Biology subjects

Kramer, O. H.

Publications and source records attributed to Kramer, O. H..

5 recordsLinked to original sources

bioRxiv2025

The Deacetylases HDAC1 and HDAC2 Safeguard BCR-ABL-positive Cells from Replication Stress-Induced Apoptosis via the Nuclear to Mitochondrial p73-NOXA Axis

Histone deacetylases (HDACs) are key epigenetic regulators that are frequently dysregulated in cancer cells. Context-specific dependencies of tumor cell survival on HDACs and the mechanisms through which HDACs determine cell stress responses by specific oncogenic pathways remain to be understood. Here we unravel how the class I deacetylases HDAC1 and HDAC2 control the fate of chronic myeloid leukemia (CML) cells undergoing DNA replication stress. Compared to normal myeloid cells (n=690), CML cells overexpress HDAC1 and HDAC2 (n=234-274). We reveal that HDAC1 and HDAC2 protect cultured and primary CML cells with the hyperactive BCR-ABL tyrosine kinase from programmed cell death through apoptosis upon DNA replication stress. Using transcriptomics and genetically defined knockdown and knockout model systems, we demonstrate that these effects depend on the transcription factor p73 and its pro-apoptotic target gene NOXA. Upon DNA replication stress, p73 binds to the NOXA promoter but only upon additional inactivation of HDAC1/HDAC2 the NOXA gene becomes transcribed. BCR-ABL translocate to the nucleus to catalyze p73 phosphorylation at tyrosine-99 for the induction of p73 and mitochondrial NOXA. Thus, the BCR-ABL oncogene creates a selective vulnerability to HDAC1/HDAC2 inhibitors, driving a cytotoxic shift in replication stress responses towards apoptosis. These data highlight HDAC1/HDAC2 as potential therapeutic targets in CML. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=138 SRC="FIGDIR/small/694110v1_ufig1.gif" ALT="Figure 1"> View larger version (15K): org.highwire.dtl.DTLVardef@1ec0c51org.highwire.dtl.DTLVardef@1cd759org.highwire.dtl.DTLVardef@17e29a8org.highwire.dtl.DTLVardef@1e0fb50_HPS_FORMAT_FIGEXP M_FIG C_FIG

cancer biology↗

bioRxiv2025

Rapid proteasomal degradation of mutant feline McDonough sarcoma-like tyrosine kinase-3 overcomes tyrosine kinase inhibitor resistance of acute myeloid leukemia cells

BackgroundFeline McDonough sarcoma (FMS)-related receptor tyrosine kinase 3 with activating internal tandem duplications (FLT3-ITD) causes acute myeloid leukemia (AML). Targeted protein degraders for FLT3 have evolved as drugs against leukemia. MethodsWe synthesized and characterized MA191 as novel von Hippel-Lindau (VHL)-based proteolysis targeting chimera (PROTAC) for FLT3. We analyzed protein expression, protein degradation mechanisms, and posttranslational modifications by immunoblot. Selective proteasome modulation, an inactive stereoisomer of MA191, and siRNA confirmed the event-driven degradation of FLT3-ITD. Hematopoietic cell survival and differentiation were determined by flow cytometry using apoptosis and cell surface markers. As models, we used cultured and primary human AML cells, FLT3 inhibitor-resistant AML cells, mature blood cells, and hematopoietic stem cells. We scrutinized the databases DepMap, GEPIA2, Hemap, and HPA to assess FLT3 expression and patient survival. Experiments with Danio rerio larvae verified in vivo anti-leukemic activity of MA191. ANOVA and Bonferroni correction were used for statistics. ResultsMA191 is a rapid nanomolar apoptosis inducer in AML cells harboring FLT3-ITD (IC50=10.16-11.6 nM; EC50=0.015-0.883 {micro}M). A stereoisomer of MA191 that cannot recruit VHL demonstrates that elimination of FLT3-ITD is superior to its inhibition. MA191 abrogates FLT3 inhibitor resistance from rebound activation of mitogen-activated kinases. Rapid depletion of FLT3-ITD by MA191 (DC50=10 nM) requires VHL, neddylation, and the pro-apoptotic BH3-only protein BIM. Reduction of FLT3-ITD by MA191 precedes apoptosis. This reveals an apoptosis-independent function of BIM on protein stability. Leukemia cells express more FLT3 than healthy cells (n=3675/n=1249) and FLT3 expression is associated with worse AML patient survival (p=0.0099). MA191 does not harm blood cells and bone marrow progenitor cells and does not disturb myeloid blood cell differentiation. In Danio rerio, MA191 halts AML cell proliferation without significant toxicity. Anti-leukemic effects of MA191 are not susceptible to anti-apoptotic effects of human stromal cells and mutations in the tyrosine kinase of FLT3-ITD that confer resistance to selective FLT3 inhibitors. Conclusionsthese insights and the disclosure of the structure of MA191 provide a framework for an improved design of PROTACs that target mutant FLT3 and are not vulnerable to extrinsic and intrinsic resistance mechanisms. Degradation kinetics appear as determinant of such resistance breakers. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=188 SRC="FIGDIR/small/660791v1_ufig1.gif" ALT="Figure 1"> View larger version (29K): org.highwire.dtl.DTLVardef@17e5988org.highwire.dtl.DTLVardef@27fbc4org.highwire.dtl.DTLVardef@1023648org.highwire.dtl.DTLVardef@39bdcb_HPS_FORMAT_FIGEXP M_FIG C_FIG

pharmacology and toxicology↗

bioRxiv2023

The protein phosphatase-2A subunit PR130 is linked to cytotoxic protein aggregate formation in mesenchymal pancreatic ductal adenocarcinoma cells

Protein phosphatase-2A (PP2A) is a major source of cellular serine/threonine phosphatase activity. PP2A B-type subunits regulate the intracellular localization and the catalytic activity of PP2A-A/PP2A-C complexes towards individual proteins. There is limited knowledge on how PP2A B-type subunits regulate biologically important functions and if these subunits determine the growth and drug responsiveness of tumor cells. Pancreatic ductal adenocarcinoma (PDAC) is a dismal disease with poor prognosis. Mesenchymal PDAC subtypes are more aggressive and metastasis-prone than epithelial subtypes. We show that mesenchymal PDAC cells express significantly higher levels of the PP2A B-type subunit PR130 and its mRNA Ppp2r3a than epithelial PDAC cells (n=38). Among 17 PP2A B-type subunits, this differential regulation is unique for Ppp2r3a and PR130. The higher levels of PR130 in mesenchymal PDAC cells are linked to their vulnerability to the PP2A inhibitor phendione. Phendione induces apoptosis and an accumulation of cytotoxic protein aggregates in such cells. These processes occur independently of the major tumor suppressor p53, which is frequently mutated in PDAC cells. Proteomic analyses reveal that phendione upregulates the chaperone heat shock protein HSP70 in mesenchymal PDAC cells. Inhibition of HSP70 promotes phendione-induced apoptosis. We additionally disclose that phendione promotes a proteasomal degradation of PR130. Genetic elimination of PR130 sensitizes mesenchymal PDAC cells to phendione-induced apoptosis and protein aggregate formation. These data illustrate pharmacologically amenable, selective dependencies of mesenchymal PDAC cells on PP2A-PR130 and HSP70. PP2A inhibition triggers a harmful accumulation of protein aggregates in neurons. This undesired mechanism might be exploited to kill mesenchymal tumor cells. O_FIG O_LINKSMALLFIG WIDTH=137 HEIGHT=200 SRC="FIGDIR/small/556106v1_ufig1.gif" ALT="Figure 1"> View larger version (40K): org.highwire.dtl.DTLVardef@c8a47forg.highwire.dtl.DTLVardef@a7abfforg.highwire.dtl.DTLVardef@d18a3org.highwire.dtl.DTLVardef@1cc9943_HPS_FORMAT_FIGEXP M_FIG C_FIG HIGHLIGHTS[tpltrtarr] The PP2A subunit PR130 is a molecular marker of mesenchymal PDAC cells [tpltrtarr]The small molecule PP2A inhibitor phendione selectively kills mesenchymal PDAC cells [tpltrtarr]Phendione decreases PR130 through proteasomes and selectively increases the heat shock protein 70 kDa in mesenchymal PDAC cells [tpltrtarr]HSP70 promotes cell survival upon inhibition of PP2A [tpltrtarr]PP2A-PR130 regulates the accumulation of cytotoxic protein aggregates in mesenchymal PDAC cells

cancer biology↗

bioRxiv2023

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

IntroductionPosttranslational 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. ObjectivesWe 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. MethodsWe 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. ResultsNovel 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. ConclusionThese 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. HighlightsO_LINovel HDACi with nanomolar activity against leukemic cells were synthesized. C_LIO_LIHDACi of the KH-series are superior to a clinical grade HDACi. C_LIO_LIHDACi of the KH-series modulate acetylation and phosphorylation of proteins. C_LIO_LIThe new HDACi KH16 regulates cell cycle arrest, apoptosis, and autophagy. C_LIO_LIApoptosis acts upstream of autophagy in KH16-treated cells. C_LI

pharmacology and toxicology↗

bioRxiv2021

NOXA expression drives synthetic lethality to RUNX1 inhibition in pancreatic cancer

Evasion from drug-induced apoptosis is a crucial mechanism of cancer treatment resistance. The pro-apoptotic protein NOXA marks an aggressive pancreatic ductal adenocarcinoma (PDAC) subtype. To identify drugs that unleash the death-inducing potential of NOXA, we performed an unbiased drug screening experiment. In NOXA-deficient isogenic cellular models we identified an inhibitor of the transcription factor heterodimer CBF{beta}/RUNX1. By genetic gain and loss of function experiments we validated that the mode of action depends on RUNX1 and NOXA. Of note, RUNX1 expression is significantly higher in PDACs compared to normal pancreas. We show that pharmacological RUNX1 inhibition significantly blocks tumor growth in vivo and in primary patient-derived PDAC organoids. Through genome wide analysis, we detected that RUNX1-loss reshapes the epigenetic landscape, which gains H3K27ac enrichment at the NOXA promoter. Our study demonstrates a previously unknown mechanism of NOXA-dependent cell death, which can be triggered pharmaceutically. Therefore, our data show a novel way to target a therapy resistant PDAC, an unmet clinical need. SignificanceRecent evidence demonstrated the existence of molecular subtypes in pancreatic ductal adenocarcinoma (PDAC), which resist all current therapies. The paucity of therapeutic options, including a complete lack of targeted therapies, underscore the urgent and unmet medical need for the identification of targets and novel treatment strategies for PDAC. Our study unravels a function of the transcription factor RUNX1 in apoptosis regulation in PDAC. We show that pharmacological RUNX1 inhibition in PDAC is feasible and leads to NOXA-dependent apoptosis. The development of targeted therapies that influence the transcriptional landscape of PDAC might have great benefits for patients who are resistant to conventional therapies. RUNX1 Inhibition as a new therapeutic intervention offers an attractive strategy for future therapies.

cancer biology↗