. Conclusion: These data reveal that HDACs are required to stabilize autophagy proteins through a suppression of apoptosis in leukemic cells. HDAC3 appears as a valid anti-cancer target for pharmacological intervention.

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“…Previous studies showed that class I HDAC inhibition reduced p53 levels [32,57–60]. Congruently, KH16 attenuates p53 levels.…”

Section: Discussionmentioning

confidence: 99%

Novel hydroxamic acid derivative induces apoptosis and constrains autophagy in leukemic cells

Fischer

Mustafa

Hausmann

et al. 2023

Preprint

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“…The increase of p21 in MV4-11 cells is not linked to an increase in p53 levels. This can be explained by sufficient levels of basal p53, and it needs to be considered that class I HDACs critically control p53 by direct lysine acetylation (Marx et al 2021 ).…”

Section: Discussionmentioning

confidence: 99%

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

et al. 2021

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Acute 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.

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“…We want to emphasize the fact that the HROC-Xenobank has, already in its establishment phase, supported “standard” in vivo studies [ 46 , 47 ], detailed molecular pathway investigations [ 48 ], biomarker studies, and more basic studies which took advantage of snap-frozen samples [ 49 ]. A first pre-clinical PDX trial has been started in-house, preceded by a dose finding study [ 34 ].…”

Section: Discussionmentioning

confidence: 99%

The HROC-Xenobank—A High Quality Assured PDX Biobank of >100 Individual Colorectal Cancer Models

Matschos

Bürtin

Kdimati

et al. 2021

Cancers

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Based on our research group’s large biobank of colorectal cancers (CRC), we here describe the ongoing activity of establishing a high quality assured PDX biobank for more than 100 individual CRC cases. This includes sufficient numbers of vitally frozen (n > 30 aliquots) and snap frozen (n > 5) backups, “ready to use”. Additionally, PDX tumor pieces were paraffin embedded. At the current time, we have completed 125 cases. This resource allows histopathological examinations, molecular characterizations, and gene expression analysis. Due to its size, different issues of interest can be addressed. Most importantly, the application of low-passage, cryopreserved, and well-characterized PDX for in vivo studies guarantees the reliability of results due to the largely preserved tumor microenvironment. All cases described were molecularly subtyped and genetic identity, in comparison to the original tumor tissue, was confirmed by fingerprint analysis. The latter excludes ambiguity errors between the PDX and the original patient tumor. A cancer hot spot mutation analysis was performed for n = 113 of the 125 cases entities. All relevant CRC molecular subtypes identified so far are represented in the Hansestadt Rostock CRC (HROC)-Xenobank. Notably, all models are available for cooperative research approaches.

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Mechanistic insights into p53‐regulated cytotoxicity of combined entinostat and irinotecan against colorectal cancer cells

Cited by 17 publications

References 58 publications

Novel hydroxamic acid derivative induces apoptosis and constrains autophagy in leukemic cells

Novel hydroxamic acid derivative induces apoptosis and constrains autophagy in leukemic cells

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

The HROC-Xenobank—A High Quality Assured PDX Biobank of >100 Individual Colorectal Cancer Models

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