Biology subjects

Heine, A.

Publications and source records attributed to Heine, A..

6 recordsLinked to original sources

bioRxiv2026

EZH2 inactivation drives MAPK-dependent vulnerability to MEK inhibition in RAS-mutant CMML

Chronic myelomonocytic leukemia (CMML) is a heterogeneous hematologic malignancy with limited therapeutic options. Although RAS and RAS-modifying mutations (RASmut) are common and associated with poor prognosis, targeting RAS signaling has shown limited clinical success. Here, we define co-occurrence of RASmut and EZH2 inactivation (EZH2inact) as a distinct molecular subgroup of CMML characterized by aggressive disease biology. Mechanistically, RASmutEZH2inact drives selective MAPK/ERK hyperactivation in mature myeloid cells and hematopoietic stem and progenitor compartments. Transcriptomic analysis of a large independent myeloid neoplasm cohort (Beat-AML) further supports this finding, demonstrating selective activation of gene signatures of MAPK/ERK activation in RASmutEZH2inact cases. Functionally, this signaling activation promotes proliferation and myelomonocytic differentiation in human and murine models. Therapeutically, this MAPK/ERK upregulation confers increased sensitivity to MEK inhibition (MEKi). In a murine RasmutEzh2inact-driven CMML model, MEKi suppresses MAPK/ERK activity and reduces leukemic burden by impairing proliferation and myelomonocytic differentiation without inducing cell death. In primary human CMML samples ex vivo, MEKi shows stronger anti-proliferative effects in RASmutEZH2inact specimens compared with RASmut samples. Drug-sensitivity data from the Beat-AML cohort further supported this. Ultimately, these findings were confirmed in a patient-derived xenograft model, where MEKi reduced the leukemic burden by selectively inhibiting the proliferation of transplanted human RASmutEZH2inact leukemic cells. In summary, our data define RASmutEZH2inact CMML as a clinically relevant subgroup with selective MAPK/ERK hyperactivation and increased sensitivity to MEKi. They provide a mechanistic explanation for the limited efficacy of MEK inhibitors in unselected CMML and support molecularly guided use of MEK-targeted therapy in this patient population.

molecular biology↗

bioRxiv2026

RAS-mutant clones drive extramedullary acute myeloid leukemia

Extramedullary acute myeloid leukemia (eAML) represents a clinically challenging manifestation of acute myeloid leukemia (AML), but its molecular drivers remain poorly defined. We performed targeted sequencing in 85 eAML biopsies, representing one of the largest molecular analyses of eAML to date. We detected mutations in RAS or RAS-modifying genes (RASMUT; NRAS, KRAS, PTPN11, CBL, and NF1) in 41% of cases, representing a significant enrichment compared to bone marrow (BM) samples of more than 1300 AML patients not selected for eAML. Analysis of paired eAML and BM specimens revealed expansion and/or de-novo appearance of RASMUT clones at the extramedullary site. Functional studies using primary murine leukemia cells and CRISPR/Cas9-engineered isogenic human leukemia cell lines demonstrated that RASMUT increase the migration and invasion of leukemic cells compared to RAS-wildtype controls. Consistently, RASMUT cells showed increased infiltration into the chorioallantoic membrane of chicken embryos and demonstrated enhanced extramedullary growth after injection into immunocompromised mice. RNA sequencing revealed increased expression of junctional adhesion molecule-like (JAML) and activation of PI3K/AKT signaling in RASMUT cells. JAML silencing and pharmacologic AKT inhibition reversed the RASMUT-driven effects on leukemic cell migration, demonstrating a causal role of the JAML-PI3K/AKT axis in RASMUT-driven eAML formation. In conclusion, these findings delineate the molecular landscape of extramedullary AML and show that RASMUT are enriched within this AML subform. They further demonstrate that RASMUT actively contribute to leukemic tissue infiltration through activation of a RASMUT-JAML-PI3K/AKT axis, highlighting AKT signaling as a potential therapeutic vulnerability in RASMUT-associated eAML.

cancer biology↗

bioRxiv2024

The CCR4/CCL17 axis drives intestinal acute Graft versus Host disease after allogeneic bone marrow transplantation

Acute-Graft-versus-Host disease (aGvHD) is a life-threatening complication after allogeneic stem-cell-transplantation. It is mediated by alloreactive T cells whose trafficking to aGvHD target organs is orchestrated by chemokines. We here asked whether CCL17 and its corresponding receptor CCR4 are involved in aGvHD development and severity. We applied an experimental mouse model of aGvHD in CCR4/CCL17 knockout mice and analyzed gut biopsies of GvHD patients. We show that the absence of CCR4 in transplanted T cells induced significantly less severe aGvHD. This was accompanied by reduced expression of Gata3. Mechanistically, only CD4+, but not CD8+CCR4-/- T cells protected from aGvHD. We next identified dendritic cells in the small intestine to produce CCL17, which selectively recruited CD4+ T cells. IL-4 production by intestinal CD4+ T cells promoted proliferation of CD8+ T cells. In line, we detected an upregulation of CCL17 and Gata3 in human aGvHD samples. Our results indicate that local CCL17 production in aGvHD target organs recruits T cells, reinforcing local tissue damage and immune cell recruitment. We identified the JAK1/2-inhibitor ruxolitinib to dampen CCL17-expression, thereby reducing GvHD severity. We here dissect a to date unknown role of the CCL17-CCR4 axis in aGvHD, which might help to develop novel therapeutic strategies.

immunology↗

bioRxiv2022

In silico, biochemical, and in vitro analysis of silvestrol binding to the DEAD box RNA helicase eIF4A reveals broad anti-pathogen potential of rocaglates across the eukaryotic tree of life

Selective inhibition of eukaryotic initiation factor 4A (eIF4A), an RNA helicase, has been proposed as a strategy to fight pathogens. Plant-derived rocaglates exhibit some of the highest specificities among eIF4A inhibitors. Sensitivity to rocaglates is determined by key amino acid (aa) residues mediating reversible clamping of the eIF4A:RNA complex. To date, no comprehensive assessment of eIF4A sensitivity to rocaglates across the eukaryotic tree of life has been performed to determine their anti-pathogenic potential. We performed an in silico analysis of the substitution patterns of six aa residues in eIF4A1 critical to rocaglate binding (human positions 158, 159, 163, 192, 195, 199), uncovering 35 pattern variants among 365 eIF4As sequenced to date. In silico molecular docking analysis of the eIF4A:RNA:rocaglate complexes of the 35 variants, modeled in a human eIF4A environment, and in vitro thermal shift assays with recombinantly expressed human eIF4A mutants, representing select natural and artificial variants, revealed that sensitivity to a natural or one of two synthetic rocaglates--silvestrol, CR-1-31-B, or zotatifin--was associated with lower inferred binding energies and higher melting temperature shifts. Helicase activities were comparable across variants and independent of sensitivity to rocaglates. In vitro testing with silvestrol validated predicted resistance based on position 163 substitutions in Caenorhabditis elegans and Leishmania amazonensis and predicted sensitivity in Aedes sp., Schistosoma mansoni, Trypanosoma brucei, Plasmodium falciparum, and Toxoplasma gondii. Our analysis shows resistance to rocaglates emerging in disparate eukaryotic clades pointing to resistance being a selective neutral trait except in rocaglate-producing Aglaia plants and their fungal parasite Ophiocordyceps. The analysis further revealed the possibility of targeting important insect, plant, animal, and human pathogens including Galleria mellonella, Ustilago maydis, Babesia ovata, and Cryptosporidium sp., with rocaglates. Finally, combined docking and thermal shift analyses might help design novel synthetic rocaglate derivatives or alternative eIF4A inhibitors to fight pathogens. Author SummaryIn the ongoing search for novel ways to fight non-viral and non-bacterial pathogens, targeting translation--the universal process of protein synthesis--to inhibit growth and cell proliferation has emerged as an attractive strategy. Here, we focused on the potential of rocaglates, a group of plant-derived compounds, to inhibit an early step in translation mediated by a RNA helicase called eIF4A. We performed a comprehensive analysis of eIF4A sequence variants to determine their potential sensitivities to rocaglates, especially in pathogens of prokaryotic, fungal, or animal origin. We complemented this in silico analysis with enzyme-based in vitro and whole pathogen in vivo experiments to confirm the sensitivity or resistance to rocaglates of specific variants of eIF4A. Our analysis provides the first comprehensive picture of rocaglate sensitivity among pathogens and establishes targeting important insect, plant, animal, and human pathogens such as wax moth larvae, a major parasite of honey bees, corn smut, a widely distributed fungal disease, Babesia, a livestock parasite that causes anemia and babesiois, and Cryptosporidium, the causative organism of cryptosporidiosis in humans, with rocaglates as a viable anti-pathogen strategy.

molecular biology↗

bioRxiv2022

Of Problems and Opportunities - How to Treat and How to not Treat Crystallographic Fragment-Screening Data

In their recent commentary in Protein Science, Jaskolski et al. analyze three randomly picked diffraction data sets from fragment-screening group depositions from the PDB and, based on that, claim that such data are principally problematic. We demonstrate here that if such data are treated properly, none of the proclaimed criticisms persist.

scientific communication and education↗

bioRxiv2020

The importance of charge in perturbing the aromatic glue stabilizing the protein-protein interface of homodimeric tRNA-guanine transglycosylase

Bacterial tRNA-guanine transglycosylase (Tgt) is involved in the biosynthesis of the modified tRNA nucleoside queuosine present in the anticodon wobble position of tRNAs specific for aspartate, asparagine, histidine and tyrosine. Inactivation of the tgt gene leads to decreased pathogenicity of Shigella bacteria. Therefore, Tgt constitutes a putative target for Shigellosis drug therapy. Since only active as homodimer, interference with dimer-interface formation may, in addition to active-site inhibition, provide further means to disable this protein. A cluster of four aromatic residues seems important to stabilize the homodimer. We mutated residues of this aromatic cluster and analyzed each exchange with respect to dimer and thermal stability or enzyme activity applying native mass spectrometry, thermal shift assay, enzyme kinetics, and X-ray crystallography. Our structural studies indicate strong influence of pH on homodimer stability. Obviously, protonation of a histidine within the aromatic cluster promotes the collapse of an essential structural motif within the dimer interface at slightly acidic pH. TOC GraphicFor table of contents use only. O_FIG_DISPLAY_L [Figure 1] M_FIG_DISPLAY C_FIG_DISPLAY

molecular biology↗