Search

Abstract Background Development of resistance to targeted therapies has tempered initial optimism that precision oncology would improve poor outcomes for cancer patients. Resistance mechanisms, however, can also confer new resistance-specific vulnerabilities, termed collateral sensitivities. Here we investigated anaplastic lymphoma kinase (ALK) inhibitor resistance in neuroblastoma, a childhood cancer frequently affected by activating ALK alterations. Methods Genome-wide forward genetic CRISPR-Cas9 based screens were performed to identify genes associated with ALK inhibitor resistance in neuroblastoma cell lines. Furthermore, the neuroblastoma cell line NBLW-R was rendered resistant by continuous exposure to ALK inhibitors. Genes identified to be associated with ALK inhibitor resistance were further investigated by generating suitable cell line models. In addition, tumor and liquid biopsy samples of four patients with ALK-mutated neuroblastomas before ALK inhibitor treatment and during tumor progression under treatment were genomically profiled. Results Both genome-wide CRISPR-Cas9-based screens and preclinical spontaneous ALKi resistance models identified NF1 loss and activating NRASQ61K mutations to confer resistance to chemically diverse ALKi. Moreover, human neuroblastomas recurrently developed de novo loss of NF1 and activating RAS mutations after ALKi treatment, leading to therapy resistance. Pathway-specific perturbations confirmed that NF1 loss and activating RAS mutations lead to RAS-MAPK signaling even in the presence of ALKi. Intriguingly, NF1 loss rendered neuroblastoma cells hypersensitive to MEK inhibition. Conclusions Our results provide a clinically relevant mechanistic model of ALKi resistance in neuroblastoma and highlight new clinically actionable collateral sensitivities in resistant cells.

Escherichia coli uses allantoin as the sole nitrogen source during anaerobic growth. In the final step of allantoin degradation, oxamic transcarbamylase (OXTCase) converts oxalurate to carbamoyl phosphate (CP) and oxamate. The activity of this enzyme was first measured in Streptococcus allantoicus in the 1960s, but no OXTCase enzyme or the encoding gene(s) have been found in any strain. This study discovered that allFGH ( fdrA , ylbE , and ylbF ) are the genes that encode the global orphan enzyme OXTCase. The three genes form an operon together with allK ( ybcF ), encoding catabolic carbamate kinase. The allFGHK operon is located directly downstream of the allECD operon that encodes enzymes for the preceding steps of OXTCase. The OXTCase kinetic parameters were analyzed using the purified protein composed of AllF-AllG-AllH (FdrA-YlbE-YlbF); for the substrate CP, K M and V max were 1.3 mM and 15.4 U/mg OXTCase, respectively, and for the substrate oxamate, they were 36.9 mM and 27.0 U/mg OXTCase. In addition, the OXTCase encoded by the three genes is a novel transcarbamylase that shows no similarity with known enzymes of the transcarbamylase family such as aspartate transcarbamylase, ornithine transcarbamylase, and YgeW transcarbamylase. The present study elucidated the anaerobic allantoin degradation pathway of E. coli . Therefore, we suggest that the genes fdrA , ylbE , and ylbF are renamed allF , allG , and allH , respectively.IMPORTANCEThe anaerobic allantoin degradation pathway of Escherichia coli includes a global orphan enzyme, oxamic transcarbamylase (OXTCase), which converts oxalurate to carbamoyl phosphate and oxamate. This study found that the allFGH ( fdrA , ylbE , and ylbF ) genes encode OXTCase. The OXTCase activity and kinetics were successfully determined with purified recombinant AllF-AllG-AllH (FdrA-YlbE-YlbF). This OXTCase is a novel transcarbamylase that shows no similarity with known enzymes of the transcarbamylase family such as aspartate transcarbamylase (ATCase), ornithine transcarbamylase (OTCase), and YgeW transcarbamylase (YTCase). In addition, OXTCase activity requires three genes, whereas ATCase is encoded by two genes, and OTCase and YTCase are encoded by a single gene. The current study discovered OXTCase, the last unknown step in allantoin degradation, and this enzyme is a new member of the transcarbamylase group that was previously unknown., Competing Interests: The authors declare no conflict of interest.

Anaerobic bacteria often use antiporters DcuB (malate/succinate antiport) or DcuA (l-aspartate/succinate antiport) for the excretion of succinate during fumarate respiration. The rumen bacterium Actinobacillus succinogenes is able to produce large amounts of succinate by fumarate respiration, using the DcuB-type transporter DcuE for l-malate/succinate antiport. Asuc_0142 was annotated as a second DcuB-type transporter. Deletion of Asuc_0142 decreased the uptake rate for l-[ 14 C]aspartate into A. succinogenes cells. Properties of transport by heterologously expressed Asuc_0142 were investigated in an Escherichia coli mutant deficient of anaerobic C4DC transporters. Expression of Asuc_0142 resulted in high uptake activity for l-[ 14 C]fumarate or l-[ 14 C]aspartate, but the former showed a strong competitive inhibition by l-aspartate. In E. coli loaded with l-[ 14 C]aspartate, [ 14 C]succinate or [ 14 C]fumarate, extracellular C4DCs initiated excretion of the intracellular substrates, with a preference for l-aspartate ex /succinate in or l-aspartate ex /fumarate in antiport. These findings indicate that Asuc_0142 represents a DcuA-type transporter for l-aspartate uptake and l-aspartate ex /C4DC in antiport, differentiating it from the DcuB-type transporter DcuE for l-malate ex /succinate in antiport. Sequence analysis and predicted structural characteristics confirm structural similarity of Asuc_0142 to DcuA, and Asuc_0142 was thus re-named as DcuA As . The bovine rumen fluid contains l-aspartate (99.6 µM), whereas fumarate and l-malate are absent. Therefore, bovine rumen colonisers depend on l-aspartate as an exogenous substrate for fumarate respiration. A. succinogenes encodes HemG (protoporphyrinogen oxidase) and PyrD (dihydroorotate dehydrogenase) for haem and pyrimidine biosynthesis. The enzymes require fumarate as an electron acceptor, suggesting an essential role for l-aspartate, DcuA As , and fumarate respiration for A. succinogenes growing in the bovine rumen.

Escherichia coli can use allantoin as its sole nitrogen source under anaerobic conditions. The ureidoglycolate produced by double release of ammonia from allantoin can flow into either the glyoxylate shunt or further catabolic transcarbamoylation. Although the former pathway is well studied, the genes of the latter (catabolic) pathway are not known. In the catabolic pathway, ureidoglycolate is finally converted to carbamoyl phosphate (CP) and oxamate, and then CP is dephosphorylated to carbamate by a catabolic carbamate kinase (CK), whereby ATP is formed. We identified the ybcF gene in a gene cluster containing fdrA - ylbE - ylbF - ybcF that is located downstream of the allDCE -operon. Reverse transcription PCR of total mRNA confirmed that the genes fdrA , ylbE , ylbF , and ybcF are co-transcribed. Deletion of ybcF caused only a slight increase in metabolic flow into the glyoxylate pathway, probably because CP was used to de novo synthesize pyrimidine and arginine. The activity of the catabolic CK was analyzed using purified YbcF protein. The V max is 1.82 U/mg YbcF for CP and 1.94 U/mg YbcF for ADP, and the K M value is 0.47 mM for CP and 0.43 mM for ADP. With these results, it was experimentally revealed that the ybcF gene of E. coli encodes catabolic CK, which completes anaerobic allantoin degradation through substrate-level phosphorylation. Therefore, we suggest renaming the ybcF gene as allK .

Metabolons represent the structural organization of proteins for metabolic or regulatory pathways. Here, the interaction of fumarase FumB, aspartase AspA, and L-tartrate dehydratase TtdAB with the C4-dicarboxylate (C4-DC) transporters DcuA, DcuB, DcuC, and the L-tartrate transporter TtdT of Escherichia coli was tested by a bacterial two-hybrid (BACTH) assay in situ, or by co-chromatography using mSPINE (membrane Streptavidin protein interaction experiment). From the general C4-DC transporters, DcuB interacted with FumB and AspA, DcuA with AspA, whereas DcuC interacted with neither FumB nor AspA. Moreover, TtdT did not interact with TtdAB. The fumB-dcuB, the dcuA-aspA, and the ttdAB-ttdT genes encoding the respective proteins colocalize on the genome and each pair of genes forms cotranscripts, whereas the dcuC gene lies alone. The data suggest the formation of DcuB/FumB and DcuB/AspA metabolons for the uptake of L-malate, or L-aspartate, and their conversion to fumarate for fumarate respiration and excretion of the product succinate. The DcuA/AspA metabolon catalyzes uptake and conversion of L-aspartate to fumarate coupled to succinate excretion. The DcuA/AspA metabolon provides ammonia at the same time for nitrogen assimilation (ammonia shuttle). On the other hand, TtdT and TtdAB are not organized in a metabolon. Reasons for the formation (DcuA/AspA, DcuB/FumB, and DcuB/AspA) or nonformation (DcuC, TtdT, and TtdAB) of metabolons are discussed based on their metabolic roles., (© The Author(s) 2022. Published by Oxford University Press on behalf of FEMS.)

Wild-type Escherichia coli was adapted to syntrophic growth with Methanobacterium formicicum for glycerol fermentation over 44 weeks. Succinate production by E. coli started to increase in the early stages of syntrophic growth. Genetic analysis of the cultured E. coli population by pooled sequencing at eight time points suggests that (i) rapid evolution occurred through repeated emergence of mutators that introduced a large number of nucleotide variants and (ii) many mutators increased to high frequencies but remained polymorphic throughout the continuous cultivation. The evolved E. coli populations exhibited gains both in fitness and succinate production, but only for growth under glycerol fermentation with M. formicicum (the condition for this laboratory evolution) and not under other growth conditions. The mutant alleles of the 69 single nucleotide polymorphisms (SNPs) identified in the adapted E. coli populations were constructed individually in the ancestral wild-type E. coli. We analyzed the phenotypic changes caused by 84 variants, including 15 nonsense variants, and found that FdrA D296Y was the most significant variant leading to increased succinate production. Transcription of fdrA was induced under anaerobic allantoin degradation conditions, and FdrA was shown to play a crucial role in oxamate production. The FdrA D296Y variant increased glyoxylate conversion to malate by accelerating oxamate production, which promotes carbon flow through the C4 branch, leading to increased succinate production. IMPORTANCE Here, we demonstrate the ability of E. coli to perform glycerol fermentation in coculture with the methanogen M. formicicum to produce succinate. We found that the production of succinate by E. coli significantly increased during successive cocultivation. Genomic DNA sequencing, evaluation of relative fitness, and construction of SNPs were performed, from which FdrA D296Y was identified as the most significant variant to enable increased succinate production by E. coli. The function of FdrA is uncertain. In this study, experiments with gene expression assays and metabolic analysis showed for the first time that FdrA could be the "orphan enzyme" oxamate:carbamoyltransferase in anaerobic allantoin degradation. Furthermore, we demonstrate that the anaerobic allantoin degradation pathway is linked to succinate production via the glyoxylate pathway during glycerol fermentation.