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. 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. 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. Novel HDACi show nanomolar activity against class I HDACs. These agents are superior to the clinically used hydroxamic acid HDACi SAHA (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 115 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 caspase-dependent processing of the autophagy proteins ULK1 and p62. These data reveal that HDACs are required to stabilize autophagy proteins through suppression of apoptosis in leukemic cells. HDAC3 appears as a valid anti-cancer target for pharmacological intervention.

Epigenetic modifiers of the histone deacetylase (HDAC) family are often dysregulated in cancer cells. Experiments with small molecule HDAC inhibitors (HDACi) have proven that HDACs are a vulnerability of transformed cells. We evaluated a novel hydroxamic acid-based HDACi (KH16; termed yanostat) in human pancreatic ductal adenocarcinoma (PDAC) cells, short- and long-term cultured colorectal cancer (CRC) cells, and retinal pigment epithelial cells. We show that KH16 induces cell cycle arrest and apoptosis, both time and dose dependently in PDAC and CRC cells. This is associated with altered expression of BCL2 family members controlling intrinsic apoptosis. Recent data illustrate that PDAC cells frequently have an altered expression of the pro-apoptotic BH3-only protein NOXA and that HDACi induce an accumulation of NOXA. Using PDAC cells with a deletion of NOXA by CRISPR-Cas9, we found that a lack of NOXA delayed apoptosis induction by KH16. These results suggest that KH16 is a new chemotype of hydroxamic acid HDACi with superior activity against solid tumor-derived cells. Thus, KH16 is a scaffold for future research on compounds with nanomolar activity against HDACs.

Checkpoint kinase‐1 (CHK1) controls DNA replication and repair. Tumor cells depend on CHK1, whose high levels are associated with worse patient prognosis. We define a bona fide proteolysis‐targeting‐chimera (PROTAC) for CHK1. PROTAC MA203 contains the type I kinase inhibitor rabusertib, which preferentially inhibits activated CHK1, and the cereblon (CRBN) ligand pomalidomide. MA203 accelerates CRBN‐dependent proteasomal degradation of CHK1 in solid tumor‐derived cells and acute leukemia cells. Chemotherapy‐induced DNA replication stress and a consequent activation of CHK1 accelerate this event‐driven process which promotes DNA damage and tumor cell apoptosis. Biochemical and cellular target engagement studies confirm the potency and selectivity of MA203. MA203 does not damage healthy differentiated and primitive hematopoietic cells, stromal cells, and retinal epithelial cells. MA203 is superior to its corresponding kinase inhibitor concerning DNA damage, dysregulation of BCL2 proteins, and apoptosis induction. These processes occur independently of the tumor‐suppressive transcription factor p53. Elimination of CHK1 protein as structural element, but not its inhibition per se, triggers a proteasomal degradation of key DNA replication and repair proteins. Genetic CHK1 elimination confirms that such newly recognized functions of CHK1 rely on functions beyond its well‐known catalytic activity. Thus, kinase‐independent functions of CHK1 can be exploited with innovative pharmacological agents.

Background: Feline 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. Methods: We 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. Results: MA191 is a rapid nanomolar apoptosis inducer in AML cells harboring FLT3-ITD (IC ₅₀ =10.16-11.6 nM; EC ₅₀ =0.015-0.883 µM). 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 (DC ₅₀ =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. Conclusions: these 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. Abstract Figure:

Background/Objectives: Acute myeloid leukemia (AML) is a hematological malignancy frequently driven by mutations in the FLT3 gene, particularly internal tandem duplications (FLT3-ITD), which contribute to aberrant cell proliferation and resistance to tyrosine kinase inhibitors (FLT3i). The limitations of current FLT3i therapies, including drug resistance, off-target effects, and poor selectivity, necessitate the development of novel therapeutic strategies. Proteolysis-targeting chimeras (PROTACs) represent a promising approach to achieving degradation of oncogenic proteins. Methods: We developed FLT3-targeting PROTACs based on the previously described compound MA49, with a focus on linker modifications to improve degradation efficiency and pharmacokinetic properties. Results: Among these, compounds MA190 and MA191, containing rigid cyclohexyl-piperidine/piperazine linkers, demonstrate superior degradation of FLT3-ITD in MV4-11 AML cells at nanomolar concentrations, achieving >95% reduction in FLT3-ITD levels, outperforming MA49. In addition to improved kinase selectivity, good solubility, and plasma stability, MA190 and MA191 also exhibit excellent metabolic stability, whereas the predecessor PROTAC MA49 was unstable in microsomal assays. In cellular assays, MA190 and MA191 induce potent apoptosis in FLT3-ITD⁺ AML cells but have minimal effects on cells with wild-type FLT3. Proteomics reveal that MA191 also degrades MAPK14 (p38α), a kinase upregulated in leukemia, in addition to FLT3. Conclusions: Dual targeting of FLT3-ITD and MAPK14 enhances proapoptotic signaling without any cytotoxic effect on normal human HEK293 cells. The co-inhibition using MA191 or a combination of doramapimod (a MAPK14 inhibitor) with a non-degrading FLT3 inhibitor result in greater caspase-3 activation than either treatment alone. This synergistic effect can be a therapeutic advantage, as several oncogenic drivers are switched off simultaneously by MA191.