C. elegans. Surprisingly, this therapeutic variation did not manifest as enhanced disaggregase activity, but rather as increased passive inhibition of aggregation of specific substrates. By exploring natural tuning of this passive Hsp104 activity, we elucidated enhanced, substrate-specific agents that counter proteotoxicity underlying neurodegeneration.The AAA+ protein disaggregase, Hsp104, increases fitness under stress by reversing stress-induced protein aggregation. Natural Hsp104 variants might exist with enhanced, selective activity against neurodegenerative disease substrates. However, natural Hsp104 variation remains largely unexplored. Here, we screened a cross-kingdom collection of Hsp104 homologs in yeast proteotoxicity models. Prokaryotic ClpG reduced TDP-43, FUS, and α-synuclein toxicity, whereas prokaryotic ClpB and hyperactive variants were ineffective. We uncovered therapeutic genetic variation among eukaryotic Hsp104 homologs that specifically antagonized TDP-43 condensation and toxicity in yeast and TDP-43 aggregation in human cells. We also uncovered distinct eukaryotic Hsp104 homologs that selectively antagonized α-synuclein condensation and toxicity in yeast and dopaminergic neurodegeneration in  Alternative protein folding and aberrant phase transitions underpin fatal neurodegenerative diseases . DiseaseSaccharomyces cerevisiae (ScHsp104) rapidly disassembles a diverse range of misfolded protein conformers, including amorphous aggregates, preamyloid oligomers, and amyloid fibers  loss-of-function phenotypes associated with sequestration of functional soluble protein into misfolded oligomers and insoluble aggregates; and (b) any toxic gain-of-function phenotypes associated with the misfolded conformers themselves . The AAAd fibers . Hsp104 d fibers . Each prd fibers . Hsp104 d fibers . Moreoveisorders , and a risorders . Hsp104 isorders . Howeverisorders .Previously, we circumvented limitations on Hsp104 disaggregase activity by developing a suite of potentiated Hsp104 variants, differing from wild-type (WT) Hsp104 by one or more missense mutations in the autoregulatory MD . These pCalcarisporiella thermophila antagonizes toxicity of TDP-43, αSyn, and polyglutamine in yeast without apparent toxic off-target effects , but not ScHsp104, reduces TDP-43 and αSyn toxicity in yeast , plants , protozoa , and chromista  , were non-toxic to yeast at 30°C or 37°C  and Salpingoeca rosetta (Sr), from chromista: Chlamydomonas reinhardtii (Cr) and Galdieria sulphuraria (Gs), and the plant Populus euphratica (Pe)), suppress TDP-43 toxicity , show at most a modest increase in nuclear TDP43-GFP compared to control strains lacking Hsp104 (Δhsp104) or expressing ScHsp104  and Thermomyces lanuginosus (Tl)) that suppress αSyn toxicity  and ATP hydrolysis  . We were surprised that ScHsp104WT reduced the proportion of cells with TDP-43 aggregates at this time point, given that it does not reduce TDP-43 aggregation in yeast . By contrast, the catalytically-inactive variant ScHsp104DPLA:DWB had a significantly increased proportion of cells with TDP-43 aggregates compared to cells expressing no Hsp104 or ScHsp104WT . Strikingly, cells expressing the potentiated variant ScHsp104A503S or the TDP-43-specific variant PeHsp104 continued to show a significantly lower percentage of cells with TDP-43 aggregates . The TDP-43-specific variant SrHsp104, meanwhile, reduced TDP-43 aggregate burden at day 1 but not day 2, suggesting an intermediate effect  . PeHsp10 motifs) . We moni motifs)  over tim motifs) . Express motifs) . At both motifs) . We also motifs) . None of motifs) , althoug motifs) . This fiin yeast . Howevere effect . Thus, wC. elegans model of Parkinson’s disease in which the dopamine transporter (dat-1) promoter is used to direct expression of αSyn to dopaminergic (DA) neurons  after 7 days post-hatching , and assessed the thermotolerance phenotypes of these strains. We observed a range of phenotypes. Specifically, 15 of 17 Hsp104s tested conferred some degree of thermotolerance above Δhsp104 alone  to yeast. Hsp104 is an essential factor for induced thermotolerance in yeast , and Hspme shock . Express04 yeast . We gene04 alone . The twophratica . Some hoely weak . Thermotrevisiae . Thermotrevisiae . Interes04 yeast  while Cl04 yeast , althougregation  while Clregation . Neverth2 = 0.24).Next, we assessed how differences in protein expression between strains may contribute to phenotypic differences. All strains mount an effective heat-shock response, as indicated by Hsp26 levels assessed by Western blot . We alsoWe next expressed and purified several Hsp104 homologs  to define their biochemical properties. Hsp104 homologs likely form hexamers , are actWT to the potentiated variant ScHsp104A503S, which exhibits elevated ATPase activity  by asseactivity . We founinactive ) and in inactive . Next, winactive . Luciferinactive  or humaninactive . The robinactive .Next, we tested the activity of several Hsp104 homologs  against an ordered amyloid substrate, semen-derived enhancer of viral infection (SEVI) . As prevMonosiga brevicollis and Chlamydomonas reinhardtii, which selectively suppress TDP-43 toxicity and foci formation in yeast, inhibit TDP-43 aggregation in vitro, whereas Hsp104 from S. cerevisiae has limited efficacy  and Hsp104DWA(KT)) in NBD1 and NBD2 to impair ATP binding, (2) conserved glutamate residues in the Walker B motifs to either alanine or glutamine (e.g. Hsp104DWB(EA) or Hsp104DWB(EQ)) in NBD1 and NBD2 to impair ATP hydrolysis, and (3) conserved tyrosines in the pore loops to alanine (e.g. Hsp104DPLA) in NBD1 and NBD2 to impair substrate threading through the central hexamer pore  ATP binding and hydrolysis, which are mediated by Walker A and Walker B motifs, respectively, and which drive conformational changes within the hexamer to support substrate translocation , and (2)mer pore . We alsomer pore .HSP104 promoter  would recapitulate the suppression of toxicity seen with the full-length homolog. We therefore co-expressed PeHsp104toxicity . Since ttoxicity , we concFor the αSyn-specific Hsp104 homologs, TtHsp104 and TlHsp104, chimeras consisting of NTD:NBD1:MD from TtHsp104 or TlHsp104 fused to NBD2:CTD from ScHsp104 were unable to reduce αSyn toxicity . To testNTDScNBD1:MD:NBD2:CTD and CtNTDScNBD1:MD:NBD2:CTD displayed enhanced thermotolerance relative to CrHsp104 and CtHsp104  fail to confer thermotolerance , although TtHsp104 and TlHsp104 (αSyn-specific variants) were exceptions . Indeed, we did not isolate any eukaryotic Hsp104 homologs capable of suppressing FUS toxicity. In contrast, potentiated Hsp104 variants typically are able to suppress toxicity of multiple toxic substrates . Naturaland disaggregase activity.The dual role of Hsp104 as a molecular chaperone capable of preventing aggregation in addition to a disaggregase capable of reversing protein aggregation has long been appreciated . Both ofWhat sequence determinants of eukaryotic Hsp104 homologs enable their toxicity-suppression phenotypes? Hsp104 homologs that selectively suppress TDP-43 toxicity or selectively suppress αSyn toxicity are more similar to each other than between groups , there are important sequence determinants throughout the other domains of the protein that enable each homolog to produce a protective phenotype. The second possible explanation is that specific domains contribute to suppression of toxicity indirectly by stabilizing the overall protein architecture of the chimeras. There are no obvious trends in the primary sequence of the homologs, however, that would clarify these two competing possibilities. Indeed, further work is needed to gain a complete understanding of how homologous Hsp104 sequences confer variable substrate-specific proteotoxicity suppression.That nearly half of the eukaryotic Hsp104 homologs we tested (8/17) were able to antagonize toxicity of either TDP-43 or αSyn  was unanticipated. There are no clear TDP-43 or αSyn homologs in the host species from which we selected Hsp104 homologs to test. Our observation that, for a given Hsp104 homolog, there was not a predictable relationship between suppression of TDP-43 or αSyn toxicity and thermotolerance, which may more closely reflect the primary in vivo function of these Hsp104 homologs, suggests one interpretation involving cryptic genetic variation . VariatiMultiple sequence alignments of Hsp104 homologs were generated with Clustal Omega . MultiplMATa, can1-100, his3-11,15, leu2-3,112, trp1-1, ura3-1, ade2-1) or the isogenic strain W303aΔhsp104 , or mammalian (pInducer20-ccdB) expression vectors via LR reactions. MbHsp104 and CrHsp104 were cloned into pNOTAG for bacterial expression through NdeI and SacI sites. Hsp104 mutants  were obtained by Quikchange mutagenesis. The specific mutants are listed in Key Resources Table. Hsp104 chimeras were cloned by overlap-extension PCR. Sequences of all Hsp104 chimeras are included in Yeast expression vectors encoding TDP-43 (pAG303GAL-TDP-43), FUS (pAG303GAL-FUS), and αSyn-GFP (pAG303GAL-αSyn-GFP and pAG304GAL-αSyn-GFP) were from Aaron Gitler . pRS313Heviously . cDNAs eGGATCCGGAAGTGGCTCAAGCGGAATGTCTGAATATATTCGGGT-3’; 5’-TGCTGCTGCCACTGCCACTGCTGATGAAGCATCTGTCTCATCCATTGCTGCTGCGTTATCTTTTGGATAGTTGACA-3’; 5’- ATCAGCAGTGGCAGTGGCAGCAGCAGCAGTCCAGAAAACATCCGA-3’; 5’- AAGTTTGTTGCGCCGGATCCCATTCCCCAGCCAGAAGACT-3’.Doxycycline-inducible TDP-43ΔNLS mammalian expression vector  was modiYeast were transformed according to standard protocols using polyethylene glycol and lithium acetate . For spoΔhsp104 yeast were transformed with the indicated Hsp104 plasmid. Transformants were grown to saturation overnight in synthetic raffinose media. Cultures were normalized, spotted onto synthetic dropout media containing glucose or galactose, and incubated at 30°C or 37°C for 2–3 d before growth was documented.W303a600 = 0.6, and 6 mL of cells were harvested. For heat-shock controls, samples were incubated at 37°C for 30 min prior to processing. Yeast lysates were extracted by incubation with 0.1M NaOH at room temperature for 5 min. Lysates were mixed with SDS sample buffer, boiled for 5 min, and subjected to Tris-HCl SDS-PAGE  followed by transfer to a PVDF membrane (Millipore). Membranes were blocked in Odyssey blocking buffer (LI-COR) for 1 hr at room temperature or overnight at 4°C. Primary antibody incubations were performed at 4°C overnight or at room temperature for 2 hr. After washing with PBST, membranes were incubated with fluorescently labeled secondary antibodies at room temperature for 1 hr, followed by washing with PBST. Proteins were detected using an Odyssey Fc Dual-Mode Imaging system. Primary antibodies used: mouse monoclonal anti-FLAG M2 (Sigma-Aldrich), rabbit polyclonal anti-TDP-43 (Proteintech), rabbit polyclonal anti-FUS , rabbit polyclonal anti-GFP (Sigma-Aldrich), mouse monoclonal 3-phosphoglycerate kinase (Novex), rabbit polyclonal anti-Hsp26 , fluorescently labeled anti-mouse and anti-rabbit secondary antibodies (Li-Cor).Yeast were grown in galactose-containing media to induce protein expression for 5 hr  or 8 hr (for strains expressing αSyn). Cultures were normalized to ODTo visualize changes in TDP-43 and αSyn localization in response to coexpression of various Hsp104s, yeast strains expressing fluorescently-tagged TDP-43 and αSyn were previously generated and described . For micCVCL_0063) and have been authenticated by the vendor and were not contaminated by mycoplasma. HEK-293T cells were maintained in Dulbecco’s modified Eagle’s medium, high glucose (Gibco) containing 10% fetal bovine serum (Life Sciences), 1% non-essential amino acids (Gibco), and 1% antibiotic-antimyotic (Gibco). Cells were plated in gelatin-coated 6-well plates at a density of 3 × 106 cells/plate 24 hr before transfection. Cells were transfected with 2 µg total DNA and 7.35 µl polyethylenimine HCl MAX transfection reagent . Wells co-transfected with mClover3-TDPΔNLS and HSP104 variants received 1 µg of each plasmid. Media was changed 6 hr post-transfection to media containing 1 µg/ml of doxycycline hyclate (Sigma-Aldrich) to induce transgene expression. Transfected cells were lifted every 24 hr over 2 days, at which point cells were analyzed by FACS (FACSAria Fusion BD). Cells were gated to have a narrow range of FCS and SSC values and to be fluorescence positive. TDP-43 aggregation was quantified by comparing the height (FITC-H) to the width (FITC-W) of the fluorescence channel using 488 nm laser and FITC filters.HEK293T cells were from ATCC , UA382 , UA383  were generated by injecting 50 ng/μl of corresponding plasmid construct into UA44  with phenotypic marker . Strains UA403 , UA404 , UA405  were generated by injecting 50 ng/μl of corresponding plasmid construct into BY250 (vtIs7 [Pdat-1::GFP]) with phenotypic marker . Three independent stable lines were created for each group. For dopaminergic neurodegeneration analyses, the transgenic animals were scored as described previously . The expression levels of α-syn and Hsp104 variants were normalized to three reference genes . No amplification was detected in NTC and NRT controls. The reference target stability was analyzed by GeNorm and passed for all reference genes listed above. All target genes were measured in triplicates for three independent transgenic lines for each sample in this study. The data analysis was performed by the Gene Expression Module of CFX Manager software.RNA isolation and RT-qPCR was performed on worms using previously published methods . BrieflyThe following primers were used for the assays:CtHsp104 Forward: GACGAAGCGTGTGCCAATACCtHsp104 Reverse: CACTTCCTGGAGCCGCTGTtHsp104 Forward: CAACTACTTCCTGCCCGAGTtHsp104 Reverse: ATCTGGACGTTGCGGTCGTTlHsp104 Forward: AACCGTCTCACCAAGCGTGTlHsp104 Reverse: GCCTCTCCGAGATAGTCCTα-syn Forward: ATGTAGGCTCCAAAACCAAGGα-syn Reverse: ACTGCTCCTCCAACATTTGTCsnb-1 Forward: CCGGATAAGACCATCTTGACGsnb-1 Reverse: GACGACTTCATCAACCTGAGCcdc-42 Forward: CCGAGAAAAATGGGTGCCTGcdc-42 Reverse: TTCTCGAGCATTCCTGGATCATtba-1 Forward: ATCTCTGCTGACAAGGCTTACtba-1 Reverse: GTACAAGAGGCAAACAGCCATΔhsp104 yeast were transformed with plasmids bearing either Hsp104 from S. cerevisiae, the indicated Hsp104 homolog, or Hsp104 mutant under the native HSP104 promoter , or an empty vector control. Transformants were selected, grown to saturation in yeast minimal media (SD-His), and then diluted to OD600 = 0.2 in fresh SD-His . Yeast were allowed to double at 30°C, after which cultures were normalized and pretreated at 37°C for 30 min to induce expression of heat-shock proteins (samples were taken here and processed for Western blot to assess Hsp104 and Hsp26 expression). Cells were then heat shocked at 50°C for the indicated time and cooled for 2 min on ice. Cultures were then diluted 1000-fold, plated on SD-His , and plates were incubated at 30°C for 2–3 days to observe viable colonies.W303aE. coli. Transformed cells were grown in 2xYT broth supplemented with 25 µg/ml chloramphenicol and 100 µg/ml ampicillin at 37°C until an OD600 of 0.4–0.6 was reached, at which point cells were cooled to 15°C. Expression was induced by addition of 1 mM isopropyl 1-thio-β-D-galactopyranoside for 15–18 hr. Cells were harvested by centrifugation , resuspended in lysis buffer ). Cells were treated on ice with 20 mg lysozyme per 1l culture and lysed by sonication. Cell debris was removed by centrifugation at 16,000 × g at 4°C for 20 min, and the supernatant was applied to Affi-Gel Blue resin (Bio-Rad). Resin was incubated with the lysates for 4 hr at 4°C with slow rotation. Resin was then washed four times with wash buffer . ScHsp104 was eluted with wash buffer supplemented with 1 M KCl. The protein was then exchanged into running buffer Q , further purified by ResourceQ anion exchange chromatography, and eluted with a linear salt gradient (50 mM-1 M NaCl). Eluted protein was then exchanged into storage buffer , snap-frozen in liquid N2, and stored at −80°C until use.TEV protease and Ulp1 were purified via standard protocols. Untagged ScHsp104 was expressed from the pNOTAG-ScHsp104 vector  and puri2, 100 mM KCl, 2.5% glycerol, 2 mM β-mercaptoethanol. To resolubilize precipitates, ammonium sulfate was removed by dialysis against this buffer, with three buffer changes. The dialyzate was filtered and applied to a 5 ml HiTrapQ column and purified with running buffer Q  and eluted with a linear gradient of buffer Q+  over 40 column volumes. Peak fractions were collected, pooled, and exchanged into size-exclusion buffer  and further purified by size-exclusion chromatography on a pre-equilibrated Superdex 200 column . Protein in size-exclusion buffer was concentrated to ~10 mg/ml, supplemented with 10% glycerol, snap-frozen in liquid N2 and stored at −80°C until use.MbHsp104 and CrHsp104 were expressed as untagged proteins by subcloning the MbHsp104 and CrHsp104 ORFs into pNOTAG through NdeI and SacI sites. Protein expression and lysis were carried out as for ScHsp104. However, MbHsp104 and CrHsp104 bound poorly to Affi-Blue resin, even after extended incubation times (data not shown). Instead, MbHsp104 and CrHsp104 were precipitated from clarified bacterial lysates by addition of solid ammonium sulfate to 40% of saturation. Precipitates were collected by centrifugation  and resuspended in buffer containing 50 mM Tris-HCl, pH 8.0, 10 mM MgCl6-tagged fusion proteins. Protein expression and lysis were carried out as for other Hsp104s, except in this case lysis buffer consisted of 40 mM HEPES-KOH pH 7.4, 500 mM KCl, 20 mM MgCl2, 2.5% glycerol, 20 mM imidazole, 2 mM β-mercaptoethanol supplemented with 5 µM pepstatin A and complete protease inhibitor tablets. Clarified lysate was loaded onto Ni-NTA resin. The resin was washed with 10 volumes of wash buffer (same formulation as lysis buffer except without protease inhibitors) and eluted in wash buffer supplemented with 350 mM imidazole. TEV protease was added to the eluted protein, and the sample was dialyzed against wash buffer containing no imidazole for 4 hr at room temperature followed by ~16 hr at 4°C. After dialysis and cleavage, the protein was loaded onto a second Ni-NTA column to remove the His6 tag and uncleaved protein. Eluted protein was pooled, concentrated, and exchanged into high salt storage buffer , snap-frozen in liquid N2, and stored at −80°C until further use.CtHsp104, TtHsp104, and TlHsp104 were expressed from pMCSG68 vector as TEV protease-cleavable His6-SUMO-tagged proteins in BL21(DE3)RIL cells. Transformed cells were grown at 37°C in Luria broth supplemented with 25 µg/ml chloramphenicol and 100 µg/ml ampicillin to an OD600 ~0.5. Cultures were cooled to 15°C, and expression was induced with 1 mM IPTG for 16 hr. Cells were harvested, resuspended in lysis buffer ), and lysed by treatment with lysozyme and sonication. Lysates were clarified by centrifugation , and incubated with Ni-NTA resin for 90 min at 4°C. Resin was washed with 10 column volumes of wash buffer  and eluted with two column volumes of elution buffer (wash buffer+300 mM imidazole). To cleave the His6-SUMO tag, Ulp1 was added at a 1:100 molar ratio, and imidazole was removed by dialysis against wash buffer. After dialysis, protein was loaded onto a 5 ml HisTrap column  and eluted with a linear imidazole gradient (20–350 mM) over 40 column volumes. Fractions containing cleaved protein were pooled, concentrated, and purified further by Resource Q  or Resource S (Sis1 and Hdj1) ion exchange chromatography. Ssa1, Hsc70, Ydj1, Hdj2, Sis1, and Hdj1 were snap-frozen in liquid N2 and stored at −80°C in elution buffer supplemented with 10% glycerol.Ssa1, Hsc70, Sis1, Ydj1, Hdj1, and Hdj2 (in pESUMO (Life Sensors)) were expressed as N-terminally His600 ~0.8. Cultures were cooled to 15°C, and expression was induced with 1 mM IPTG for 16 hr. Cells were harvested, resuspended in lysis buffer ), and lysed by treatment with lysozyme and sonication. Lysates were clarified by centrifugation . Cleared lysate was added to a 50% slurry of amylose resin (New England Biolabs) in lysis buffer and incubated at 4°C for 30 min to bind MBP-TDP-43. The resin was washed with lysis buffer, and eluted MBP-TEV with elution buffer . Purified fractions were pooled, snap-frozen in liquid N2, and stored at −80°C until use.TDP-43 was expressed with an N-terminal, TEV-cleavable maltose binding protein (MBP) tag in BL21(DE3)RIL cells. Transformed cells were grown at 37°C in Luria broth supplemented with 25 µg/ml chloramphenicol and 100 µg/ml ampicillin to an OD2 at room temperature and elution of Hsp104 was monitored by both absorbance at 280 nm and refractive index. For TtHsp104, Superdex 200 5/150 column was used, and 1 mM ATP was added to the running buffer to promote oligomerization. For TlHsp104, the presence of higher order species at the leading edge of the elution peak raised the observed molecular weight, although elution times for TlHsp104 are consistent with hexamers observed for other Hsp104 homologs under identical conditions.Molecular weights of Hsp104 homologs (30 µM monomer) were determined using multi-angle light scattering coupled with refractive interferometric detection (Wyatt Technology Corporation) and a Superdex 200 10/300 size-exclusion column. The column was equilibrated with 40 mM HEPES-KOH, 140 mM KCl, and 20 mM MgClHsp104 (0.25 µM monomer) was incubated with ATP (1 mM) for 5 min at the indicated temperatures in buffer . ATPase activity was determined by inorganic phosphate release using a malachite green phosphate detection kit (Innova). Background hydrolysis was determined at time zero and subtracted.Luciferase disaggregation and reactivation was performed as described . Aggrega2 microplate reader with excitation and emission filters set to 440 nm and 482 nm, respectively. To assess fibril morphology by negative stain EM, reaction aliquots were spotted on Formvar carbon-coated grids (EM Sciences) and stained with 2% uranyl acetate. Samples were visualized using a JEOL-1010 electron microscope.SEVI remodeling was performed as previously described . SEVI fi2, 1 mM DTT) without agitation. Aggregation was monitored by turbidity (A395 nm) in a TECAN Infinite M1000 plate reader. In some reactions, either ScHsp104, MbHsp104, or CrHsp104 were added (6 µM) with or without ATP (5 mM) and regeneration system (1 mM creatine phosphate and 0.25 µM creatine kinase). We verified that neither Hsp104 nor ATP affected cleavage of the MBP tag by Western blot  aggregates is associated with neurodegenerative diseases such as Parkinson's disease and amyotrophic lateral sclerosis (ALS). Bacteria and fungi, but not mammalian cells, contain specialized AAA+ chaperones of the Hsp104 type that have the ability to dissociate fibrillar aggregates. Here, the authors investigated natural homologues of Hsp104 from species across multiple kingdoms that can suppress cellular toxicity arising from aggregation of disease-associated proteins, such as TDP-43, α-synuclein and FUS. Using natural variants of Hsp104, they identified homologs that prevented aggregation without showing previously observed off-target effects. These findings may help in developing new therapeutic strategies.Decision letter after peer review:eLife. Your article has been reviewed by three peer reviewers, one of whom is a member of our Board of Reviewing Editors, and the evaluation has been overseen by Huda Zoghbi as the Senior Editor. The following individual involved in review of your submission has agreed to reveal their identity: David Li-Kroeger (Reviewer #3).Thank you for submitting your article "Therapeutic genetic variation revealed in diverse Hsp104 homologs" for consideration by The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.eLife". Please let us know if you would like to pursue this option. As the editors have judged that your manuscript is of interest, but as described below that additional experiments are required before it is published, we would like to draw your attention to changes in our revision policy that we have made in response to COVID-19 (https://elifesciences.org/articles/57162). First, because many researchers have temporarily lost access to the labs, we will give authors as much time as they need to submit revised manuscripts. We are also offering, if you choose, to post the manuscript to bioRxiv  along with this decision letter and a formal designation that the manuscript is "in revision at eLife, subject to the following revisions:The reviewers and the reviewing editor agree that this study should be considered for publication in Essential revisions:1) The authors need to more carefully interpret protein levels in their experiments. Throughout the text, the authors conclude that the levels of Hsp104, ɑ-synuclein, and TDP-43 do not change enough to affect their preferred interpretations. However, levels of the Hsp104 variants vary over at least an order of magnitude . The authors need to consider Hsp104 variant levels when arguing that one Hsp104 variant is more effective than another (e.g. subsection “Diverse Hsp104 homologs selectively suppress TDP-43 toxicity and aggregation in yeast”).Furthermore, some Hsp104 variants reduce expression of toxic proteins . The text needs to properly acknowledge that alterations in toxic protein expression could be a mechanism by which some Hsp104 variants suppress toxicity .2) Some experiments lack important controls. In Figure 4, the authors should also analyze worms lacking both ɑ-synuclein and Hsp104 as well as worms expressing the Hsp104 variants alone. This control will help the reader to understand: (a) whether the loss of DA neurons is ɑ-synuclein-specific, and (b) whether the Hsp104-dependent rescue of DA neurons is ɑ-synuclein-specific. In Figure 5G,H, the authors should include un-aggregated luciferase controls so the reader can better understand the magnitude of luciferase refolding induced by these Hsp104 variants.S. cerevisiae Hsp104 as well as Hsp104 from one of the more exotic species.3) The result that these Hsp104 variants could suppress toxicity by a disaggregase-independent mechanism is a compelling finding that requires an additional control. Because many of these Hsp104 variants have not been biochemically characterized before this work, the authors should demonstrate that the canonical walker A, pore loop, and walker B mutations used in Figure 6 disrupt disaggregase activity in the expected manner. The authors could use the same luciferase assay from Figure 5G,5H with wild-type, walker A mutant, pore loop mutant, and walker B mutant variants of 4) Subsection "Hsp104 homologs prevent TDP-43 aggregation in human cells":a) Regarding the observation that ScHsp104WT also reduced the proportion of HEK293T cells with TDP-43 aggregation at 24-hours post transfection while it did not in yeast cells , the authors did not address if this is just a protein artefact in utilizing mClover3-TDP-43ΔNLS instead of TDP-43-GFP, or if it reflects differences in cellular environments or factors related to Hsp104 activity that are unaccounted for. It should be emphasized that the latter possibility requires attention as it raises the problem of relevance in using the yeast model to approximate the human cell, despite the authors' justification by referencing such practice by other groups. It is thus prudent that the authors repeat some of the TDP-43 experiments for ScHsp104WT in yeast cells using mClover3-TDP-43ΔNLS instead of TDP-43-GFP, for example.b) In tracking the TDP-43 foci formation at 24-hours versus 48-hours post transfection, the authors did not address as to why the magnitude of increase in % cells with foci from 24-hours to 48-hours displayed by no-Hsp104 (vector-only) control is drastically lower compared to all other cells co-expressing a Hsp104 homologue. One explanation is the unaccounted protein overexpression-induced stress / toxicity experienced by cells co-transfected with an Hsp104 homologue that is absent in the vector-only (no Hsp104) control, which will likely make the results more difficult to interpret.c) Western blots for samples at 48-hours post transfection is missing.C. elegans":5) Subsection "αSyn-selective Hsp104 homologs prevent dopaminergic neurodegeneration in C. elegans model (TDP-43 toxicity in C. elegans model: https://www.ncbi.nlm.nih.gov/pubmed/20530643). This data is currently missing from the manuscript.a) Regarding the interpretation of lack of neuroprotection by CtHsp104 due to target promiscuity, this argument will be convincing once CtHsp104 is shown to also fail in providing neuroprotection against TDP-43 toxicity compared to Hsp104 homologues that are specific against TDP-43 using the 6) Subsection "Differential suppression of proteotoxicity by Hsp104 homologs is not due to changes in disaggregase activity":a) Regarding the authors' statement on potentiated Hsp104 disaggregases having elevated ATPase and disaggregase activities: Even though this has been shown previously, ScHsp104(A503S) or another potentiated mutant should be included in the biochemistry assays presented as a positive control, which will validate the experimental procedures employed and importantly, to provide conclusive evidence that the natural Hsp104 homologues are indeed "potentiated" differently compared to the artificially potentiated mutants.b) In Figure 5C, the authors should also include Western blots showing normal induction of Hsp26 or other heat shock-inducible proteins as confirmation that the yeast strains used are not compromised in heat shock response.c) In Figure 5E, the authors should label the individual rescuer and non-rescuer Hsp104 homologues; the current figure is not very informative.d) Although the authors presented extensive biochemistry data showing the ATPase and disaggregase activity of Hsp104 homologues, there is no discussion in regard to the oligomerization state, which can be assessed using a variety of experimental techniques, including native PAGE, size exclusion chromatography, analytical ultracentrifugation, negative-stain electron microscopy, etc. In fact, examination of oligomeric state becomes particularly important in the authors' investigation of Hsp104 domains that contribute to suppression of aggregate toxicity, as chimeric proteins or isolated cognate domains may not form the expected hexameric form. Furthermore, data pertaining to the particular oligomeric state of the chimeras / cognate domains are expected to provide valuable information in establishing the molecular / structural basis of any retention of loss of toxicity suppression activity.7) Subsection "ATPase-independent passive chaperone activity underlies suppression of TDP-43 and αSyn toxicity by Hsp104 homologs":a) Regarding the authors' claim of Hsp104 homologues in suppressing aggregate toxicity via passive chaperone activity: Although the authors have provided extensive, thorough yeast genetics data showing that neither the ATPase nor substrate translocation activity is required for aggregate toxicity suppression, to claim passive chaperone activity being the molecular basis requires additional experimental proof, such as comparing the refolding rate of thermally denatured reporter proteins (e.g. luciferase) in the presence or absence of Hsp104 homologues, their respective chimeric proteins or isolated cognate domains.8) In subsection “Diverse Hsp104 homologs selectively suppress TDP-43 toxicity and aggregation in yeast” and subsection “Distinct Hsp104 homologs selectively suppress αSyn toxicity and inclusion formation in yeast”, referring to the data In Figure 1—figure supplement 3: Suppression of TDP-43 or αSyn toxicity by select Hsp104 homologs is a substrate-specific effect . The authors make the claim that "MbHsp104, SrHsp104, CrHsp104, PeHsp104, and GsHsp104, which all suppress TDP-43 toxicity, do not suppress αSyn nor FUS toxicity" (panel A) and that "TtHsp104 and TlHsp104 do not suppress TDP43 nor FUS toxicity" panel B and that "other Hsp104s investigated in this study do not suppress toxicity of TDP-43 (left), αSyn (middle), or FUS (right)" (panel C). While most of the data here support the authors conclusions, the wording "does not suppress" is a very strong statement. The data is not completely convincing. For example, in panel C, for the FUS overexpression (right panel) ChtHsp104 and StHsp140 seem to show some suppression of toxicity. Similarly, MtHsp104, StHsp104 and TaHsp104 for α-Syn maybe show some slight suppression. I don't think this changes the nature of the conclusion; however, the authors should be a little more cautious in their choice of wording and/or better discuss the data if they feel that there is compelling evidence that absolutely no suppression is shown.9) In subsection “Diverse Hsp104 homologs selectively suppress TDP-43 toxicity and aggregation in yeast”: the authors state "Hsp104 homologs were consistently expressed,.… ". The data on the Western Blot for Hsp104  is not convincing. The levels of protein look variable. Perhaps a quantification of replicates could be added to show whether Hsp104::flag protein levels are indeed consistent? Similarly, in Figure 2 panel C the Western blot could also be more convincing with replication and quantification. In Figure 5C, the Western Blot and the text that refer to it show variation in the levels of expression as well. For example, ScHsp104, TtHsp104 and GsHsp104 show much higher protein levels than StHsp104, MtHsp104 and ChtHsp104 yet the text claims: "These differences are not due to differences in expression, as all homologs were consistently expressed, as confirmed by Western blot ". ScHsp104, TtHsp104 and GsHsp104 are among the highest rescue of thermotolerance. Contrast this with Figure 7—figure supplement 1, where the expression levels look much more consistent, with only CtHsp104 ∆N from the HSP104 promoter (panel F) showing lowered expression. The chimeras presented in Figure 8—figure supplement 1 also show some variation in levels. Here, the claim in the text is not as strong , however the figure caption states "Chimeric Hsp104s and proteotoxic substrates are consistently expressed. In addition, the authors discuss the variation in regard to the results  fail to confer thermotolerance"). In summary, the differences in expression levels for these experiments should be better quantified if possible and should be discussed in more depth regarding possible effects on the data, especially given the reliance on ectopic expression throughout the manuscript. Essential revisions:1) The authors need to more carefully interpret protein levels in their experiments. Throughout the text, the authors conclude that the levels of Hsp104, ɑ-synuclein, and TDP-43 do not change enough to affect their preferred interpretations. However, levels of the Hsp104 variants vary over at least an order of magnitude . The authors need to consider Hsp104 variant levels when arguing that one Hsp104 variant is more effective than another (e.g. subsection “Diverse Hsp104 homologs selectively suppress TDP-43 toxicity and aggregation in yeast”).Furthermore, some Hsp104 variants reduce expression of toxic proteins . The text needs to properly acknowledge that alterations in toxic protein expression could be a mechanism by which some Hsp104 variants suppress toxicity .We thank the reviewers for raising this concern regarding how varying protein levels affect our interpretations. We have quantified three independent replicates of the blots shown in Figure 1D and Figure 2C. For the blot shown in Figure 1D, we find that neither Hsp104-FLAG nor TDP-43 levels vary significantly across strains. We have added bar charts showing this quantification to the manuscript as Figure 1E, F. Similarly, for the blot shown in Figure 2C, neither Hsp104-FLAG nor αSyn-GFP levels vary significantly across strains. We have added bar charts reflecting this quantification to the manuscript as Figure 2D, E.Sc, Pe, and Sr) have increased TDP-43 expression. These findings suggest that Hsp104 homologs do not decrease TDP-43 foci formation by lowering TDP-43 expression. We have added these data to Figure 3C, D.Similarly, we also quantified three independent replicates of the blot shown in Figure 3B (which now includes samples taken 24hours and 48hours post-transfection) for Hsp104-FLAG and mClover3-TDP43∆NLS. We find that no sample (with the exception of no Hsp104 control) is significantly deficient in Hsp104 expression, although PeHsp104 expression is significantly increased at both day 1 and day 2 post-transfection. We also find that at day 1 post-transfection, none of the conditions have significantly different TDP-43 expression. Likewise, at day 2, none of the conditions shows diminished TDP-43 expression, although some conditions .We have also quantified three independent replicates of the blot shown in Figure 5C  and added this quantification to the manuscript as Figure 5G. Here, we find that some Hsp104 homologs are expressed at significantly lower levels than ScHsp104-FLAG. However, we find that Hsp104-FLAG levels are a poor predictor of thermotolerance performance  does not contribute significantly to the observed phenotypes.2) Some experiments lack important controls. In Figure 4, the authors should also analyze worms lacking both ɑ-synuclein and Hsp104 as well as worms expressing the Hsp104 variants alone. This control will help the reader to understand: (a) whether the loss of DA neurons is ɑ-synuclein-specific, and (b) whether the Hsp104-dependent rescue of DA neurons is ɑ-synuclein-specific.We have added data in Figure 4—figure supplement 1 showing that expression of either GFP alone or GFP plus Hsp104 homologs alone in dopaminergic neurons is not intrinsically toxic . Therefore, the dopaminergic neurodegeneration phenotype is α-synuclein-specific and Hsp104 homologs mitigate this α-synuclein-mediated neurodegeneration.In Figure 5G,H, the authors should include un-aggregated luciferase controls so the reader can better understand the magnitude of luciferase refolding induced by these Hsp104 variants.We now note in subsection “Differential suppression of proteotoxicity by Hsp104 homologs is not due to changes in disaggregase activity” that when combined with Hsp70 and Hsp40, ScHsp104 typically recovers ~10-30% of the unaggregated, native luciferase activity in these experiments .3) The result that these Hsp104 variants could suppress toxicity by a disaggregase-independent mechanism is a compelling finding that requires an additional control. Because many of these Hsp104 variants have not been biochemically characterized before this work, the authors should demonstrate that the canonical walker A, pore loop, and walker B mutations used in Figure 6 disrupt disaggregase activity in the expected manner. The authors could use the same luciferase assay from Figure 5G,5H with wild-type, walker A mutant, pore loop mutant, and walker B mutant variants of S. cerevisiae Hsp104 as well as Hsp104 from one of the more exotic species.S. cerevisiae Hsp104 disaggregase activity has been exhaustively characterized in previous work, see for example: . Furthermore, this loss of disaggregase activity is reflected by thermotolerance defects in vivo . Thus, defective thermotolerance is a reliable reporter of mutations that impair disaggregase activity, which is why we employed it in Figure 7—figure supplement 1I to show that Walker A, Walker B, and pore-loop mutants shown in Figure 6  are defective in disaggregase activity. To address the reviewers’ concern that we characterize the biochemical effects of these mutants directly, we purified Walker A, Walker B, and pore-loop mutants for the Hsp104 homolog from Calcarisporiella thermophila and characterized their in vitro disaggregase activity against luciferase aggregates. As expected, Walker A, Walker B, and pore-loop mutants were all severely impaired in disaggregase activity. We have added these data to Figure 7—figure supplement 1J.The inactivating effect of Walker A, pore-loop, and Walker B mutations on 4) Subsection "Hsp104 homologs prevent TDP-43 aggregation in human cells":a) Regarding the observation that ScHsp104WT also reduced the proportion of HEK293T cells with TDP-43 aggregation at 24-hours post transfection while it did not in yeast cells , the authors did not address if this is just a protein artefact in utilizing mClover3-TDP-43ΔNLS instead of TDP-43-GFP, or if it reflects differences in cellular environments or factors related to Hsp104 activity that are unaccounted for. It should be emphasized that the latter possibility requires attention as it raises the problem of relevance in using the yeast model to approximate the human cell, despite the authors' justification by referencing such practice by other groups. It is thus prudent that the authors repeat some of the TDP-43 experiments for ScHsp104WT in yeast cells using mClover3-TDP-43ΔNLS instead of TDP-43-GFP, for example.We have shown that ScHsp104 has limited ability to antagonize TDP-43 aggregation in yeast  , in human cells after 48hours of expression , and at the pure protein level  . These data from multiple settings (using different TDP-43 constructs) make us confident that ScHsp104 does not effectively antagonize TDP-43 aggregation. The reviewers draw attention to the reduced proportion of cells with TDP-43 aggregates after 24hours when ScHsp104 is expressed . This finding is interesting, but ultimately after longer incubation times, ScHsp104 is unable to antagonize TDP-43 aggregation in human cells  just as it has limited ability to antagonize TDP-43 aggregation in yeast  or at the pure protein level . Thus, we suggest that there is no reason to doubt the relevance of the yeast model for human cells. Importantly, our yeast model  of TDP-43 aggregation and toxicity has empowered the discovery of several key genetic modifiers of TDP-43 aggregation and toxicity, which have translated to fly, mouse, and neuronal models of ALS/FTD . Indeed, this yeast model sparked the discovery of intermediate-length, polyglutamine expansions (~27-33 glutamines) in ataxin 2 as a common risk factor for ALS . Based on these findings that originated from our yeast model, ataxin 2 antisense oligonucleotides are now in clinical development by Biogen/Ionis to treat ALS b) In tracking the TDP-43 foci formation at 24-hours versus 48-hours post transfection, the authors did not address as to why the magnitude of increase in % cells with foci from 24-hours to 48-hours displayed by no-Hsp104 (vector-only) control is drastically lower compared to all other cells co-expressing a Hsp104 homologue. One explanation is the unaccounted protein overexpression-induced stress / toxicity experienced by cells co-transfected with an Hsp104 homologue that is absent in the vector-only (no Hsp104) control, which will likely make the results more difficult to interpret.A503S, which show increases of ~3.7% and ~4.7%, respectively. It is unclear why the increase is more pronounced from 24hours to 48hours for DPLA:DWB (~12.2%), ScHsp104 (~8.3%), and SrHsp104 (~6.5%), but we note that we have observed no overt toxicity from expressing TDP-43 or the Hsp104 variants in HEK293T cells over this short timeframe. This finding corroborates several prior studies, which found that expression of Hsp104 in mammalian cells is not toxic . Indeed, Hsp104 expression can be neuroprotective in mammalian models of neurodegeneration . In the present study, we have shown that expression of Hsp104 homologues in dopaminergic neurons of C. elegans is not toxic . Regardless, a key result from our experiments is that Hsp104A503S and PeHsp104 consistently and significantly reduce TDP-43 aggregation in human cells (HEK293T cells) .In response to the reviewers' comment, we reanalyzed the raw data  for Figure 3E to ensured that we gated the data to account for any shifts in expression. Hence, the graphs in Figure 3E have now been updated. We note that reviewers are not fully correct, the increase in % cells with TDP-43 aggregates from 24-hours to 48-hours displayed by no-Hsp104 (vector-only) control is not drastically lower compared to all other cells co-expressing a Hsp104 homologue. For example, for the no Hsp104 control the increase is ~3.7%, which is comparable to PeHsp104 and Hsp104c) Western blots for samples at 48-hr post transfection is missing.We have added Western blots from samples at 48hours post-transfection, along with quantification of the blots for Hsp104 and mClover3-TDP-43∆NLS (at both 24hours and 48hours post-transfection) to Figure 3B. Please refer to our response to point 1 above for further discussion of these data.5) Subsection "αSyn-selective Hsp104 homologs prevent dopaminergic neurodegeneration in C. elegans":a) Regarding the interpretation of lack of neuroprotection by CtHsp104 due to target promiscuity, this argument will be convincing once CtHsp104 is shown to also fail in providing neuroprotection against TDP-43 toxicity compared to Hsp104 homologues that are specific against TDP-43 using the C. elegans model (TDP-43 toxicity in C. elegans model: https://www.ncbi.nlm.nih.gov/pubmed/20530643). This data is currently missing from the manuscript.We thank the reviewers for suggesting we consider how CtHsp104 behaves against TDP-43 in a worm model. However, it is not trivial for us to establish and characterize a new worm model, especially under the prevailing conditions of the pandemic. Nonetheless, to address this issue we have instead assessed CtHsp104 activity against TDP-43 aggregation in HEK293T cells using the same experimental protocol as in Figure 3. We find that CtHsp104 does not inhibit TDP-43 aggregation in HEK293T cells and in fact exacerbates aggregation. We have added these data to a new Figure 4—figure supplement 3. Our data show that CtHsp104, which is not substrate-specific, may have limited efficacy in animal model systems.6) Subsection "Differential suppression of proteotoxicity by Hsp104 homologs is not due to changes in disaggregase activity":a) Regarding the authors' statement on potentiated Hsp104 disaggregases having elevated ATPase and disaggregase activities: Even though this has been shown previously, ScHsp104(A503S) or another potentiated mutant should be included in the biochemistry assays presented as a positive control, which will validate the experimental procedures employed and importantly, to provide conclusive evidence that the natural Hsp104 homologues are indeed "potentiated" differently compared to the artificially potentiated mutants.A503S  showing that ScHsp104A503S is enhanced in ATPase and disaggregase activity compared to WT ScHsp104 as established previously . We note, however, that we have already provided conclusive genetic evidence that the natural Hsp104 homologs are potentiated differently than the artificially potentiated ScHsp104 variants . Thus, DWA, DWB, or DPLA mutations eliminate the ability of ScHsp104A503S to rescue TDP-43 and a-synuclein toxicity in yeast , whereas these mutations typically do not affect rescue of TDP-43 and a-synuclein toxicity by Hsp104 homologues in yeast . Since DWA mutations eliminate ATP binding, DWB mutations eliminate ATP hydrolysis, and DPLA eliminates pore-loop binding to substrate, these results strongly suggest that the Hsp104 homologs rescue TDP-43 and a-synuclein toxicity by a different mechanism than ScHsp104A503S.We have added data comparing ATPase activity and luciferase disaggregation activity of WT ScHsp104 and ScHsp104b) In Figure 5C, the authors should also include Western blots showing normal induction of Hsp26 or other heat shock-inducible proteins as confirmation that the yeast strains used are not compromised in heat shock response.We thank the reviewers for this suggestion and have added an Hsp26 blot to Figure 5E . These data show that the yeast strains we use for thermotolerance induce Hsp26 after incubation at 37°C and thus are able to mount a robust heat shock response.c) In Figure 5E, the authors should label the individual rescuer and non-rescuer Hsp104 homologues; the current figure is not very informative.We thank the reviewers for bringing up this issue of clarity. We have amended Figure 5E  so that each data point is now color-coded by the Hsp104 homolog it represents.d) Although the authors presented extensive biochemistry data showing the ATPase and disaggregase activity of Hsp104 homologues, there is no discussion in regard to the oligomerization state, which can be assessed using a variety of experimental techniques, including native PAGE, size exclusion chromatography, analytical ultracentrifugation, negative-stain electron microscopy, etc.Hsp104 is only functional as an ATPase or disaggregase as a hexamer . Thus, ATPase activity and disaggregase activity serve as strong evidence that the various Hsp104 homologues form functional hexamers . We have previously shown that CtHsp104 forms hexamers under these conditions . We have now added size-exclusion chromatography coupled to multi-angle light Scattering (SEC-MALS) data to the manuscript  demonstrating that the other Hsp104 homologs we purified  are likely hexameric.In fact, examination of oligomeric state becomes particularly important in the authors' investigation of Hsp104 domains that contribute to suppression of aggregate toxicity, as chimeric proteins or isolated cognate domains may not form the expected hexameric form. Furthermore, data pertaining to the particular oligomeric state of the chimeras / cognate domains are expected to provide valuable information in establishing the molecular / structural basis of any retention of loss of toxicity suppression activity.E. coli Hsp104) can form hexamers that possess robust disaggregase activity in vitro and in vivo . Moreover, we have assessed the thermotolerance activity of all the chimeras , which provides an accurate proxy for disaggregase activity. In many cases, the chimeras display partial thermotolerance activity , but in other cases they do not, which indicates impaired functionality with respect to thermotolerance. Possible causes of reduced functionality are dysregulated interdomain interactions, reduced hexamerization, reduced ATPase activity, or reduced ability to collaborate with yeast Hsp70. We suspect that the latter possibility applies to several chimeras (e.g. Sr-Sc chimeras and Ct-Sc chimeras) that lose thermotolerance activity when they do not possess the ScHsp104 middle domain, the primary site of Hsp70 interaction . In some cases, chimeras are robustly expressed, provide no thermotolerance but rescue TDP-43 toxicity (e.g. CrNTD:NBD1:MDScNBD2:CTD). Importantly, these findings provide further evidence that rescue of TDP-43 toxicity by this chimera does not depend on disaggregase activity.As noted in the manuscript, the isolated domains of Hsp104 and homologs are all anticipated to be monomeric based on previous studies . We have not purified the very many chimeric proteins assessed in these studies, as this would be a very large endeavor. However, prior studies indicate that chimeric proteins formed between Hsp104 and ClpB (7) Subsection "ATPase-independent passive chaperone activity underlies suppression of TDP-43 and αSyn toxicity by Hsp104 homologs":a) Regarding the authors' claim of Hsp104 homologues in suppressing aggregate toxicity via passive chaperone activity: Although the authors have provided extensive, thorough yeast genetics data showing that neither the ATPase nor substrate translocation activity is required for aggregate toxicity suppression, to claim passive chaperone activity being the molecular basis requires additional experimental proof, such as comparing the refolding rate of thermally denatured reporter proteins (e.g. luciferase) in the presence or absence of Hsp104 homologues, their respective chimeric proteins or isolated cognate domains.By passive chaperone activity we simply meant passive inhibition of aggregation. Our data  suggest that Hsp104 homologs inhibit aggregation of TDP-43. However, we do not believe that the homologs are necessarily refolding TDP-43. We appreciate that “chaperone activity” may suggest assisting native folding, whereas we simply meant inhibition of aggregation. To avoid any confusion, we have removed the “passive chaperone” terminology from the manuscript and now simply refer to passive inhibition of aggregation.8) In subsection “Diverse Hsp104 homologs selectively suppress TDP-43 toxicity and aggregation in yeast” and subsection “Distinct Hsp104 homologs selectively suppress αSyn toxicity and inclusion formation in yeast”, referring to the data In Figure 1—figure supplement 3: Suppression of TDP-43 or αSyn toxicity by select Hsp104 homologs is a substrate-specific effect . The authors make the claim that "MbHsp104, SrHsp104, CrHsp104, PeHsp104, and GsHsp104, which all suppress TDP-43 toxicity, do not suppress αSyn nor FUS toxicity" (panel A) and that "TtHsp104 and TlHsp104 do not suppress TDP43 nor FUS toxicity" panel B and that "other Hsp104s investigated in this study do not suppress toxicity of TDP-43 (left), αSyn (middle), or FUS (right)" (panel C). While most of the data here support the authors conclusions, the wording "does not suppress" is a very strong statement. The data is not completely convincing. For example, in panel C, for the FUS overexpression (right panel) ChtHsp104 and StHsp140 seem to show some suppression of toxicity. Similarly, MtHsp104, StHsp104 and TaHsp104 for α-Syn maybe show some slight suppression. I don't think this changes the nature of the conclusion; however, the authors should be a little more cautious in their choice of wording and/or better discuss the data if they feel that there is compelling evidence that absolutely no suppression is shown.We have altered the wording of the manuscript at these junctures from “do not suppress” to “have minimal effect on”.9) In subsection “Diverse Hsp104 homologs selectively suppress TDP-43 toxicity and aggregation in yeast”: the authors state "Hsp104 homologs were consistently expressed,.… ". The data on the Western Blot for Hsp104  is not convincing. The levels of protein look variable. Perhaps a quantification of replicates could be added to show whether Hsp104::flag protein levels are indeed consistent? Similarly, in Figure 2 panel C the Western blot could also be more convincing with replication and quantification. In Figure 5C, the Western Blot and the text that refer to it show variation in the levels of expression as well. For example, ScHsp104, TtHsp104 and GsHsp104 show much higher protein levels than StHsp104, MtHsp104 and ChtHsp104 yet the text claims: "These differences are not due to differences in expression, as all homologs were consistently expressed, as confirmed by Western blot ". ScHsp104, TtHsp104 and GsHsp104 are among the highest rescue of thermotolerance. Contrast this with Figure 7—figure supplement 1, where the expression levels look much more consistent, with only CtHsp104 ∆N from the HSP104 promoter (panel F) showing lowered expression. The chimeras presented in Figure 8—figure supplement 1 also show some variation in levels. Here, the claim in the text is not as strong , however the figure caption states "Chimeric Hsp104s and proteotoxic substrates are consistently expressed. In addition, the authors discuss the variation in regard to the results  fail to confer thermotolerance"). In summary, the differences in expression levels for these experiments should be better quantified if possible and should be discussed in more depth regarding possible effects on the data, especially given the reliance on ectopic expression throughout the manuscript.We have quantified blots in Figure 1, Figure 2, and Figure 5 to be able to more substantively demonstrate that varying Hsp104 levels are not a significant contributor to our observed phenotypes . These findings suggest that the minor variations in chimera expression are also not a major factor.


KRAS, TP53) and microsatellite status were also preserved in the respective cell line derivates. In conclusion, the four examined newly established cell lines exhibited a phenotype and genotype closely recapitulating their parental tumor. Hence, newly established cancer cell lines may be useful models for further pharmacogenomic studies.Human cancer cell lines are frequently used as model systems to study molecular mechanisms and genetic changes in cancer. However, the model is repeatedly criticized for its lack of proximity to original patient tumors. Therefore, understanding to what extent cell lines cultured under artificial conditions reflect the phenotypic and genomic profiles of their corresponding parental tumors is crucial when analyzing their biological properties. To directly compare molecular alterations between patient tumors and derived cell lines, we have established new cancer cell lines from four patients with gastrointestinal tumors. Tumor entities comprised esophageal cancer, colon cancer, rectal cancer and pancreatic cancer. Phenotype and genotype of both patient tumors and derived low-passage cell lines were characterized by immunohistochemistry (22 different antibodies), array-based comparative genomic hybridization and targeted next generation sequencing (48-gene panel). The immunophenotype was highly consistent between patient tumors and derived cell lines; the expression of most markers in cell lines was concordant with the respective parental tumor and characteristic for the respective tumor entities in general. The chromosomal aberration patterns of the parental tumors were largely maintained in the cell lines and the distribution of gains and losses was typical for the respective cancer entity, despite a few distinct differences. Cancer gene mutations (e.g.,  Since the establishment of the cervical carcinoma cell line HeLa in 1951, human cancer cell lines have widely been used as model systems to study molecular mechanisms and genetic changes in cancer3. The advantages of established “immortal” cell lines as two-dimensional in vitro cancer models are obvious: easy to maintain and to (molecularly) manipulate, fast growing (short doubling time), cheap, broadly and easily available, potentially animal protecting and represent a source for unlimited production of specific tumor DNA, RNA and protein4. Cancer cell lines represent pure populations of cancer cells, facilitating the detection of molecular genetic changes, in particular homozygous deletions. However, the cell line model is repeatedly criticized due to its limited reflection of in vivo solid tumors which are not simply pure cancer cells but include a variety of other cell types such as inflammatory cells, stromal cells and blood vessels. Furthermore, cross-contamination of cell lines, microbial contamination, misidentification of the tissue of origin and over-passaging are critical issues, in particular with respect to continuous long-term cell lines6.Cancer is a heterogeneous disease in terms of both phenotype and genotype. Various types of tumors with different histomorphologies, genetic and epigenetic alterations, and clinical courses exist9. Interestingly, more frequently used cell lines often had less genomic similarities to primary tumors than rarely used cell lines9. Given that tumorigenesis can be viewed as an evolutionary process, this illustrates that cell lines can apparently diverge from their primary tumor counterparts over time during pro-longed culture in vitro. How fast and to which extent this genomic evolution occurs is influenced by the inherent genomic instability of the tumor cells and the selective pressures such as culture conditions (as opposed to host microenvironment) they are subject to. For instance, Mouradov et al. identified an overrepresentation of CTNNB1 mutations and microsatellite instability in established colon cancer cell lines compared to patient tumors8. These genomic alterations might have been acquired or selected for in culture or might eventually be related to the preferential outgrowth of particular genotypes, given that establishment of new cell lines remains a challenge and only about 10–15% of CRCs give rise to cell lines8. Ince et al. reported that improved culture conditions produce cell lines with genotypes and phenotypes more closely resembling primary tumors10. Hence, a culture context dependent genomic evolution when propagating tumor cells outside their host environment cannot be denied12. This is corroborated by more recent findings from Ben-Uri et al., showing that during patient-derived xenograft (PDX) passaging particular copy number alterations (CNAs) were acquired that differed from the ones seen in patients, while certain CNAs characteristic for and recurrently observed in patient tumors gradually disappeared during PDX passaging such as the gain of chromosome arm 8q (harbors the MYC oncogene on 8q24.3) in breast cancers13. Moreover, considerable genomic heterogeneity has been identified between established cancer cell lines cultured independently in different laboratories, indicating ongoing genomic evolution15. Concordantly, data from single cell cloning showed single cell derived clones of established CRC cell lines select for but cannot fully maintain the genotype of the parental cell population16. All these findings highlight the critical role of culture conditions, prolonged passaging and the degree of genomic instability in shaping the evolution of cancer cell lines genomes and phenotypes away from patient tumors. Furthermore, this underlines the need for the establishment of new patient-derived models such as patient-derived cell lines (PDCLs), PDXs or patient-derived organoids (PDOs), which may better reflect the biologic properties of the tumors they were derived from. Considerably large biobanks of PDOs have been established for several tumor entities, leadingly by Clevers et al., and data comparing phenotype and genotype of these PDOs with corresponding patient tumors suggest a high overlap21. Establishment of new PDCL remains more difficult and hence data are sparser. Sample numbers of successful outgrowths are usually small, and most studies report the establishment of one or a few new cell lines, often along with some molecular or functional characterization of the newly generated cell lines. However, a direct comparison with the patient tumor from which the cell line was derived from is only rarely provided. We consider a thorough molecular characterization of new patient-derived models along with the tumors they were derived from crucial since the genotype can directly influence transcriptional changes and is correlated with drug response . To address challenges with current cancer cell lines, such as the lack of data on the tumors of origin and cancer subtypes that are missing or poorly represented, the Human Cancer Models Initiative (HCMI) has been established22. The HCMI is an international effort to generate, genomically characterize and annotate the new cancer cell models as a resource for the scientific community.Large-scale comparisons of the genomic profiles of established cell lines and primary patient tumors from The Cancer Genome Atlas (TCGA) project showed for colon cancer and ovarian high-grade serous cancer that overall established cell lines resemble the genomic changes and molecular subtypes observed in patient tumors fairly well, despite some incongruenceHere, we report the establishment of four new PDCLs from different tumors of the digestive system including esophageal cancer, colon cancer, rectal cancer and pancreatic ductal adenocarcinoma (PDAC). We performed a direct side-by-side comparison with the respective parental tumors to determine to what extent the newly established low-passage cell lines reflect the phenotypic, genomic and genetic characteristics of the tumor from which they have been derived. To this end, both parental tumors and corresponding derived cell lines were analyzed by immunohistochemistry, array comparative hybridization (aCGH) and targeted next generation sequencing (TruSeq Amplicon 48-gene panel). This allowed us to assess how faithfully newly established PDCLs model their tumors of origin.24. All patients gave informed consent prior to surgery, and the study was approved by the local ethics committee of the Medical Faculty Mannheim of Heidelberg University (2012-293N-MA) and by the institutional review board of the National Institutes of Health (OHSRP #13221/MTA #41443). All experiments were performed in accordance with relevant guidelines and regulations.Fresh tumor samples for cell culture were acquired from patients who underwent surgery for a tumor of the digestive system at University Medical Center Mannheim, Germany. Samples included in this study were taken from an esophageal carcinoma (liver metastasis), a colonic adenocarcinoma, a rectal adenocarcinoma and a pancreatic ductal adenocarcinoma, diagnosed between 2012 and 2015. Corresponding formalin-fixed paraffin-embedded (FFPE) parental tumor tissue was obtained from the archive of the Institute of Pathology, University Medical Center Mannheim, Germany. In addition to clinical diagnosis and the pathology report, the diagnosis was histologically confirmed by two pathologists . Tumor diagnosis and staging was performed according to the WHO classification and the American Joint Committee on Cancer (AJCC)/Union for International Cancer Control (UICC) staging system25. Briefly, fresh tumor tissues were cleaned from surrounding connective tissue and necrotic and/or hemorrhagic areas, and subsequently finely minced into cubes of approximately 1 mm3 using two scalpels. Neither enzymatic nor mechanic dissociation of the tumor cells was performed. Basal culture media were as follows: Dulbecco's Modified Eagle Medium  or DMEM/keratinocyte serum free medium mixed at a ratio of 2 to 1 . Basal media were supplemented with fetal bovine serum , penicillin  and gentamicin (2.5 mg/ml). The cells were cultured at 37 °C in a humidified atmosphere of 5% CO2 in air, and the medium was replaced every third day. All experiments for this study were performed on cell lines between the first and fourth passage. Cell lines tested negative for mycoplasma contamination and were authenticated using short-tandem repeat (STR) profiling.To establish cell lines, fresh tumor tissues were processed by the outgrowth method as described previously26. The following loci were used: amelogenin, CSF1PO, D5S818, D7S820, D13S317, D16S359, TH01, TPOX, and vWA. For cell line authentication, the Cellosaurus STR Similarity Search Tool  was used27.STR profiling was done according to the recommendations of the Standard for human cell line authentication established by the American Type Culture Collection Standards Development Organization Workgroupg for 10 min and supernatant (culture medium) was discarded. Cell pellets were re-suspended 1:1 in 3% (w/v) agar agar . Once cooled down and solidified, the cells agar mixture was placed in a tissue embedding cassette and subsequently formalin-fixed and paraffin-embedded for further processing.Cultured adherent cells were dissociated using Trypsin–EDTA (0.25%) . Cell suspensions of cultured cells were spun down at 30029. The parameters were determined by visual assessment of two pathologists . The H score was calculated according to the following formula, ranging from 0 to 300:Two to three µm sections from tissue blocks  or cell blocks (derived cell lines) were mounted on Superfrost Plus microscope slides . After deparaffinization and re-hydration, heat-induced antigen retrieval (HIAR) was performed in Novocastra Epitope Retrieval Solution pH 9 or pH 6  in a water bath at 95 °C for 20 min (pH 9) or 40 min (pH 6), respectively, followed by incubation of sections with primary antibodies for 30 min at room temperature .30. All samples were evaluated based on 3-µm hematoxylin eosin (H&E) stained sections and the tumor regions for DNA isolation were marked for macrodissection. Tumor cell contents of the parental tumors were 50% (patient 1 & 2), 40% (patient 3) and 20% (patient 4), respectively. DNA concentration and purity were measured by a NanoDrop 1000 Spectrophotometer . In addition, double-stranded DNA was quantified by a Qubit 3.0 fluorometer  using the Qubit dsDNA BR (Broad Range) Assay Kit (Life Technologies). DNA integrity was evaluated on 1% agarose gels stained with Gel-Red . BIOMED-2 control gene PCR assay of differently sized amplicons  was performed to visualize DNA fragment size distribution and to test amplifiability and integrity of DNA samples31. In addition, amplifiability of the isolated FFPE DNA was assessed by a qPCR assay  and delta Ct values were determined against the provided control DNA (ACD1) and as additional reference against human genomic DNA .DNA was isolated from fresh or frozen cells using the DNeasy Blood and Tissue Mini Kit . From FFPE tumor tissue , DNA isolation was done as described previously32. Sex-matched human genomic DNA  was used as reference. Labeled DNA was hybridized to SurePrint G3 CGH 8X60K microarrays (Agilent). Microarrays were scanned with microarray scanner G2565BA (Agilent) and microarray images were processed by Feature Extraction software version 10.7.3.1 (Agilent). Data were visualized and analyzed using Nexus Copy Number software version 9.0 . Clustering was done using the average linkage hierarchical clustering algorithm. The percentage of genome detected concordantly as gain, loss or neutral between parental tumor and derived cell line was calculated .DNA isolated from fresh/frozen cells was enzymatically labeled using the SureTag Labeling System  and DNA from FFPE tumor tissue was labeled using the Genomic DNA ULS Labeling Kit (Agilent) as described previouslyABL1, AKT1, ALK, APC, ATM, BRAF, CDH1, CDKN2A, CSF1R, CTNNB1, EGFR, ERBB2, ERBB4, FBXW7, FGFR1, FGFR2, FGFR3, FLT3, GNA11, GNAQ, GNAS, HNF1A, HRAS, IDH1, JAK2, JAK3, KDR, KIT, KRAS, MET, MLH1, MPL, NOTCH1, NPM1, NRAS, PDGFRA, PIK3CA, PTEN, PTPN11, RB1, RET, SMAD4, SMARCB1, SMO, SRC, STK11, TP53, VHL. The PhiX control library (Illumina) was spiked in each run at a final concentration of 10 pM to estimate the sequencing error rate. The pooled libraries were paired-end (2 × 151) sequenced on a MiSeq instrument (Illumina). The mean read depth for targeted regions (mean coverage) was ~ 1400×. Alignment and variant calling were done using the BaseSpace TruSeq Amplicon App, Version 3.0.0 (Illumina) using the somatic variant caller. Annotation was done based on RefSeqGene (https://www.ncbi.nlm.nih.gov/refseq/rsg/) and variants were further evaluated based on dbSNP 147 , COSMIC database  and ClinVar database35. Only non-synonymous variants were considered. Specific filtering criteria included (1) passed variant caller filters, (2) read depth ≥ 10 with a fraction of alternative reads ≥ 0.1, (3) ExAC frequency < 0.001%. Reads were visualized using the Integrative Genomics Viewer 37.Libraries of fresh and FFPE isolated DNA (250 ng) were prepared using the TruSeq Amplicon—Cancer Panel (Illumina). The TruSeq Amplicon—Cancer Panel spans mutational hotspots in > 35 kilobases (kb) of target genomic sequence in the following genes: We attempted to characterize how well genomic and phenotypic characteristics of patient tumors are maintained in cell lines compared to their tumor of origin. Towards this goal, we aimed to generate primary cancer cell lines from different gastrointestinal carcinomas using patient specimens Fig. .Figure 1We succeeded in establishing new primary cell lines from four patients comprising the following tumor entities: esophageal carcinoma, colon carcinoma, rectal carcinoma and pancreatic cancer Table . The majThe newly established cell lines mainly grew as adherent, i.e., anchorage-dependent, monolayers in vitro, showing epithelial cell morphology and retaining some characteristic features of the respective parental tumors such as gland formation Fig. . InteresNext, we aimed to determine the similarity of the immunophenotype of parental tumors and derived cell lines. To this end, we applied a panel of 22 immunohistochemical markers. The panel included intermediate filaments , adhesion molecules , lineage-specific transcription factors , glycoproteins/mucins with differential expression across tumors , a neuroendocrine marker (chromogranin A), a growth factor receptor (HER2/neu), a cell cycle checkpoint protein (cyclin D1), and a proliferation marker (Ki-67). Furthermore, DNA mismatch repair proteins  and TP53 were included.Overall, the newly established cell lines retained most of the immunophenotypic properties of the tumors they had been derived from, resulting in a strongly positive correlation as indicated by Pearson’s correlation coefficients r close to 1 ranging from 0.90 to 0.97 Fig.  and S2. Taken together, our immunophenotypic profiling the newly derived cell lines revealed that the expression profiles of cell lines are largely shared with the tumor of origin and characteristic of the respective tumor types in general. These cell lines hence recapitulate overall the histologic and immunophenotypic features of the tumors they were derived from, despite certain differences.To compare genome-wide DNA copy number changes of newly established cell lines and corresponding primary tumors, we performed aCGH analyses. The copy number profiles showed a non-random distribution of gains and losses that was characteristic for the respective tumor entities and similar, though not identical between parental tumors and tumor derived cell lines Fig. . ParentaTP53 and KRAS were consistently present in both primary tumor samples and the derived cell line 41. SATB2, a newer marker for colonic differentiation, was more robust and consistently expressed in both parental CRCs and corresponding derived cell lines, underlining the diagnostic superiority for this marker compared to CDX243.Here, we compared the genomic and phenotypic characteristics of newly generated, patient derived low-passage cell lines (passages 1–4) with their respective tumors of origin to assess in a direct manner how well patient tumor characteristics are maintained in culture. All newly established cell lines presented here grew in an epithelium-like pattern and largely retained the original tumor immunophenotype in vitro. The human epithelial nature was confirmed by cytokeratin expression in three out of four tumors 45. Clustering  of copy number data separated microsatellite stable tumors from the microsatellite unstable CRC while paired tumor and cell line samples clustered together, demonstrating the similarity of their respective chromosomal aberration patterns. However, despite the overall similarity of the CNA patterns between parental tumor and corresponding cell lines, certain differences could be observed. Overall, signal to noise ratios tended to be lower in the parental tumors compared to the cell lines. This lower sensitivity in copy number detection is owed to the dilution of tumor cell DNA by DNA from intermingled stromal and inflammatory cells. This is most obvious in PDAC, which usually  has an extensive stromal component and is also evident in the TCGA PDAC cohort46. In our study, the PDAC-derived cell line was mainly characterized by losses of chromosomes and chromosome arms 6, 8p, 17p (harbors TP53) and 18q (harbors SMAD4) along with gains of chromosome arms 8q (harbors the MYC gene) and 17q, consistent with previous reports47. However, only a fraction of 57% of the gains and losses in the cell line could be detected in the primary tumor, which had a tumor cell content of only 20%. The gain of 8q (harbors MYC), observable in the primary tumor and in the cell line, has been proposed as a metastatic driver in pancreatic ductal adenocarcinoma48. Patient 4 from our cohort indeed developed liver metastases within three months after surgery.Comparative analysis of chromosomal aneuploidy patterns by aCGH revealed generally consistent gain and loss patterns of parental tumors and derived cell line, reflective of the typical aneuploidy distributions observed in the respective tumor entities50. However, a loss of 9p and gain of 9q (possibly due to isochromosome formation), which do not typically occur in colorectal cancer, were solely detected in the cell line sample. One possible explanation for the discrepancy could be spatial intratumor copy number heterogeneity and the selective outgrowth of a tumor subclone that was underrepresented in the primary tumor52. Alternatively, this alteration could have been acquired during or induced by the culture process. Indeed, Greshock et al. have previously reported the loss of 9p, along with other copy number changes, as recurrent cell line-specific alterations53. A loss of 9p21.3 (CDKN2A locus) has also been described in the immortalization process of normal nasopharyngeal epithelial cells and may eventually confer a growth advantage in vitro under certain circumstances54. However, genomic alterations acquired during the culture process usually take time to be present in most of the cells .In contrast, the cell line derived from the tumor of patient 2  showed two differences that cannot necessarily be explained by a higher detection limit in patient tumors. The primary tumor and the derived cell line were characterized by gains of 7 and 20 along with a loss of 18q, a CNA pattern typical for CRC55. The microsatellite instable CRC and its derived cell line only displayed the lowest number of copy number alterations, which included a gain of chromosome 7, an event typically observed in CRC50.The highest CNA burden was observed in the least differentiated tumor . This is not surprising as CNA burden has previously been reported to be associated with more aggressive tumor behaviorKRAS or BRAF in CRC) were invariably present in both primary tumor sample and derived cell line18. Only in the MSI cell line minor discrepancies for mutations in PTEN and TP53 could be observed, likely attributable to the increased mutation rate as a consequence of MSI. We would like to emphasize that this did not affect mutations in driver genes such as BRAF or KRAS. This is in line with data from single cell subcloning of established CRC cell lines SW480 and HT29, which showed consistent mutations of KRAS and BRAF, respectively, across all established single cell clones—despite ongoing karyotype evolution/chromosomal instability16.As shown for colorectal cancer derived PDOs, mutations in known driver genes or genes of interest in the respective tumor entity (e.g., In conclusion, our low-passage cell lines closely recapitulated tumor-characteristic immunophenotypic profiles and maintained treatment-relevant genomic features including MSI status, key mutations of driver genes and tumor-type specific copy number alterations. Hence, this newly established cell line panel in conjunction with patient history could help to gain valuable insight into the mechanisms of drug sensitivity or resistance of tumors. These findings support further exploration of patient-derived cell lines as an ex vivo platform in cancer research and to personalize anticancer treatment.Supplementary Information.


EGFR mutation is EGFR‐tyrosine kinase inhibitors (TKIs). However, most patients will develop acquired resistance after 9–13 months. This study investigated novel molecular mechanisms of acquired resistance to EGFR‐TKIs to identify a potential new treatment for EGFR‐TKI resistant NSCLC patients.The first‐line standard treatment of non‐small cell lung cancer (NSCLC) with MET, PTEN, or EGFR expression changes. Next‐generation sequencing was performed and identified differentially expressed genes between two cell lines and examined the genes with GO and KEGG pathway database analyses. We also examined the molecular alterations in COSMIC and GDSC databases and performed hazard predictions using SIFT, PolyPhen‐2, Mutation Taster, and CADD.We established an EGFR‐TKI resistant cell line (HCC827‐TR) by culturing the HCC827‐P cell line through continuous erlotinib culture. We used Sanger sequencing, RT‐PCR, and western blot to rule out known resistance mechanisms in HCC827‐TR cells, including EGFR‐T790M and FGF2 as a differentially expressed gene with a G101T point mutation in HCC827‐TR cells that showed high mutation frequency and hazard score. HCC827‐TR cells showed elevated FGF2 compared to parental cells. It is noteworthy that treatment with the FGFR inhibitor AZD4547 could restore the sensitivity of HCC872‐TR cells to erlotinib.Our results identified FGF2 gene mutation. Targeted inhibition of the FGF2/FGFR signaling pathway may effectively restore the sensitivity of the resistant cells to erlotinib. These results suggest a novel treatment strategy for EGFR‐TKI resistant NSCLC patients.An erlotinib‐resistant cell line HCC827‐TR was successfully constructed and we identified the EGFR‐TKI resistance mechanism involving the Significant findings of the study: Identifies a novel molecular mechanism for EGFR‐TKI acquired resistance.What this study adds: A potential novel strategy for the treatment of EGFR‐TKI resistant NSCLC patients. Non‐small cell lung cancer (NSCLC) accounts for approximately 80% of all lung cancer cases,EGFR mutation is the most common genetic variant (50%–60%) in lung adenocarcinoma patients in East Asia. The IPASS studyEGFR mutation is an important strong predictor of the clinical efficacy of EGFR‐TKIs in lung adenocarcinoma. The deletion of exon 19 and the L858R point mutation in exon 21 are the most common EGFR mutation types and confer sensitivity to EGFR‐TKIs. Several large randomized phase III clinical trials, such as First‐SIGNA,EGFR sensitive mutations is significantly better than that with traditional chemotherapy. Meanwhile, the side effects can be well controlled as compared to those of chemotherapy. Accordingly, EGFR‐TKI has become a standard first‐line treatment for advanced NSCLC with EGFR‐TKI‐sensitive mutations.EGFR mutation, the overwhelming majority of patients acquire resistance after 9–13 months, leading to disease progression.EGFR mutation of T790M that occurs in exon 20 and accounts for 50%–60% of all cases.MET amplification,EGFR mutant gene,PTEN deletionDespite the initial beneficial effects of EGFR‐TKI in patients with The purpose of this study was to first establish an EGFR‐TKI drug‐resistant cell line and screen for resistance mechanisms. We then conducted high throughput whole exon sequencing of the primary cell line and drug‐resistant cell line to identify and examine potential differentially expressed genes that might lead to drug resistance. We also performed targeted inhibition of the identified gene signal downstream pathway in tumor cells to observe changes of drug resistance to confirm the significance for further clinical study.2. The cells were subcultured when they reached 80%–90% adherence on the culture dishes and showed good morphology.HCC827‐P cells were obtained from an American type culture collection and cultured in RPMI‐1640 medium at 37°C in a saturated humidity atmosphere containing 5% COHCC827‐P cells were cultured in a medium containing erlotinib at 1 nmol/L. When the cells showed viability similar to that of cells without erlotinib, we gradually increased the concentration of erlotinib until it reached 1 μmol/L. The entire induction period lasted approximately eight months.4 cells in each well. After incubation at 37°C for 24 hours, different concentrations of erlotinib were added into the wells in triplicate. The half maximal inhibitory concentration (IC50) was calculated using curve regression analysis.Cells were plated into 96‐well plates at 200 μL cell suspension containing approximately 1.0 × 10DNA was extracted from HCC827‐P and HCC827‐TR cell lines using the TRIzol Reagent Kit . Four dNTPs were added to the PCR reaction system. Fluorescence labeling was performed, and the samples were tested with urea‐modified polyacrylamide gel electrophoresis.GAPDH using the 2−ΔΔCt method.Total RNA was extracted using the TRIzol Reagent Kit ; cDNA reverse transcription was performed using the Reverse Transcription Kit  as per the manufacturer's instructions. RT‐PCR was performed using Quantitative PCR Kit . The primer pairs are shown in Table Cells were lysed, and total protein was extracted. The proteins were separated with sodium dodecyl sulfate polyacrylamide gel electrophoresis and were then transferred to a PVDF membrane. After blocking, the membranes were incubated with primary antibodies (GAPDH (sc‐47 724) from Santacruz, Delaware Ave, USA; PTEN (9559L), FGF2 (3196S), FGFR (3471S), FRS2 (3864L) from cell signaling, Boston, USA) at 4°C overnight and then incubated with secondary antibody. Protein bands were analyzed using the Chemi Doc XRS System .Total DNA was extracted using the TRIzol Reagent Kit. Genomic DNA was randomly fragmented into DNA fragments of 180–250 bp. A DNA library was prepared, and liquid phase hybridized with biotin‐labeled probes. We captured the exons of target genes and performed single molecule amplification. The whole exon region of two cell lines were detected and contrasted with the Agilent‐V6 chip capture system in Illumina HiSeq2500 sequencing platform (150 bp paired end).5 cells/well) and treated with various concentrations of erlotinib alone, or in combination with AZD4547 in triplicate. After being cultured and collected, they were added to an Eppendorf tube with 5 μL Lysol phosphatidyl ethanolamine , 5 μL 7‐amino‐actinomycin D , and 400 μL loading buffer; the cells were analyzed for apoptotic populations using flow cytometry .Cells were plated in six‐well plates  using HCC827‐P as described in the Methods section. Phase contrast microscopy demonstrated that HCC827‐P and HCC827‐TR showed an epithelioid monolayer arrangement. HCC827‐P had clear boundaries and were arranged regular with round or oval shapes, a large nucleus and attached to each other   in a concentration‐dependent manner , but there was significant statistically differences between the other groups    and MET (P = 0.049); these were altered, but the difference was not significant. Similarly, the expressions of PTEN in the two cell lines also showed no statistical difference (P = 0.456)  in the HCC827‐TR cell line was 581, and 228 have been reported in the dbSNP database. Single sample alteration type statistics showed that the highest alterations incidences were G > A, C > T. The least common alterations were A > C and T > G  in HCC827‐TR cells was 10; they were increased in five regions and reduced in five regions, with a total length of 9 611 185 bp and 1 523 874 bp, respectively. The CNVs were not located in any presently known gene loci that could lead to drug resistance. There were five chromosomes with CNVs, including chromosomes 4, 6, 9, 12, and 21  and FGFR2 (P = 0.165) in the two cell lines , while the expression levels of FGFR2 in the two cell lines were not significantly different  signaling pathways are activated with the phosphorylation of key kinases AKT and ERK, respectively. We compared the levels of P‐AKT and P‐ERK in the two cell lines with or without 1 μmol/L erlotinib. They significantly decreased in HCC827‐P cells treated with 1 μmol/L erlotinib; however, in HCC827‐TR cells, the level of P‐ERK decreased slightly, and P‐AKT showed almost no change after treatment with 1 μmol/L erlotinib compared with the control , regardless of the concentration of AZD4547; the results were similar in HCC827‐TR cells . When we compared the two combined treatment groups (E0.1 + A5 and E0.1 + A10), although the inhibition rate showed a statistical difference, the difference was not significant in HCC827‐P cells (P = 0.048), and no significant difference was observed in the HCC827‐TR cells (P = 0.085)  in HCC827‐P and HCC827‐TR cell lines  or 1 μmol/L (E1) erlotinib combined with 5 μmol/L (A5) or 10 μmol/L (A10) AZD4547 (FGF2/FGFR inhibitor). MTS assay in HCC827‐P cells revealed a significant difference in the inhibition rate between the single drug group and the combined groups (P = 0.000) and HCC827‐TR (P = 0.000) cells, the rise level of the apoptotic index in HCC827‐TR cells treated with E1 + A10 was significantly higher than that in HCC827‐P cell lines  HCC827‐P cell line and established HCC827‐TR resistant cells. MTS assays revealed that IC50 of HCC827‐P cells was about 0.1 μmol/L, while that of HCC827‐TR cells was at least 10 μmol/L, indicating that its resistance was 100 times higher than that of the primary cells. These results confirmed that the HCC827‐TR cell line was an EGFR‐TKI resistant cell line.The mechanism of EGFR‐TKI resistance has been defined at around 60%–70%, classified into the secondary mutations in EGFR at the DNA level; the results revealed that both HCC827‐TR and HCC827‐P cell lines did not contain the T790M mutation. Therefore, the mechanism of resistance was not related to the secondary mutations. Meanwhile, the expressions of proto‐oncogene MET and anti‐oncogene PTEN in two cell lines were not significantly different.Sanger sequencing was used to examine secondary mutations in FGF2 for subsequent studies. Previous studies have shown that FGF2 can promote the growth of malignant tumors via autocrine and paracrine pathways and that it plays an important role in tumor migration.et al.et al.NGS was used to examine whole exons of the two cell lines, and after a series of bioinformatics analyses, we selected FGF2 resulted in substitution of glycine by valine. We did not detect any difference in the mRNA expression of FGF2 in the two cell lines; however, FGF2 protein expression was significantly higher in HCC827‐TR cell lines compared with that in the parental cells. Moreover, the expression of FGFR showed no significant differences between the two cells, both in the mRNA and protein levels. We believe that the point mutation in FGF2 may result in the conformational changes or altered binding sites of the translated protein, resulting in hindered degradation of FGF2. High levels of FGF2 binding with FGFR can activate the signaling pathway and induce cell proliferation and resistance. In addition, the expressions of the P‐AKT and P‐ERK were significantly downregulated in response to 1 μmol/L erlotinib in HCC827‐P cells, while the continuous activation of P‐AKT and P‐ERK was still present in the HCC827‐TR cell line after the addition of erlotinib. This suggests that the FGF2‐FGFR continuous activation of the downstream network signal pathway through bypass and the growth of the tumor cells is not restricted by the inhibitory effect of EGFR‐TKI. However, this is only a hypothesis, and further experiments are needed to verify this possibility.The point mutation of G101T in exon 1 in http://clinicaltrials.gov website, 26 FGFR‐targeted agents are currently under investigation in clinical trials, of which 13 agents are being tested in phase I trials, 10 agents are being investigated in phase II trials, AZD4547 and erdafitinib are in phase III, although no drugs have been approved for lung cancer, some studies have shown positive results.The fibroblast growth factor receptor pathway plays a key role in signal transduction in lung cancer; it can activate multiple signal transduction pathways, including rat sarcoma (RAS) kinase and MAPK, which in addition to performing proliferative functions, controls cellular processes, such as cell cycle progression, migration, metabolism, and differentiation.FGF2 mutation is an important mechanism of resistance in HCC827‐TR cells. Our apoptosis results also confirmed this conclusion.AZD‐4547 is a highly selective FGFR inhibitor; a patient‐derived xenograft model of FGFR1‐amplified lung cancer was highly sensitive to AZD4547 as single agent.In conclusion, we successfully constructed an erlotinib‐resistant cell line HCC827‐TR and identified EGFR‐TKI resistance mechanism involving FGF2 gene mutation. It is noteworthy that the targeted inhibition of the FGF2/FGFR signaling pathway could effectively restore the sensitivity of the resistant cells to erlotinib. These results not only expand our understanding of the molecular mechanisms underlying drug resistance in NSCLC cancers, but also suggest a novel treatment strategy for EGFR‐TKI resistant NSCLC patients.No authors have financial or other contractual agreements that might cause conflicts of interest.Table S1 Primers used for RT‐PCR.Click here for additional data file.Table S2 The mutation frequency of differentiated genes in HCC827‐TR cell lines.Click here for additional data file.