Combined chemosensitivity and chromatin profiling prioritizes drug combinations in CLL.

The Bruton tyrosine kinase (BTK) inhibitor ibrutinib has substantially improved therapeutic options for chronic lymphocytic leukemia (CLL). Although ibrutinib is not curative, it has a profound effect on CLL cells and may create new pharmacologically exploitable vulnerabilities. To identify such vulnerabilities, we developed a systematic approach that combines epigenome profiling (charting the gene-regulatory basis of cell state) with single-cell chemosensitivity profiling (quantifying cell-type-specific drug response) and bioinformatic data integration. By applying our method to a cohort of matched patient samples collected before and during ibrutinib therapy, we identified characteristic ibrutinib-induced changes that provide a starting point for the rational design of ibrutinib combination therapies. Specifically, we observed and validated preferential sensitivity to proteasome, PLK1, and mTOR inhibitors during ibrutinib treatment. More generally, our study establishes a broadly applicable method for investigating treatment-specific vulnerabilities by integrating the complementary perspectives of epigenetic cell states and phenotypic drug responses in primary patient samples.

A Domino 10-Step Total Synthesis of FR252921 and Its Analogues, Complex Macrocyclic Immunosuppressants.

FR252921, FR252922, and FR256523 are a family of potent macrocyclic polyene immunosuppressive agents with a novel mode of action. However, the lack of an efficient and flexible synthesis has hindered further biological studies, mostly due to the fact that the natural products appear to be kinetic isomers regarding the triene moiety. Herein, we report the development and application of an unprecedented, unique domino Suzuki-Miyaura/4π-electrocyclic ring-opening macrocyclization, resulting in a concise, unified, and stereoselective synthetic route to these complex targets in only 10 steps. This in turn enables ready access to a range of unnatural analogues, among which several compounds showed inhibition of T-lymphocyte proliferation at levels equal or superior to those of the natural products themselves.

Anthranoyl-CoA monooxygenase/reductase from Azoarcus evansii possesses both FMN and FAD in two distinct and independent active sites.

Anthranoyl-CoA monooxygenase/reductase (ACMR) participates in an unusual pathway for the degradation of aromatic compounds in Azoarcus evansii. It catalyzes the monooxygenation of anthranoyl-CoA to 5-hydroxyl-2-aminobenzoyl-CoA and the subsequent reduction to the dearomatized product 2-amino-5-oxo-cyclohex-1-ene-1-carbonyl-CoA. The two reactions occur in separate domains, termed the monooxygenase and reductase domain. Both domains were reported to utilize FAD as a cofactor for hydroxylation and reduction, respectively. We have heterologously expressed ACMR in Escherichia coli BL21 and found that the monooxygenase domain contains FAD. However, the reductase domain utilizes FMN and not FAD for the reduction of the intermediate 5-hydroxyl-2-aminobenzoyl-CoA. A homology model for the reductase domain predicted a topology similar to the Old Yellow Enzyme family, which exclusively bind FMN, in accordance with our results. Binding studies with 2-aminobenzoyl-CoA (AbCoA) and p-hydroxybenzaldehyde (pHB) as probes for the monooxygenase and reductase domain, respectively, indicated that two functionally distinct and independent active sites exist. Given the homodimeric quartenary structure of ACMR and the compact shape of the dimer as determined by small-angle X-ray scattering experiments we propose that the monooxygenase and reductase domain of opposite peptide chains are involved in the transformation of anthranoyl-CoA to 2-amino-5-oxo-cyclohex-1-ene-1-carbonyl-CoA.

High-throughput drug screening identifies the ATR-CHK1 pathway as a therapeutic vulnerability of CALR mutated hematopoietic cells.

Mutations of calreticulin (CALR) are the second most prevalent driver mutations in essential thrombocythemia and primary myelofibrosis. To identify potential targeted therapies for CALR mutated myeloproliferative neoplasms, we searched for small molecules that selectively inhibit the growth of CALR mutated cells using high-throughput drug screening. We investigated 89 172 compounds using isogenic cell lines carrying CALR mutations and identified synthetic lethality with compounds targeting the ATR-CHK1 pathway. The selective inhibitory effect of these compounds was validated in a co-culture assay of CALR mutated and wild-type cells. Of the tested compounds, CHK1 inhibitors potently depleted CALR mutated cells, allowing wild-type cell dominance in the co-culture over time. Neither CALR deficient cells nor JAK2V617F mutated cells showed hypersensitivity to ATR-CHK1 inhibition, thus suggesting specificity for the oncogenic activation by the mutant CALR. CHK1 inhibitors induced replication stress in CALR mutated cells revealed by elevated pan-nuclear staining for γH2AX and hyperphosphorylation of RPA2. This was accompanied by S-phase cell cycle arrest due to incomplete DNA replication. Transcriptomic and phosphoproteomic analyses revealed a replication stress signature caused by oncogenic CALR, suggesting an intrinsic vulnerability to CHK1 perturbation. This study reveals the ATR-CHK1 pathway as a potential therapeutic target in CALR mutated hematopoietic cells.

The metabolic growth limitations of petite cells lacking the mitochondrial genome.

Eukaryotic cells can survive the loss of their mitochondrial genome, but consequently suffer from severe growth defects. 'Petite yeasts', characterized by mitochondrial genome loss, are instrumental for studying mitochondrial function and physiology. However, the molecular cause of their reduced growth rate remains an open question. Here we show that petite cells suffer from an insufficient capacity to synthesize glutamate, glutamine, leucine and arginine, negatively impacting their growth. Using a combination of molecular genetics and omics approaches, we demonstrate the evolution of fast growth overcomes these amino acid deficiencies, by alleviating a perturbation in mitochondrial iron metabolism and by restoring a defect in the mitochondrial tricarboxylic acid cycle, caused by aconitase inhibition. Our results hence explain the slow growth of mitochondrial genome-deficient cells with a partial auxotrophy in four amino acids that results from distorted iron metabolism and an inhibited tricarboxylic acid cycle.

Systematic characterization of BAF mutations provides insights into intracomplex synthetic lethalities in human cancers.

Aberrations in genes coding for subunits of the BRG1/BRM associated factor (BAF) chromatin remodeling complexes are highly abundant in human cancers. Currently, it is not understood how these mostly loss-of-function mutations contribute to cancer development and how they can be targeted therapeutically. The cancer-type-specific occurrence patterns of certain subunit mutations suggest subunit-specific effects on BAF complex function, possibly by the formation of aberrant residual complexes. Here, we systematically characterize the effects of individual subunit loss on complex composition, chromatin accessibility and gene expression in a panel of knockout cell lines deficient for 22 BAF subunits. We observe strong, specific and sometimes discordant alterations dependent on the targeted subunit and show that these explain intracomplex codependencies, including the synthetic lethal interactions SMARCA4-ARID2, SMARCA4-ACTB and SMARCC1-SMARCC2. These data provide insights into the role of different BAF subcomplexes in genome-wide chromatin organization and suggest approaches to therapeutically target BAF-mutant cancers.

Mechanism of cargo-directed Atg8 conjugation during selective autophagy.

Selective autophagy is mediated by cargo receptors that link the cargo to the isolation membrane via interactions with Atg8 proteins. Atg8 proteins are localized to the membrane in an ubiquitin-like conjugation reaction, but how this conjugation is coupled to the presence of the cargo is unclear. Here we show that the S. cerevisiae Atg19, Atg34 and the human p62, Optineurin and NDP52 cargo receptors interact with the E3-like enzyme Atg12~Atg5-Atg16, which stimulates Atg8 conjugation. The interaction of Atg19 with the Atg12~Atg5-Atg16 complex is mediated by its Atg8-interacting motifs (AIMs). We identify the AIM-binding sites in the Atg5 subunit and mutation of these sites impairs selective autophagy. In a reconstituted system the recruitment of the E3 to the prApe1 cargo is sufficient to drive accumulation of conjugated Atg8 at the cargo. The interaction of the Atg12~Atg5-Atg16 complex and Atg8 with Atg19 is mutually exclusive, which may confer directionality to the system.